**Biomarkers and Nutrients in Musculoskeletal Disorders**

Editors

**C ´esar Calvo-Lobo Ricardo Becerro de Bengoa Vallejo Marta Elena Losa-Iglesias Marta San-Antol´ın David Rodr´ıguez Sanz Daniel L ´opez-L ´opez**

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This is a reprint of articles from the Special Issue published online in the open access journal *Nutrients* (ISSN 2072-6643) (available at: https://www.mdpi.com/journal/nutrients/special issues/ biomarkers musculoskeletal).

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## **Contents**


### *Editorial* **Biomarkers and Nutrients in Musculoskeletal Disorders**

**César Calvo-Lobo 1, Ricardo Becerro-de-Bengoa-Vallejo 1, Marta Elena Losa-Iglesias 2, David Rodríguez-Sanz 1,\*, Daniel López-López <sup>3</sup> and Marta San-Antolín <sup>4</sup>**


Worldwide, the burden of musculoskeletal disorders is increasing with great variations between-countries, which makes it difficult for policymakers to provide resources and adequate interventions in order to provide for their appropriate management [1]. Thus, musculoskeletal disorders remain a public health problem and their incidence and trend is increasing for some specific conditions [2].

Indeed, nutrients and biological biomarkers play a key role in the prognosis, diagnosis and health status of patients suffering from musculoskeletal conditions, being the main indicators for understanding biological processes as well as tailoring therapeutic interventions and nutritional programs in patients with musculoskeletal disorders [3].

Thus, the purpose of this Special Issue was to provide an update on the state of the art, through current reviews as well as new insights and interventions, about the main role of nutrients and biomarkers in patients who suffer from musculoskeletal conditions from a multidisciplinary point of view. According to the aim of this Special Issue, a total of six papers summarized in Tables 1 and 2 were published between May and December, 2020, in Nutrients.

Among these publications, three papers were reviews providing an update of various topics including the current metabolic impact of COVID-19 pandemic confinement secondary to diet modifications and the reduction of physical activity [4]; the influence of nutrients for osteoporosis prevention in a specific population of patients suffering from inflammatory bowel disease [5]; and the relationship of nutrient and metabolic status with spinal muscular atrophy [6].

In addition, three papers were original research studies supporting key information from a multidisciplinary approach about the effects of curcumin and resveratrol on satellite cells as well as biomarkers for muscle regeneration process [7]; the fat rich diet effect on acetylcholine spontaneous release produced in mice neuromuscular junctions revealing key aspects for the relationship myofascial pain syndrome and nutrients or biomarkers [8]; and the effects of supplementation with vitamin D and L-cysteine on the biomarkers of musculoskeletal conditions in high-fat-diet–fed mice with vitamin D deficiency [9].

The main characteristics of the reviews provided in this Special Issue are presented in Table 1, updating the state of the art of the available scientific evidence on nutrients and biomarkers in musculoskeletal conditions about trending topics such as the effects of the COVID-19 pandemic, the prevention of osteoporosis related to inflammatory bowel disease, as well as the influence of nutrients and biomarkers in spinal muscular atrophy [4–6].

Firstly, Martínez-Ferran et al. [4] provided an interesting review which detailed the key metabolic consequences of the COVID-19 confinement, such as increased insulin resistance, body and abdominal fat, as well as inflammatory cytokines, which are linked to metabolic

**Citation:** Calvo-Lobo, C.; Becerro-de-Bengoa-Vallejo, R.; Losa-Iglesias, M.E.; Rodríguez-Sanz, D.; López-López, D.; San-Antolín, M. Biomarkers and Nutrients in Musculoskeletal Disorders. *Nutrients* **2021**, *13*, 283. https://doi.org/ 10.3390/nu13020283

Received: 14 January 2021 Accepted: 18 January 2021 Published: 20 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

syndrome development and the appearance of musculoskeletal disorders, among others. The authors encouraged researchers to explore positive energy balance as a plausible mechanism for these nutritional and musculoskeletal disorders, and suggested that the restriction of calorie intake could prevent the COVID-19 confinement impact associated with physical inactivity.

**Table 1.** The characteristics of the review studies included in the Special Issue about nutrients and biomarkers in musculoskeletal diseases.


Secondly, Ratajczak et al. [5] reviewed the influence of nutrients in preventing osteoporosis and low bone mineral density in patients who suffered from inflammatory bowel disease, i.e. ulcerative colitis and Crohn's disease. Vitamin D and calcium were the most commonly studied nutrients for bone mineral density. In addition, vitamins such as A, B12, C and K, calcium, folic acid, magnesium, phosphorus, zinc, sodium, selenium and copper were implicated in bone mass formation. These patients seemed to commonly consume inadequate amounts of these vitamins and minerals, impairing absorption and disturbing nutritional status with an increase in the risk of osteoporosis. Therefore, the authors also encouraged researchers to develop nutritional guidelines for patients suffering from inflammatory bowel diseases to prevent osteoporosis.

Lastly, the review carried out by Li et al. [6] highlighted the relationship between nutrient and metabolic status and neurodegenerative diseases. Concretely, various fatty acids' metabolic alterations and glucose tolerance impairment were linked to spinal muscular atrophy. Thus, nutritional support as well as monitoring of biomarkers and nutrients may play a key role in patients who suffer from spinal muscular atrophy. Furthermore, metabolomics may provide a promising support as therapeutic targets or specific biomarkers for metabolic alterations and for the quantification of specific metabolites in patients who suffer from this neurodegenerative disease.

The principal characteristics of the original research reports published in this Special Issue are presented in Table 2, providing new insights and interventions about the key role of nutrients and biomarkers in patients who suffer from musculoskeletal conditions, dealing with specific main topics such as the importance of satellite cells in the process of muscle regeneration, the release of acetylcholine in the neuromuscular junction for the myofascial pain syndrome development, as well as nutritional supplementation for musculoskeletal disorders [7–9].

**Table 2.** Characteristics of the original research reports included in the Special Issue about nutrients and biomarkers in musculoskeletal diseases.


First, Mañas-García et al. [7] carried out an excellent experimental laboratory study detailing the curcumin and resveratrol effects on satellite cells by analyzing muscle regeneration biomarkers. Resveratrol and curcumin supplementation in the immobilized muscles of mice elicited an increase in muscle satellite cells. Curcumin treatment for reloaded muscles improved the cross-sectional area of hybrid muscle fibers and sirtuin-1 activity, while resveratrol treatment for reloaded muscles improved the cross-sectional area of fast-twitch muscle fibers, sirtuin-1 content and progenitor muscle cell count. In addition, curcumin and resveratrol treatment for unloaded muscles improved the satellite cell number. The authors encourage the use of curcumin and resveratrol due to their potential clinical effects against muscle disuse and atrophy to optimize the muscle regeneration process.

Second, Gimenez-Donoso et al. [8] performed an outstanding experimental laboratory study which supported novel findings in a prevalent musculoskeletal condition such as myofascial pain syndrome, showing the first relationship in the research literature between this syndrome and nutritional biomarker status due to a fat rich diet producing acetylcholine spontaneous release in neuromuscular junction of mice. Spontaneous acetylcholine release at the neuromuscular junction was previously proposed as the key mechanism activating a vicious circle which perpetuates myofascial pain syndrome as a group of motor, sensitive and autonomic signs and symptoms originating from myofascial trigger points. This experimental laboratory study was carried out in male Swiss mice evaluating intramuscular adipocytes with Sudan-III and the plaque noise of the neuromuscular junction suggesting spontaneous acetylcholine release by electromyography. An increased plaque noise was presented after the interruption of the proposed diets. Thus, supplementation by a hypercaloric diet increased spontaneous neurotransmission in the neuromuscular junction promoting the development of myofascial trigger points.

Finally, Parsanathan et al. [9] performed an experimental laboratory study in high-fatdiet–fed mice with vitamin D deficiency, showing that vitamin D and L-cysteine cosupplementation mitigated biomarkers of musculoskeletal conditions. Vitamin D and L-cysteine cosupplementation provided beneficial effects for gene expression regarding myogenic biomarkers. Therefore, cosupplementation of vitamin D and L-cysteine improved the skeletal muscle biomarkers of musculoskeletal conditions more than monotherapy in vitamin D deficient high-fat-diet mice.

Future studies should address the role of ultrasound imaging as a promising tool for nutrient and biomarker status in musculoskeletal disorders [10]. The analysis of muscle tissue by sonoelastography may provide an alternative tool to indirectly evaluate sarcopenia and the lack of skeletal muscle mass from both qualitative and quantitative points of view, presenting a sensitivity of 77.3%, a specificity of 100% and a diagnostic accuracy of 87.5% [11]. The expression of mediators of neoangiogenesis and vascular density of the synovial characteristics in patients with rheumatoid arthritis provided a good correlation (*r* = 0.73) with the vascular area at histological level, linked to the cellular profile and pro-inflammatory cytokines, which provided considerable validity for using these measurements as objective assessments of synovial inflammation in clinical practice [12]. In addition, ultrasound analysis of the echo-texture, echo-intensity and echovariation of the muscle tissue suggested promising results as biomarkers in neuromuscular pathologies such as amyotrophic lateral sclerosis [13]. Finally, the ultrasound software for the evaluation of muscle glycogen in the skeletal muscle may be decisive in detailing the musculoskeletal status and risk of injury in sport performance with promising results in this field, being a validated software with respect to muscle biopsy (*r* = 0.81) that allowed a non-invasive assessment of muscle glycogen [14,15].

In conclusion, the main findings of this Special Issue summarize the current scientific evidence available about nutrients and biomarkers in musculoskeletal diseases related to the metabolic impairment secondary to COVID-19 confinement, the prevention of osteoporosis for patients suffering from inflammatory bowel diseases, and nutritional issues in patients who suffer from spinal muscular atrophy, as well as new insights from experimental animal models on pharmacological agents to enhance muscle tissue regeneration, spontaneous acetylcholine release in the neuromuscular junction of mice by hypercaloric diet supplementation increasing spontaneous neurotransmission and the consequent activation of myofascial trigger points to originate myofascial pain syndrome, and the improvement of myogenic biomarkers of musculoskeletal conditions and gene expression due to vitamin D and L-cysteine cosupplementation. Future studies should investigate the role of ultrasound imaging as a promising tool for nutrient and biomarker status in musculoskeletal disorders.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Metabolic Impacts of Confinement during the COVID-19 Pandemic Due to Modified Diet and Physical Activity Habits**

#### **María Martinez-Ferran 1, Fernando de la Guía-Galipienso 2,3,4, Fabián Sanchis-Gomar 5,6 and Helios Pareja-Galeano 1,\***


Received: 2 April 2020; Accepted: 20 May 2020; Published: 26 May 2020

**Abstract:** While the detrimental effects of a chronic positive energy balance due to a sedentary lifestyle have been well established, the impacts of a short period of abruptly reduced physical activity and overeating arising from strict confinement due to the COVID-19 pandemic will soon start to emerge. To reasonably anticipate major consequences according to the available evidence, we hereby review the literature for studies that have explored the health impacts of several weeks of a reduction in physical activity and daily step-count combined with modified eating habits. These studies identify as main metabolic consequences increases in insulin resistance, total body fat, abdominal fat and inflammatory cytokines. All these factors have been strongly associated with the development of metabolic syndrome, which in turn increases the risk of multiple chronic diseases. A plausible mechanism involved in these impacts could be a positive energy balance promoted by maintaining usual dietary intake while reducing energy expenditure. This means that just as calorie intake restriction could help mitigate the deleterious impacts of a bout of physical inactivity, overeating under conditions of home confinement is very likely to exacerbate these consequences. Moreover, hypertension, diabetes, and cardiovascular disease have been identified as potential risk factors for more severely ill patients with COVID-19. Thus, adequate control of metabolic disorders could be important to reduce the risk of severe COVID-19.

**Keywords:** COVID-19; acute sedentary lifestyle; step reduction; positive energy balance; metabolic consequences; insulin resistance; metabolic syndrome; sarcopenia

#### **1. Introduction**

The current COVID-19 pandemic has led governments of the mainly affected countries to impose strict confinement rules on their citizens. These include measures such as working from home and closing schools, shops, restaurants and any business or service considered non-essential in order to slow down the spread of the contagion and thereby prevent the collapse of health care systems. These measures have, however, had their impacts on the general health of the population because of both exercise restrictions and effects on diet. Exercise restrictions have been the consequence of closed gyms and sports centers, restrictions on walking distance, lack of space and infrastructure of homes for

physical exercise, and lack of technical knowledge of the population on appropriate training routines. Effects on nutrition include limited access to shops, a poorer quality of food products due to the already visible impacts on family income, and overeating. Before this pandemic, insufficient physical inactivity was already described as a global public health problem, with over a quarter of all adults not undertaking the levels of physical activity required for good health [1]. As a result of the current situation in which many people are confined to their homes, physical activity and exercise levels drastically decline while dietary habits remain unchanged or fail to offset this inactivity producing a positive energy balance. There is strong epidemiological evidence that a chronic sedentary lifestyle is detrimental for health [2,3]. Likewise, there is evidence that such negative effects persist even when performing physical exercise programs, revealing that it is just as important to reduce sitting times as it is to lengthen exercising periods [4].

Exercise plays a fundamental role in the prevention of most chronic diseases. Our body needs a relatively long period to benefit from the healthy adaptations that exercise generates, modulated by different molecular mechanisms such as epigenetics, metabolic modulation or reduced inflammation [5–13]. Unfortunately, it requires only a period of a few days to reverse these adaptations, and the body returns to a physiological situation similar to baseline or even worse [2]. This means that trying to maintain an active lifestyle during home quarantine is essential to avoid physical consequences and this approach may also help mitigate the psychological impacts of confinement, especially among the elderly [14–16].

To analyze the consequences of physical inactivity and an acute positive energy balance due to changes in eating habits, different models have been employed [17,18]. Nevertheless, these interventions do not reflect the current home confinement situation, in which daily physical activity is drastically reduced and there is a tendency to eat more and worse affecting the risk of metabolic-associated chronic diseases such as cardiovascular diseases (CVD) across a large part of the world population (Figure 1). This paper therefore reviews evidence of the metabolic-health impacts of a short period of a reduction in physical activity and a tendency to overeat.

**Figure 1.** Consequences of overfeeding and reduced physical activity.

#### **2. Methods**

Electronic databases (Medline, EMBASE, and Web of Science) were searched without language restrictions to identify all reports on metabolic-related alterations, physical inactivity and overfeeding. Inclusion criteria were: (i) publication in a peer-reviewed journal, (ii) human study, (iii) studies examining the impacts of acute physical inactivity, and (iv) studies examining the impacts of acute changes in dietary habits. Reasons for exclusion were: (i) studies in non-adult subjects, (ii) no control group, (iii) data reported not usable.

#### **3. Results and Discussion**

#### *3.1. Metabolic Consequences of Acute Physical Inactivity in Healthy Adults*

In this section, we review the impact of acute physical inactivity on glycemic control, inflammatory markers, body composition and cardiorespiratory fitness (CRF) in healthy young adults (Table 1).

Pedersen et al. [19,20] employed a step reduction model to examine the consequences of current lifestyles involving prolonged periods of inactivity interspersed with short episodes of low to moderate physical activity. In their investigation, participants were instructed to reduce daily steps by taking lifts instead of stairs and using their cars instead of walking or cycling.

The two following studies were conducted on healthy young men who performed less than 2 h of regular exercise per week and walked more than 3500 steps per day. During the interventions, participants maintained their usual dietary habits. In the first study, participants were recruited for 2 sub-studies [19]. In the first sub-study [19], eight participants (27.1 (5.7) years; body mass index (BMI) 22.9 (4.0) kg/m2) reduced their activity from 6203 steps/day (5135–7271) to 1394 steps/day (1261–1528) for three weeks. Results included a significant increase in the area under the curve (AUC) for plasma insulin during an oral glucose tolerance test (OGTT) from baseline to the third week. In the second sub-study of the first study [19], ten subjects (23.8 (4.6) years; BMI 22.1 (2.1) kg/m2) reduced their mean activity level of 10,501 steps/day (8755–12,247) to 1344 steps/day (1272–1416) for 2 weeks. In this study, both plasma insulin AUC and plasma C-peptide levels increased significantly after the inactivity period. In the oral fat tolerance test, AUCs for plasma insulin, C-peptide and triglycerides (TG) also increased significantly. Further, while total fat mass (FM) remained unchanged, intra-abdominal FM increased (by 7%) after the two weeks of step reduction, and this was accompanied by a significant reduction in total free fat mass. In the second study [20], ten young healthy males (23.8 (1.5) years; BMI 22.1 (0.7) kg/m2) reduced their daily steps from a baseline value of 10,501 (808) to 1344 (33) for two weeks. This step reduction led to a decreased glucose infusion rate due to a reduction in peripheral insulin sensitivity, and a concurrent decrease in insulin-provoked muscle Akt phosphorylation with no effect on endogenous glucose production in the liver. In contrast to the results of study 2 [19], there was no significant change in total FM after step reduction, but leg lean mass was lower. Finally, two weeks of physical inactivity did not produce changes in plasma levels of TG, free fatty acids (FFA), glucose, insulin or C-peptide.

Interestingly, the findings of a similar study indicated that just three days of step reduction from 12,956 (769) to 4319 (256) steps/day led to impaired glycemic control in twelve healthy active participants (8 men, 4 women; 29 (1) years; 23.6 (0.9) kg/m2) [21]. In response to this short intervention, the authors also noted increased postprandial glucose levels, increased fasting plasma insulin and C-peptide responses to OGTT, along with increased insulin resistance and diminished insulin sensitivity.

Maximal aerobic capacity (VO2max) is a major predictor of functional capacity and is the gold standard indicator of CRF. A person's VO2max also reflects pulmonary and muscle function, nutritional status or the health state of other organ systems. While higher CRF is associated with better health, lower CRF is associated with increased mortality independently of other risk factors [2]. Several studies [20,22–24] have determined the effects of an acute period of inactivity on VO2max. Results indicated that following two weeks of physical inactivity VO2max was reduced when participants maintained their normal diet [20,24], when calorie intake was restricted [23] or when intake was increased by 50% kcal [22]. In two of these studies, nevertheless, it was found that VO2max returned to baseline after normal physical activity was resumed [22,24].

**Table 1.** Summary of the studies reviewed examining the e ffects of acute physical inactivity and/or overfeeding.




Area under curve (AUC), alanine transaminase (ALT), body mass (BM), body mass index (BMI), cardiorespiratory fitness (CRF), continuous glucose monitoring (CGM), C-reactive protein (CRP), exercise (EX), fat mass (FM), females (F), first-degree relatives with type 2 diabetes (FDR+ve), first-degree relatives without type 2 diabetes (FDR−ve), free fatty acids (FFA), glucose infusion rate (GIR), high density lipoprotein cholesterol (HDL-c), homeostatic model assessment for insulin resistance (HOMA-IR), hyperinsulinemic-euglycemic during clamp (H-E), LDL-C (low-density lipoprotein cholesterol), males (M), maximal voluntary contraction (MVC), muscle protein synthesis (MPS), non-esterified fatty acids (NEFA), oral glucose tolerance test (OGTT), physical activity (PA), short physical performance battery (SPPB), soluble intercellular adhesion molecule (sICAM), step reduction (SR), triglycerides (TG), total cholesterol (TC),tumornecrosisfactoralpha(TNF-α),whitebloodcells(WBC),ྥnosignificantchange,↑significantincrease,↓significantdecrease,>significantlyhigher,<significantlylower.

Collectively, the above data indicate that a reduction in daily physical activity of three days, two weeks or three weeks impairs glycemic control. In addition, two weeks of step reduction increased FM and reduced lean mass, and also had a negative impact on VO2max.

#### *3.2. Metabolic Consequences of Acute Physical Inactivity in Overweight Adults*

In the previous articles, interventions were conducted on subjects within the normal weight range. Additionally, similar step reduction interventions have been tested on both overweight and normal weight populations to compare the consequences of acute physical inactivity (Table 1).

To examine whether reduced physical activity in adults with central overweight could lead to a rapid decline in metabolic and inflammatory homeostasis compared to what happens in lean counterparts, Dixon et al. [25] performed a study in which individuals were subjected to a week of step reduction to below 4000 steps/day. The participants of this study were nine active men with central overweight (49 (1.0) years; BMI 29.3 (1.2) kg/m2) and nine active lean men (51.5 (1.4) years; BMI 29.3 (1.2) kg/m2) who maintained their normal diet. In the former group, subjects had significantly higher total FM and abdominal FM levels before the intervention (these variables were not nevertheless determined after the intervention). Results indicated that, while insulin and glucose AUC responses to an OGTT and fasting TG concentrations increased in both groups, in the overweight group, glucose and insulin AUC, TG and C-reactive protein (CRP) and alanine transaminase were all higher before the intervention and remained so throughout. No changes were produced in total cholesterol, low-density lipoprotein (LDL) or high-density lipoprotein (HDL) cholesterol over the intervention period and neither did differences emerge between groups.

In another study, the metabolic consequences of a drop in physical activity from a daily step-count of <10,000 to 1500 for 14 days were examined in 45 active healthy participants who continued with their usual diet [24]. Of these 45 participants, 16 had first-degree relatives with type 2 diabetes (10 females, 6 males; 40 (14) years; BMI 27 (5) kg/m2) and 29 did not (18 females, 11 males; 33 (13) years; BMI 24 (3)). Those in the former group had a significantly higher BMI classified as "overweight" and greater waist and hip circumferences, although there were no significant differences in FM. Both groups experienced a significant reduction in insulin sensitivity accompanied by a significant decrease in glucose and insulin AUC. Although both groups showed a reduction in muscle insulin sensitivity, the overweight group displayed a lower sensitivity. The period of reduced physical activity significantly lowered VO2max across the study population without differences between groups. Total lean mass and lower limb lean mass decreased significantly and there was a significant increase in total FM and liver fat; those with overweight accumulated more android fat (1.5%) after step reduction. Lipid profiles were also modified in that higher total cholesterol, LDL-cholesterol and TG were recorded after step reduction, a greater TG increase being detected in the overweight subjects. All variables returned to baseline values 14 days after the subjects resumed their usual physical activity. After resuming normal activity, the overweight group engaged in lower amounts of vigorous activity and had lower insulin sensitivity.

According to the findings of a study by Bowden et al. [29], obese individuals with metabolic syndrome had lower CRF (measured as VO2max peak) than both non-obese subjects without metabolic syndrome and non-obese individuals with metabolic syndrome, the latter being the most sedentary population. The authors also found that higher VO2max peak, lesser sedentary time and average daily METS were correlated with lower liver fat. Results suggested that high levels of CRF in the overweight and obese population significantly reduced or eliminated the elevated risk of CVD and all-cause mortality. This indicates that CRF changes the relationship between body fat and its prognosis. We should underscore that many of the benefits of improved CRF are derived from an increase in physical activity [30].

In conclusion, in these studies a reduction in acute physical activity negatively influenced glycaemia control and lipid profile (TG, total cholesterol, LDL-cholesterol). Regarding body composition, step reduction increased FM, liver fat mass and reduced lean mass in both overweight and normal weight subjects. However, in those overweight, consequences were usually somewhat greater.

#### *3.3. Detrimental Health E*ff*ects of an Acute Sedentary Lifestyle in the Elderly*

The prevalence of sarcopenia is high among the elderly. A loss of skeletal muscle mass and strength has several repercussions on health, and all conditions in which muscle activity is reduced can lead to sarcopenia [31]. Moreover, ageing is associated with abdominal obesity, an important contributor to insulin resistance and metabolic syndrome, along with a higher level of proinflammatory cytokines [32]. Accordingly, drastic decreases in physical activity could have worse consequences in elderly subjects by accelerating the ageing process and the appearance of age-related diseases. As an example, the two studies described below examine the impacts of a step reduction intervention in elderly subjects on glycemic control, body composition, inflammatory parameters and CRF (Table 1).

Breen et al. (2013) conducted a study in healthy older adults (5 males, 5 females; 72.3 (1.0) years; BMI 29.0 (1.8)) who were moderately active (>3500 steps/day). During the intervention, participants reduced their daily step-count by approximately 76% of habitual levels while maintaining their dietary habits. After this period, insulin resistance was increased and postprandial insulin sensitivity was reduced. Fasting insulin concentrations and its peak plasma concentrations at 30 min of OGTT were greater after step reduction and AUC for plasma glucose and insulin during OGTT increased. The body composition data revealed that after step reduction, body fat percentage increased and skeletal leg muscle mass was significantly reduced. Further findings were postprandial rates of myofibrillar protein synthesis reduced by approximately 26% after the intervention with no difference in postabsorptive rates.

A similar step-reduction intervention was conducted in 22 moderately active older adults (12 males: 69 (3) years, BMI 27.3 (4.6): 10 females; 70 (5) years, BMI 27.7 (5.1)) [28]. Participants reduced their daily step-count by 70% and maintained their usual dietary habits. Body composition variables remained unchanged. However, the authors reported that just a week of step reduction led to increased insulin resistance and reduced insulin sensitivity. Moreover, glucose and insulin AUC were elevated as were fasting plasma glucose and insulin concentration during OGTT. Also observed was a reduction in muscle protein synthesis. What it is interesting to point out is that after the step reduction protocol, participants were reassessed after 14 days of return to their habitual step-count. In this examination it was confirmed, however, that glycemic control and inflammatory markers had not recovered. In contrast, in a younger study population (36 (14) years) changes in metabolic variables produced were reversed when normal physical activity levels were recovered [28].

In both studies conducted in older subjects, plasma concentrations of inflammatory markers (TNF-α and CRP) were significantly increased after the step reduction intervention [27,28]. In the second study [28], IL-6 was also increased and after returning to normal activity, inflammatory markers were still elevated. In contrast, in the studies conducted in young individuals, no changes were produced in inflammatory parameters [20,22,25,26].

In summary, acute physical inactivity led to impaired glycemic control, increased inflammation and reduced muscle protein synthesis. Inactivity may also reduce fat free mass while increasing FM. In addition, recovering normal levels of activity in the elderly could be harder compared to younger subjects.

#### *3.4. Metabolic E*ff*ects of Acute Physical Inactivity plus Overfeeding*

Energy balance is the state in which energy intake equals energy expenditure. A positive energy balance, whereby energy intake exceeds expenditure, can lead to weight gain due to increased body fat [33]. In the studies described in the previous section, participants kept up their usual dietary intake while reducing energy expenditure, resulting in a positive energy balance that may have contributed to the observed metabolic changes [19,20,24,25]. In this section, we review the impact of physical inactivity when added to a diet intervention (Table 1).

In a study of the effects of two weeks of step reduction combined with overfeeding, nine healthy young men (24.3 (3.3) years; BMI 21.6 (2.5) kg/m2) undertook 14 days of step reduction from 10,278 (2399) to 1521 (488) steps/day and increased their daily total energy intake by 50% kcal [22]. This study showed that insulin sensitivity reduction occurs after three days of inactivity and overfeeding. Clamp-derived insulin sensitivity was reduced after 14 days of inactivity and oral glucose tolerance remained unaffected. The insulin response to OGTT increased after the first and the second week. Body composition was affected by the intervention, in that body weight was higher due to an increase in total FM (1.5 (0.5) kg; *p* < 0.05), and in android, gynoid and visceral fat. Finally, plasma levels of leptin and adiponectin increased after the intervention and no changes in TG or FFA were observed. The authors of this study also reported that after 16 days of returning to an uncontrolled free-living environment, body weight and adiposity were still elevated while remaining variables returned to baseline values.

Interestingly, one study explored whether metabolic dysfunction caused by inactivity might be blunted by energy restriction [23]. Ten physically active men and women (24 (1) years) reduced their daily steps from 10,000 to 5000 for 10 days. Participants completed two periods of physical inactivity while consuming either a control diet (16% kcal from protein, 64% kcal from carbohydrate, 20% kcal from fat) or a higher-protein diet (30% kcal from protein, 50% kcal from carbohydrate, and 20% kcal from fat) in a randomized crossover design. In both diets, energy intake was decreased by 15–20% of total energy expenditure to offset the reduction in energy expenditure (400 kcal/day). As a positive control condition, a group of subjects from the initial sample (n = 5) repeated the same protocol of inactivity in association with overfeeding (35% kcal). The results of this study revealed that when diet was controlled, body fat was not altered by physical inactivity and body weight was significantly reduced; abdominal FM was also lowered. In contrast, when overfeeding was accompanied by inactivity, both body weight and body fat went up. Further, when overfeeding was combined with inactivity, the authors observed increases in fasting blood glucose, plasma insulin, plasma c-peptide, insulin resistance, and 2 h postprandial glucose and insulin concentrations. However, when the diet was controlled none of these changes were produced.

Other authors have looked at what happens when a reduction in physical activity and overfeeding are accompanied by an exercise intervention [26]. Over one week, 26 physically active men (25 (7) years; BMI 23.8 (2.5)) were randomly assigned to two groups. In both groups, physical activity was restricted to under 4000 steps/day and energy intake increased (50% kcal) while individuals in one of the groups undertook 45 min of daily treadmill running at 70% of VO2max. In both groups, increases were recorded in body weight, waist/hip circumference and lean mass. In the group of subjects who did not train, insulin sensitivity and B-cell function were reduced and the insulin response to OGTT was increased. This group also showed an increase in total cholesterol and adiponectin. However, the addition of physical exercise was able to abolish these changes.

Taken together, these findings indicate that energy balance plays a key role in the metabolic consequences of acute physical inactivity. Accordingly, while overeating could worsen its repercussions, energy restriction could help avoid its impacts. Although physical exercise seems to improve glycemic control, a positive energy balance still affects body composition. In the studies reported, however, calorie intake was controlled, so it is unknown whether reduced physical activity may have led to reduced energy intake.

Physical activity plays an important role in energy balance, and subjects engaging in higher levels of physical activity may have improved sensitivity of the appetite control system [34]. Evidence shows that there is weak coupling between energy intake and expenditure in individuals displaying low levels of daily physical activity, but strong coupling with high levels of physical activity [35].

In 1956, Mayer [36] described that calorie intake increases with activity only within a certain zone of "normal activity". This author also confirmed that below a level of physical activity or so-called "sedentary zone", a further decrease in activity is not followed by a decrease in food intake. Recent investigations have also shown that a reduction in physical activity is not usually offset by a reduction in energy intake, resulting in a positive energy balance [37,38]. Further, it has been proposed that sedentary activities not only reduce energy expenditure, but they also promote increased food intake. Hence activities such as watching television or cognitive tasks stimulate food intake such that the sensations of satiety and fullness are ignored leading to overconsumption [39]. This evidence suggests it is highly likely that a significant reduction in physical activity will not be accompanied by a reduction in energy intake and this will result in a positive energy balance likely worsening the metabolic effects of sedentary behavior.

#### *3.5. Manipulating Dietary Intake to O*ff*set the Metabolic Impacts of Confinement*

While it is important to remain active to avoid the problems an acute sedentary lifestyle brings, what about our attitude to food during this period of confinement? On the one hand, there may be overfeeding, while on the other, calorie intake may be restricted due to reduced activity or physical inactivity. Restricted calorie intake could be an optimal option for our current situation. This means a diet lower in a given percentage of calories than our regular diet, but that, nevertheless, is balanced to include all the necessary nutrients. Research in some animals has shown that the intake of up to 40% fewer calories has an impressive positive effect on markers of disease and ageing [18]. In humans, a few randomized controlled clinical trials have examined the effects of calorie restriction on health. The findings of a study performed in healthy subjects who underwent a three-month period of calorie restriction, i.e., five consecutive days per week of a fasting-mimicking diet low in calories, proteins, and sugars but high in unsaturated fatty acids, were reduced BMI, trunk, and total body fat [40]. Likewise, reductions were recorded in blood pressure, triglycerides, total and low-density lipoprotein cholesterol, C-reactive protein, and insulin-like growth factor 1 (IGF-1). The authors concluded that cycles of a five-day fasting-mimicking diet were safe, feasible, and effective in reducing risk factors associated with metabolic-related diseases.

In a two-year follow-up clinical trial, the effect of two years of 15% calorie restriction was assessed in healthy individuals. Results indicated an average weight loss of 8.7 kg (70% was body fat) in the calorie restriction group versus an average gain of 1.8 kg in the control group. Further, subjects in the restricted calorie intake group showed a 10% reduction in the metabolic rate of sleep, associated with reduced levels of reactive oxygen species and thyroid activity (reduced T3 and T4), which are biomarkers of aging [41]. In another study carried out in eighteen healthy and physically active subjects, the effects of caloric restriction close to 40% of the standard calorie intake for six weeks were assessed [42]. Diet was based on three days of severe restriction (600–800 Kcal) per week and normal intake for the rest of the week. Results indicated considerable weight loss including reduced fat mass (mostly android) and a less appreciable effect on fat-free mass. Hence, the option of calorie restriction should be considered with caution due to the lack of evidence in humans.

The key during this period of confinement would be a balanced diet comprising all the necessary nutrients, including healthy fats with balanced levels of sugar and cholesterol. During confinement, low-calorie diets should not be recommended, as they are not effective in the long term, and do not provide sufficient energy for a person in this situation of staying at home. Carbohydrates are an appropriate source of energy and are needed daily, mainly if associated with aerobic exercise. Foods rich in carbohydrates with a low glycemic index (whole grains, brown rice, vegetables, legumes, fruits, etc.) and proteins are an essential part of the diet, especially during this period of greater inactivity and we should avoid carbohydrates with a high glycemic index such as sugars, sweets, or bread. Foods rich in proteins with a lower percentage of fat such as chicken and turkey meat, fish, cooked eggs, fresh cheeses, legumes (soy), as well as dairy products such as yogurt and cottage cheese, are recommended because proteins have a stimulating effect on metabolism and are involved in the elimination of fats. Therefore, the combination of an adequately balanced diet and regular physical exercise, should serve to maintain a stable metabolic balance.

#### *3.6. Physical Activity to Mitigate the Metabolic Impacts of Confinement*

In many countries, an undesirable effect of the COVID-19 pandemic is restricted outdoor physical exercise. Recommendations include indoor walking every 2 h in order to stimulate both cardiovascular and musculoskeletal systems. Aerobic exercise, such as jogging at home if there is enough space, is highly recommended, as well as performing flexo-joint extension (shoulders, elbows, wrists, back, hip, knees, and ankles) and strength, flexibility and stretching exercises of the main muscle groups. A central-question is: what type of training is appropriate for an individual with metabolic syndrome? In a meta-analysis examining the effects of aerobic exercise training, strength training, or both combined, on cardiovascular risk factors in patients with metabolic syndrome, it was observed that aerobic training improved waist circumference, fasting blood glucose, HDL-cholesterol, triglycerides, diastolic blood pressure, and VO2peak [43]. No changes were related to strength training alone, probably due to the limited data available. Accordingly, high-intensity aerobic training performed over more than 12 weeks (3 days/week), shows the most marked effects on cardiovascular risk factors [43]. Inactivity slows the metabolic benefits of exercise, while exercise improves postprandial lipemia levels, glucose tolerance, and insulin sensitivity, all of which are risk factors for CVD. Another study highlights that physical inactivity (e.g., sitting 13.5 h/day and walking fewer than 4000 steps a day) provokes resistance to metabolic improvements that usually result from an acute episode of aerobic exercise, emphasizing that exercise, a heart-healthy diet, and an active lifestyle should be combined to achieve a healthy cardiometabolic profile [44].

Another added problem during this period of home confinement at is that the only form of exercise for many people is walking up and down the corridor of their house. Thus, the question arising is how many steps per day are recommended? Taking more steps per day (8000 vs. 4000 steps per day) is associated with lower all-cause mortality but a significant association has not been found between step intensity and mortality after adjusting for the total number of steps per day [45]. It is essential to walk as much as possible regardless of intensity, since muscle is an endocrine organ that modulates the production of substances according to their activity, requiring minimal muscle activation to obtain benefits [13]. During confinement, it is very likely that physical activity drops drastically possibly resulting in more hours of bed rest, with the consequent loss of muscle mass and its impaired function, and increases in glucose intolerance. Nutrition will play an even more significant role at this challenging moment and must include a consumption in the range of 1.4–2.0 g/kg per day protein to protect from the consequences of muscle inactivity [46,47]. Protein intake should be customized according to different factors, such as the type of population (young or older) [48], energy status, the quality of protein intake or the mode and intensity of exercise [46].

An exercise program for the confinement period has been proposed [49]. Recommendations include increasing the frequency of exercise to 5–7 days per week, 200–400 min of aerobic training and 2–3 days of resistance training. Mobility should be included every day as well as balance and coordination distributed through different training. This should be done at least two times per week. For older people, moderate intensity exercise is recommended during quarantine. Exercise may be performed without any specific training materials. Resistance training can be done through body weight exercises, such as squats, push-ups or sit-ups. Household items such as water bottles or packets of food can be used as weights. Different examples of aerobic exercise are dancing, stair climbs and walking or running on the spot. Balance exercises could be performed via stepping over obstacles or walking along a straight line marked out on the floor [49,50]. Additionally, yoga or traditional Tai Ji Quan can be considered as they do not require any equipment or large space [50].

Resistance training has been demonstrated to reduce the loss of muscle mass and muscle strength [51–53] and improve bone density [54], metabolic health and insulin resistance [53]. Resistance training should be properly designed for older adults following principles of individualization, periodization, and progression [53]. However, minimally supervised home-based training has also been shown to be a safe and effective method of increasing body muscle strength [51]. Resistance training can be useful to fight against the metabolic and physical consequences of COVID-19. However, exercise programs should consider other important components: aerobic, balance, coordination and mobility [49].

#### **4. Limitations**

As the main limitation of the studies reviewed, with the exception of three studies [24,26,28] (n = 45, n = 26, n = 22, respectively), most had <20 participants, so statistical power was low. Participants were mainly males (n = 119) and there was a lower number of females (n = 51). Additionally, none of the studies reported on sex differences, an aspect that would be interesting to explore. Nevertheless, as results exist for both sexes, we believe that recommendations can be followed by both.

Another aspect we should highlight is the fact that not all the articles measured the same variables. Although all examined glycemic control, not all analyzed CRF, body composition, lipid profile or inflammatory factors. These variables could have provided much evidence of the deleterious impact of step reduction. We found no study including a short term step reduction intervention in obese persons or people with metabolic syndrome. According to the high prevalence of both syndromes and their pathogens, it is probably that in these populations, the health impacts of short term physical inactivity will be worse.

In most of the studies reviewed, while physical activity was reduced, participants maintained their dietary habits and, as a result, their energy balance was positive [19,20,24,25,27,28]. Another three articles added overfeeding to the intervention [22,23,26], and just one examined calorie restriction [23]. In these studies, most deleterious consequences were avoided. So it is unknown if the negative repercussions of acute physical inactivity are derived from the inactivity itself, the positive energy balance or from both aspects. According to the current evidence, home exercise as well as a healthy balanced diet avoiding overeating could be a good strategy to mitigate the impacts of acute physical inactivity. Further research on this topic is needed.

#### **5. Conclusions**

Metabolic syndrome, also known as "insulin resistance syndrome", is defined as "a constellation of interconnected physiological, biochemical, clinical, and metabolic factors that directly increases the risk of atherosclerotic CVD, type 2 diabetes mellitus, and all-cause mortality" [55]. Metabolic syndrome is strongly linked to insulin resistance, oxidative stress, inflammation, obesity, endothelial dysfunction and CVD [56]. In turn, physical inactivity has been related to every described risk factor for metabolic syndrome: dyslipidemia, hypertension, hyperglycemia, visceral obesity, and prothrombotic and proinflammatory events (Figure 2).

Insulin resistance is a central component of metabolic syndrome [56] and while high levels of daily physical activity can prevent insulin resistance, physical inactivity is a primary cause of insulin resistance and a loss of insulin sensitivity in skeletal muscle [2]. In the studies reviewed, it was observed that just two weeks of physical inactivity and a positive energy balance can increase insulin resistance and modify glycemic control [19–28]. Obesity and visceral obesity are also central components of metabolic syndrome [55,56] with an important role in CVD [57] through different mechanisms such as insulin resistance and the induction of a proinflammatory state [58]. The studies reviewed detected increases in body fat [22–24,27] and abdominal fat mass [19,22,24] after just one or two weeks of step reduction associated with a positive energy balance.

The role of inflammation in the pathogenesis of metabolic syndrome and CVD has been well documented [55,56,59]. In the studies conducted in elderly subjects reviewed here, it was confirmed that two weeks of physical inactivity led to increases in TNF-α, IL6 and CRP [27,28]. TNF-α and IL-6 are cytokines with endocrine, autocrine and paracrine functions, and their gene expression is increased in the adipocytes, macrophages and lymphocytes of obese individuals [56]. TNF-α acts locally on adipocytes and reduces insulin sensitivity via different mechanisms, increases FFA levels through the induction of lipolysis, and inhibits adiponectin release [60]. This cytokine also attenuates nitric oxide-mediated vasodilation and is involved in the vascular pathology of metabolic syndrome, atherosclerosis and coronary disease [56]. IL-6 creates insulin resistance in the liver and enhances the hepatic synthesis of acute phase proteins such as CRP and fibrinogen. CRP shows high correlation with metabolic syndrome, diabetes and CDV, and fibrinogen leads to a prothrombotic state [56,60]. IL-6 also promotes the expression of adhesion molecules by endothelial cells and activates the local renin-angiotensin system, whose activation contributes to metabolic syndrome development [56,60].

**Figure 2.** Consequences of a short-term reduction in physical activity.

Acute sedentarism can be deleterious for health though other mechanisms. It has been reported that short term physical inactivity lowers VO2max [20,22–24] and also reduces lean mass and fat free mass, with greater impacts on the lower body [19,20,24,27]. Both low skeletal muscle mass and maximal aerobic capacity (VO2max) are biomarkers associated with a shorter life expectancy [2]. The impact of this reduction in muscle mass could be especially important in the elderly, due to a higher prevalence of sarcopenia and its health impacts in these subjects. Moreover, sarcopenia combined with obesity (sarcopenic obesity) has been linked to a worse metabolic impact and increased risk of mortality [61].

More research is needed to examine whether energy restriction could avoid the consequences of acute physical inactivity as suggested in one of the articles reviewed [23]. Nevertheless, there is evidence to support that at lower levels of physical activity, energy intake is dysregulated leading to a positive energy balance [35–38]. Maintaining food glycemic control is a specific measure in patients with diabetes to help reduce infection risk and severity. In effect, it has been also recommended that attention be paid to nutrition and adequate protein intake, along with exercise to improve immunity [62]. Short-term physical inactivity and a positive energy balance can have several consequences for health related to reduced insulin sensitivity, higher total body and central fat, and a proinflammatory state,

which are all central risk factors for metabolic syndrome. For the elderly, consequences could be worse, increasing the risk of developing sarcopenic obesity.

According to recent evidence, adequate control of metabolic disorders is important to reduce the risk of severe COVID-19. We should try to avoid the deleterious consequences of physical inactivity and positive energy balance by maintaining physical activity and exercise levels in a safe home environment and adhering to a healthy diet. Of course, this is also important for people without metabolic disorders to avoid the reported deleterious effect of physical inactivity and positive energy balance, which may prompt the development of metabolic syndrome and its comorbidities. COVID-19 varies from a mild self-limiting flu-like illness to full-blown pneumonia, respiratory failure and death [62]. Hypertension, diabetes, and CVD have been identified as potential risk factors for the more severely ill patients. In addition, COVID-19 could also enhance damage to the heart in patients with CVD [63,64]. As obesity increases, so does risk of chronic disease related to metabolic syndrome and based on recent data, obese individuals are also being considered at high risk for severe complications of COVID-19 [65,66].

Accordingly, our recommendations during this period of confinement are to avoid overeating by following a healthy balanced diet. This diet should be based on carbohydrates with a low glycemic index, such as vegetables, legumes or fruits, healthy fats and food rich in proteins with a lower percentage of fat. Moreover, calorie intake may be restricted due to reduced activity or physical inactivity. This nutrition recommendation should be combined with an adequate daily physical activity program designed by sports science specialists to prevent metabolic-related health problems. This program should consider different components: resistance, aerobic, mobility, coordination and balance. The recommended frequency of training is 5–7 days per week, including at least 2–3 days of resistance training.

Once COVID-19 transmission is controlled, different countries are setting new regulations in which exercising in the street is allowed. Each set of regulation may have different rules, in terms of time, or number of people training together. Depending on the country, personal training or in small groups might be permitted. However, reopening of gym or sport centers to the population at large could take longer. For this reason, resistance training should continue at home as recommended here, as well as exercise for mobility, coordination and balance. As indicated by the studies reviewed in this article, short term physical inactivity can lead to a reduction in CFR and also in muscle mass. In some countries this inactivity has been much longer. Accordingly, people must consider that their fitness level is lower than before confinement if they begin to exercise in the street, such as running. Counselling by a sports science specialists could be useful to avoid injuries. Likewise, it is imperative that all the actions we carry out comply with the social distancing recommended by the health authorities.

**Author Contributions:** M.M.-F. and H.P.-G. designed the study; M.M.-F. and F.S.-G. analyzed the data; M.M.-F., F.d.l.G.-G. and F.S.-G. interpreted the results of experiments; M.M.-F. and F.d.l.G.-G. prepared the figures and tables; M.M.-F., F.S.-G. and H.P.-G. drafted the manuscript; H.P.-G., F.S.-G. and F.d.l.G.-G. edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Helios Pareja-Galeano is supported by a grant from Universidad Europea de Madrid (#2019/UEM01).

**Acknowledgments:** Fabian Sanchis-Gomar is supported by a postdoctoral contract granted by "Subprograma Atracció de Talent - Contractes Postdoctorals de la Universitat de València."

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Nutrients in the Prevention of Osteoporosis in Patients with Inflammatory Bowel Diseases**

#### **Alicja Ewa Ratajczak \*, Anna Maria Rychter, Agnieszka Zawada, Agnieszka Dobrowolska and Iwona Krela-Ka ´zmierczak \***

Department of Gastroenterology, Dietetics and Internal Diseases, Poznan University of Medical Sciences, 49 Przybyszewskiego Street, 60-355 Poznan, Poland; a.m.rychter@gmail.com (A.M.R.); aga.zawada@gmail.com (A.Z.); agdob@ump.edu.pl (A.D.)

**\*** Correspondence: alicjaewaratajczak@gmail.com (A.E.R.); krela@op.pl (I.K.-K.); Tel.: +48-667-385-996 (A.E.R.); +48-8691-343 (I.K.-K.); Fax: +48-8691-686 (A.E.R.)

Received: 12 May 2020; Accepted: 3 June 2020; Published: 6 June 2020

**Abstract:** The chronic character of inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, results in various complications. One of them is osteoporosis, manifested by low bone mineral density, which leads to an increased risk of fractures. The aetiology of low bone mineral density is multifactorial and includes both diet and nutritional status. Calcium and vitamin D are the most often discussed nutrients with regard to bone mineral density. Moreover, vitamins A, K, C, B12; folic acid; calcium; phosphorus; magnesium; sodium; zinc; copper; and selenium are also involved in the formation of bone mass. Patients suffering from inflammatory bowel diseases frequently consume inadequate amounts of the aforementioned minerals and vitamins or their absorption is disturbed, resulting innutritional deficiency and an increased risk of osteoporosis. Thus, nutritional guidelines for inflammatory bowel disease patients should comprise information concerning the prevention of osteoporosis.

**Keywords:** bowel diseases; diet; osteoporosis; bone density; nutrients

#### **1. Introduction**

Inflammatory bowel diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC), are chronic conditions, the aetiology of which is not entirely known. Possible risk factors comprise genetic predisposition, immunological disorders, and environmental conditions [1]. Furthermore, the chronic character of these diseases causes extra-intestinal complications such as osteoporosis, which is manifested by low bone mineral density, resulting in an increased risk of fractures [2].

The World Health Organisation (WHO) classifies Bone Mineral Density (BMD) on the basis of Dual-Energy X-ray Absorptiometry (DEXA)—i.e., the gold standard in the diagnosis of osteoporosis:


Risk factors of osteoporosis in IBD include early age onset; steroid therapy; malnutrition; low body mass; hormonal disorders, including a decreased oestrogen level; and malabsorption causing a nutritional deficiency, particularly in terms of vitamin D and calcium. However, osteoporosis may also stem from genetic factors. In the course of IBD, pro-inflammatory molecules TNF-α, IL-1β, IL-6, and IL-17 levels are elevated and lead to increased bone resorption, causing a decrease in bone mineral density [4]. In fact, low bone mineral density, osteopenia and osteoporosis were found in

22–77%, 32–36%, and 7–15% of IBD patients, respectively [1]. Additionally, low BMD is observed more frequently in CD than UC patients [5].

An important risk factor for BMD loss in IBD patients is the use of certain medications. Corticosteroids (CS) are a group of medications whose prolonged use is associated with side effects involving bone tissue [5]. CS increase apoptosis and decrease the formation of osteoblasts and promote osteoclastogenesis. Moreover, CS influence calcium balance (decreasing calcium absorption in the intestine and renal resorption) and the neuroendocrine system. The data indicate that following the first year of steroid therapy, bone mass may decrease by about 12%, and 2–3% per year in the following year. In addition, CS use decreases muscle mass, which leads to an elevated risk of fractures [6,7]. Corticosteroids also affect the receptor activator of nuclear factor κβ/receptor activator of nuclear factor κβ ligand/osteoprotegerin (RANK/RANKL/OPG) pathway. In fact, CS increase the expression of RANKL, which binds with the RANK receptor in osteoclasts, which in turn elevates their differentiation and activation. Moreover, corticosteroids decrease the level of OPG responsible for the inhibition of RANKL activity [8]. According to research studies, therapy with steroids was associated with a lower BMD in IBD patients [9]. Nevertheless, different results obtained in other studies may stem from differences in the methodology of the research. Wada et al. reported that steroid therapy was a risk factor for low BMD for UC patients but not for CD patients [10]. In our study, we also demonstrated a correlation between a cumulative prednisolone dose, administered in the course of the disease, and the lumbar spine (L2–L4) T-score, the femoral neck (FN) T-score, and the Z-score in UC patients. However, the above-mentioned association was not found in CD patients [5]. It is generally accepted that osteoporosis affects 30–50% of subjects treated with CS. Therefore, in order to prevent steroid-related osteoporosis, the BMD should be evaluated before and after every year of CS treatment. Additionally, the supplementation of calcium (1200 mg/day) and vitamin D (800 IU) should also be recommended [11].

As far as Infliximab (IFX) is concerned, research studies suggest that IFX treatment in CD patients increased the N-terminal telopeptide of type I collagen [12]. However, another study showed no difference in the level of N-terminal telopeptide in type I collagen, although it was pointed out that the bone-specific alkaline phosphatase and total osteocalcin were elevated in CD patients who had used IFX [13]. The BMD and bone mineral content of the lumbar spine increased in CD patients treated by IFX [14]. Lima et al. reported the association between azathioprine and infliximab use and low BMD in a patient with CD [15]. However, another study revealed that IFX did not affect BMD [16]. Krajcovicova et al. showed that a combined therapy with anti-TNFα and azathioprine improved the lumbar spine BMD by –3% per year, whereas corticosteroid therapy reduced the BMD by –3% per year [17]. Moreover, Hoffmann et al. reported that immunomodulatory treatment was associated with a BMD decrease [18], although another study revealed that biological and immunomodulatory treatment did not affect the risk of hip fracture [19].

The use of monoclonal antibodies in osteoporosis associated with IBD is the subject of several studies. Bone metabolism at the osteoclast/osteoblast level is regulated by the following factors belonging to the tumour necrosis factor superfamily: receptor activator of nuclear factor kappa B (κB; RANK), its ligand (receptor activator of nuclear factor κB ligand, RANKL), and osteoprotegerin (RANKL/RANK/OPG). Krela-Ka´zmierczak I. et al. showed that low OPG levels may be associated with osteoporosis in UC. Nevertheless, in CD patients increased RANKL levels were observed [20,21]. Interleukin 6 can modulate BMD via the OPG/sRANKL system in the femoral neck, which can cause a loss of bone, particularly in the course of CD. Therefore, these studies may contribute to the development and implementation of new secondary osteoporosis therapy in the course of IBD [22]. It is worth emphasizing the effects of biological therapy employed in IBD on bones. Although studies focusing on the effects of anti-TNF-α therapy on bone metabolism in IBD are limited, it was observed that IFX treatment induced beneficial effects on bone metabolism in IBD patients [23,24], which suggests that monoclonal antibodies constitute an important factor in the treatment of osteoporosis. Understanding the role of RANKL and sclerostin in bone cell biology entirely changes the therapeutic perspective. Sclerostin, an antagonist of the Wnt pathway, plays a key role in bone formation and is mainly secreted by osteocytes. In fact, high levels of RANKL and sclerostin were detected in osteoporosis, leading to the production of antibodies which are able to neutralize their activity. Denosumab (anti-RANKL antibody) is a fully human monoclonal antibody which inhibits osteoclastic-medicated bone resorption by binding to osteoblast-produced RANKL. Romosozumab, a monoclonal antibody binding sclerostin, increases bone formation and decreases bone resorption. Denosumab and romosozumab present promising results in the treatment of postmenopausal osteoporosis [25]. Therefore, the use of these antibodies in the therapy of secondary osteoporosis in IBD patients requires further research.

Diet and nutritional status constitute risk factors in the development of osteoporosis. Micro and macronutrients, vitamins, and mineral components intake is often below the recommended values, particularly when the disease is active. Hence, malnutrition and deficiency in nutrients essential to bone mineralization can affect patients with inflammatory bowel diseases [26].

#### **2. Proteins, Fats, and Carbohydrates Intake in IBD and the Risk of Osteoporosis Development**

The European Society for Clinical Nutrition and Metabolism (ESPEN) recommends supplying 1.2–1.5 g protein/kg body weight in active IBD. Patients in remission should consume 1 g protein/kg body weight. Improper diet and the loss of nutrients in the gastrointestinal tract leads to an increase in fat mass, as well as a decrease in free fat mass in patients suffering from IBD. On the basis of these guidelines, there is no special diet in the remission period [27]. The recommended macronutrient intake values for adults are listed below (Table 1) [28,29].


**Table 1.** References of macronutrient consumption.

\* DRI—Dietary Reference Intake [28]; \*\* RDA—Recommended Dietary Allowance [29].

#### *2.1. Protein*

Protein provides about 50% of bone volume, 1/3 of bone mass and forms the bone matrix, therefore its supply determines the maintenance of bone mass in adults [30]. On the other hand, an excessive intake of protein causes increased calcium excretion and negative calcium balance [31].

Moderate or severe hypoalbuminemia was diagnosed in 17% and 24% of IBD patients, respectively. In patients with albumin deficiency following surgical procedures, mortality within 30 days was higher than in patients with normal albumin levels [32]. Protein-energy wasting is observed more frequently in IBD patients than in healthy individuals, and the same observation can be made in Crohn's disease patients as compared to subjects suffering from ulcerative colitis [33]. There were no statistically significant differences between men with ulcerative colitis in remission and healthy subjects [34].

One of the research studies showed a statistically significant difference in protein intake between men over 63 years old with and without a femur fracture. Every 3% increase in protein daily intake caused a 20% decreased risk of femur fractures [35]. What is more, the percentage of protein in the daily caloric content of middle aged women (65–72 years old) correlated negatively with a femoral neck. Additionally, a protein intake higher than 1.2 g/kg body weight was associated with lower BMD in the femoral neck and lumbar spine [36].

#### *2.2. Fats*

There was no statistically significant differences in the intake of total fat, saturated fat, and mono–and poly–unsaturated fat between healthy individuals and CU patients [34]. The European Society for Clinical Nutrition and Metabolism does not recommend n-3 fatty acid supplementation for maintaining remission in IBD [27].

The development of osteoporosis was correlated with the type of fatty acids. In fact, the risk of fractures was increased by n-3 and saturated fatty acid intake and it was decreased by monounsaturated and n-6 fatty acids. Dong et al. demonstrated that the supplementation of eicosapentaenoic and docosahexaenoic acid (1.2 g per day) did not result in statistically significant differences in osteocalcin and bone-specific alkaline phosphatase [37]. Furthermore, n-3 fatty acid supplementation did not significantly alter bone-specific alkaline phosphatase and type I collagen-telopeptide levels, although it decreased the osteocalcin level [38]. The twenty-four-week supplementation of n-3 fatty acid in combination with aerobic exercise reduced chronic inflammation and increased BMD in postmenopausal women [39].

#### *2.3. Carbohydrates*

Carbohydrates supply energy for host cells and the intestinal microbiome. Fermentable carbohydrates (especially mono–and di–saccharides) and fibre constitute important factors in inflammatory bowel diseases [40].

A meta-analysis demonstrated that a low-FODMAP diet (low-Fermentable Oligosaccharides, Disaccharides, Monosaccharides, and Polyol) decreased the incidence of abdominal pain, nausea, weakness, and flatulence, although no change in the rate of constipation was observed [41]. Patients with IBD in remission were supplied with insufficient amounts of carbohydrates relative to recommendations [42]. "National Health and Nutrition Examination Survey" research demonstrated a negative association between energy density (a diet rich in carbohydrates, sugar, fat, and saturated fatty acid) and BMD in the femur and femoral neck [43]. In animals, a high fructose diet decreased transepithelial active calcium ion transport and the 1.25(OH)2D3 level by about 30–40% [44].

On the other hand, the fructose content in breast milk triggered an increased BMC (Bone Mineral Content) in a 6-month-old infant compared to the first month of the baby's life. Nevertheless, lactose and glucose did not cause changes in BMC [45]. Another study pointed to the association between carbonated beverages consumption, which frequently are sources of sugar, and fractures [46]. In fact, a meta-analysis showed that the consumption of non-alcoholic beverages was negatively associated with calcium supply [47]. Additionally, fibre possibly prevents a decrease in BMD, as water-soluble fibre intake causes increased calcium retention in bones [48].

#### **3. The Importance of Vitamin Intake in IBD and Bone Mineral Density**

#### *3.1. Vitamin D*

Vitamin D comprises a group of chemical compounds such as ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). The active forms of vitamin D are 1.25(OH)2D and 24.25(OH)2D. Vitamin D acts directly and indirectly on the bone [49], regulates calcium absorption in the gastrointestinal tract, determines the proper serum calcium level, influences osteoblasts mineralization and differentiation, and decreases the synthesis of parathormone and the reabsorption of phosphates from bones. Vitamin D can be found in fatty sea fish, cod liver oil, and yolk [49,50].

IBD patients are a group presenting a higher risk of vitamin deficiency and, therefore, should control their 25(OH)D serum level. The recommended vitamin D supplementation for healthy adults is 800–2000 IU from September to April [51]. According to the available data, vitamin D serum level was lower than 30 ng/mL in IBD, but without statistically significant differences compared to the control group. It may stem from the low exposure to sunlight of the whole population and malabsorption caused by the inflammation in the gastrointestinal tract associated with IBD [52]. The deficiency was observed in 52% of children with IBD [53]. A sufficient vitamin D level was found more often in patients with a non-inflammatory disease activity; however, the serum concentration level was not correlated with any inflammatory markers [54]. In terms of IBD patients, a vitamin D dose of 1820 IU was sufficient to maintain the normal 25(OH)D serum level [55].

Although the supplementation of vitamin D decreased the risk of fractures in general as well as the risk of femoral neck fractures, it did not alter the risk of osteoporosis [56]. In elderly patients, the 1-year supplementation of vitamin D (12,000, 24,000, or 48,000 IU per month) did not change the hip BMD but decreased the parathormone level [57]. A meta-analysis showed the supplementation of vitamin D in patients over 50 years of age was associated with an increased risk of fractures. The dose of 1200 mg calcium and minimum 800 IU of vitamin D was more effective than lower doses [58]. However, a high dose (4000 IU and more) of vitamin D decreased the volumetric BMD in healthy adults. The study did not reveal significant differences in bone strength [59].

#### *3.2. Vitamin C (Ascorbic Acid)*

Bone tissue is 90% made of collagen, and vitamin C is a necessary antioxidant for its synthesis [60]. Moreover, it is possible that ascorbic acid participates in the synthesis of osteoblasts and influences osteoclast differentiation [61]. In animals, vitamin C deficiency increases osteoclast and decreases bone formation [62].

Since patients with IBD fear the symptoms of the disease, they often eliminate vegetables and fruits from their diets. This, in turn, may lead to an insufficient vitamin C intake and to the deterioration of the patients' health. Children, the elderly, and patients with the malabsorption syndrome are particularly exposed to this deficiency. Ascorbic acid can be found in citrus fruits, potatoes, parsley, and berries without seeds; thus, their consumption should be encouraged in this patient group [63]. In fact, patients with inflammatory bowel diseases consumed significantly less fruits and fewer vegetables; hence, they presented lower serum vitamin C than healthy individuals [64]. Patients suffering from ulcerative colitis consumed a similar amount of vitamin C in comparison with the healthy subjects; however, the INQ (Index of Nutritional Quality) of UC patients was significantly lower than that of healthy adults [65]. What is more, children with IBD presented a significantly lower intake of vitamin C than healthy children [66]. Enteral nutrition decreased the serum vitamin C level in children with active Crohn's disease, whereas glutamine supplementation did not influence the ascorbic acid level [67].

Sahni et al. estimated vitamin C intake on the basis of the Food Frequency Questionnaire. Following a 17-year-long observation period, subjects who consumed the highest amount of vitamin C suffered femoral neck and non-spine fractures less frequently than individuals reporting the lowest intake. The authors suggested that vitamin C intake in the elderly can be beneficial to bone health [60]. Additionally, daily vitamin C intake was associated with BMD in postmenopausal women. Patients with osteoporosis consumed a smaller amount of ascorbic acid than women not suffering from osteoporosis [68]. On the other hand, an American study presented a lack of association between vitamin C intake (from food and supplements) and BMD in postmenopausal women. In fact, long-term vitamin C supplementation was connected with a higher BMD in 55 to 64-year-old women [69]. Furthermore, although it was just a pilot study comprising 34 subjects, it showed that anti-oxidative vitamins influenced BMD, vitamin E (600 mg), and vitamin C (1000 mg) with and without resistance training, as well as preventing a decrease in BMD [70]. Kim et al. reported that a higher vitamin C intake was associated with a lower risk of osteoporosis development in subjects over 50 years of age; however, this was only in patients reporting little physical activity [71].

#### *3.3. Vitamin B12 and Folic Acid*

Vitamins B9 (folic acid) and B12 (cobalamin) are water-soluble vitamins. Folic acid is necessary for DNA methylation, whereas cobalamin is a coenzyme found in many biochemical reactions in the human body, e.g., in the metabolization of folic acid, or DNA synthesis. Their deficiency causes megaloblastic anaemia as well as nervous system disorders [72]. Vitamin B12 and folic acid are involved in the remethylation of homocysteine to methionine; therefore, a deficiency in these vitamins leads to hyperhomocysteinemia. It is vital to bear in mind that the serum homocysteine level is correlated

to osteoporotic fractures [73]. A deficiency in folic acid and vitamin B12 is due to insufficient intake, abnormal absorption, or increasing demand. Furthermore, cobalamin connects with an intrinsic factor, allowing for the absorption of vitamins in the ileum, and folic acid is absorbed in the duodenum and jejunum. Both of these vitamins are not absorbed in the large intestine. The risk of B9 or B12 deficiency is lower in ulcerative colitis than in Crohn's disease, which may stem from the fact that CD can affect all parts of the gastrointestinal tract, including the small intestine, whereas inflammation in UC predominantly affects the large intestine [74].

A meta-analysis showed that the serum folic acid level was lower in IBD patients than in healthy individuals. This could be accounted for by an insufficient intake, higher utilization, or taking medications. The authors recommended the routine supplementation of folic acid for colorectal cancer prevention. However, the study did not demonstrate a significant difference in serum B12 levels between the patients and the control group [72].

Kim et al. pointed out a negative correlation between the serum homocysteine level and BMD in women under 50 years of age [75]. Women with a normal homocysteine concentration consumed a larger amount of vitamin B12 than women with a higher level. In the same group, subjects with a normal lumbar spine BMD presented a lower serum homocysteine level and higher folic acid concentration in erythrocyte than women with low BMD or osteoporosis [76]. There was no correlation between the serum vitamin B12 level and the risk of fractures in women and men. However, an association was found between homocysteine concentration and the risk of a femoral neck fracture, particularly in women [77]. Salari et al. investigated the influence of folic acid supplementation (1 mg/day) on the serum osteocalcin and homocysteine level in postmenopausal women. They observed a significant difference in osteocalcin concentration between the study and control group after 6 months. It was found that the serum homocysteine level decreased in both groups, although the changes were not significant within the groups or between them [78]. Vitamin B9 and B12 supplementation was demonstrated to decrease homocysteine concentration and increase serum folic acid and cobalamin levels. Serum alkaline phosphatase and C-terminal cross-linking collagen I telopeptide levels did not change significantly [79].

Hyperhomocysteinemia (diagnosed in 60% of patients) was associated with osteoporosis and low BMD in CD patients, whereas bone disorders affected 90% of the subjects [80].

#### *3.4. Vitamin A*

Vitamin A is vital for growth; vision; and osteoclastogenesis stimulation, which further influences BMD. A total of 16% of non-adult patients with IBD presented vitamin A deficiency [81].

There was no statistically significant difference in vitamin A intake between men suffering form UC and the control group [34]. In fact, vitamin A or beta-carotene supplementation within a 15-year period did not increase the risk of bone fractures [82]. The retinol serum level correlated negatively with the BMD of the lumbar spine and femoral neck in postmenopausal women [83]. A meta-analysis showed that a high vitamin A level but not beta-carotene intake increased the risk of a femoral neck fracture. Additionally, a high and a low serum vitamin A level increased the risk of a femoral neck fracture. The authors did not observe a significant difference in total fractures depending on the supply and vitamin A concentration [84].

#### *3.5. Vitamin K*

The enzyme participating in the synthesis of glutamic acid (Gla) found in osteocalcin created in osteoblast and in the matrix bone protein is vitamin K. It occurs in two forms: K1 (phylloquinone) and K2 (menaquinone, MK-n) [85].

The study, conducted among postmenopausal Japanese women with osteoporosis, indicated that the administration of risedronic acid with vitamin K (45 g/day) was not more beneficial than administration without vitamin K supplementation. In fact, there was no statistically significant difference in fractures within the studied group [86]. Another study demonstrated that supplementation of menaquinone decreased carboxylated osteocalcin to a undercarboxylated osteocalcin ratio in young adults (47 ± 14 years old) and in postmenopausal women [87]. However, the supplementation of 360 ng of vitamin K (MK-7) for 12 months did not change bone mass as compared to the control group. The authors observed an increase in the serum carboxylated osteocalcin and a decrease in the undercarboxylated osteocalcin level in the study group in comparison with the placebo group [88].

#### **4. Minerals and Bone Mineral Density in IBD**

#### *4.1. Calcium and Phosphate*

Calcium (Ca) is responsible for proper BMD, blood coagulation, and the proper functioning of the cardiovascular system. In the human body, more than 99% of Ca is stored in bones. Therefore, a decreased serum calcium level leads to its release from bones and causes bone tissue resorption [50]. Furthermore, an insufficient calcium intake causes hormonal disorders, leading to a higher risk of fractures. Calcium can be found in such sources as milk, dairy products, and green leafy vegetables [89]. Additionally, the human body contains about 700 g of phosphorus (P), which is mainly stored in bones (80–90%). Hence, both its excessive and inadequate intake can develop osteoporosis. Phosphorus deficiency, or its insufficient supply to calcium supply ratio, causes bone resorption and inhibits bone mineralization and bone formation. On the other hand, an oversupply of P, particularly with insufficient Ca intake, results in excessive parathormone excretion and the loss of bone mass [90].

The insufficient intake of calcium was estimated in 80–86% of IBD patients, who avoid milk and dairy products due to lactose intolerance [91]. Patients with IBD have lower calcium and phosphate levels in comparison with healthy individuals [52].

Another cause of calcium malabsorption is the use of steroids as well as the occurrence of diarrhoea. The supplementation of calcium in a 1000–1500 mg/day dose is recommended for most patients with inflammatory bowel diseases. Furthermore, patients treated with steroids require calcium and vitamin D supplementation [91]. Calcium intake was correlated negatively with the femoral neck BMD but not with the lumbar spine BMD in IBD patients [92]. Premenopausal women suffering from IBD consumed insufficient amounts of calcium and vitamin D, and their intake of Ca and vitamin D was correlated [93]. Moreover, a low calcium serum level was observed in patients more frequently than in the control group, although it was insignificant. Additionally, the Ca serum level was negatively correlated with steroids [52]. A meta-analysis demonstrated that calcium supplementation without other substances (for example, vitamin D) did not alter the risk of femoral neck fractures in both sexes [94]. The study revealed that an increased intake of calcium by every 300 mg decreased the risk of fractures, although it was nonlinear. The highest risk was found in the intake below 751 mg of calcium. The fracture risk was unchanged in the intakes of more than 1137 mg and 882–996 mg of calcium [56]. Gutiérrez et al. demonstrated that a one-week diet rich in phosphorus (1677 ± 167 mg/day) increased Fibroblast Growth Factor 23 (FGF23), osteocalcin, and osteopontin levels. The aforementioned results suggest that a phosphorus-rich diet negatively affects health [95], and that women over 45 years of age, both with and without osteoporosis, consume similar amounts of calcium. Thus, Ca intake was not associated with the incidence of fractures [96].

#### *4.2. Magnesium*

Magnesium (Mg) is absorbed in the small intestine, and its absorption ranges from 30% to 80%. Bones store about 60% of the total body magnesium. The main sources of Mg are legumes, seeds, nuts, almonds, spinach, and buckwheat. Not only is this element responsible for the stability and permeability of cell membranes but it also maintains the DNA double helix integrity and regulates the activity of about 300 enzymes [97]. On the other hand, magnesium deficiency causes decreased osteoblast and osteoclast activity, resulting in bone metabolism disorders [98]. Chronic hypomagnesemia leads to the disturbance of parathyroid hormone production, leading to hypocalcaemia [99].

Patients with UC and CD consumed a lower amount of Mg than healthy adults. CD patients consumed 60–63% of the daily magnesium requirement [65,100]. Magnesium intake correlated with BMD, with a stronger correlation found in men than in women [98]. Postmenopausal women who consumed 422.5 mg and more of Mg per day presented a higher hip and total body BMD by 3% and 2%, respectively, than the individuals supplying <206.5 mg Mg/day. No association was observed between magnesium intake and the risk of fractures. On the other hand, a high magnesium dose was associated with a higher risk of forearm and wrist fractures in comparison with a low Mg intake. The authors paid attention to the subjects with a high supply of magnesium, since they reported much physical activity, which increases the frequency of falls [101]. The supplementation of magnesium (106 mg) and calcium (1200 mg) for 4 weeks in postmenopausal women did not change the serum parathyroid hormone level both in the study and the control group. However, the supplementation increased the serum CTX (C-terminal telopeptide) level—i.e., a bone resorption marker [102]. A conducted meta-analysis indicated that a high magnesium intake was not associated with a lower risk of hip fractures. On the other hand, magnesium dose was connected with the hip and femoral neck BMD, although no association was found with the lumbar spine BMD [103].

#### *4.3. Sodium (Na)*

The absorption of water and electrolytes, including sodium (Na), takes place in the colon. The lymphatic function of the large intestine can be impaired in the course of the mucosal inflammation [104].

In spite of the fact that the human body contains as much as 105 g of sodium, the intake of Na in the population is still too high, with some people consuming 9–12 g salt per day, which results in numerous disorders and also affects bones [105]. Na is known to improve taste and preserve products [106]; it is usually found in salt (40% of mass), meat and its preparations, grains, milk, and dairy products. This element constitutes the main extracellular cation excreted in urine and sweat. Moreover, sodium is responsible for the maintenance of the acid-base balance, cell work, and the transmission of nerve impulses. Although a normal sodium Na<sup>+</sup> level is 135–145 mmol, both too high and too low Na concentration levels constitute a threat to health and life. In fact, hypernatremia causes weakness, headache, vomiting, loss of appetite, weak nerve reflexes, and cardiac disorders. On the other hand, hyponatremia induces neuromuscular excitability, confusion, and cardiac arrest.

Patients with UC in remission consumed non-significantly lower sodium amounts than healthy individuals [34]. The sodium intake was lower in malnourished subjects than in properly nourished patients [107].

The Korea National Health and Nutrition Examination Survey indicated that osteoporosis was observed more frequently in postmenopausal women consuming ≥ 4001 mg of salt per day than in those consuming ≤ 2000 mg/day. A salt intake of ≥ 5001 mg was associated with a higher risk of osteoporosis in the femoral neck compared to the consumption of ≤ 2000 mg/day [108]. A sodium-rich diet (11.2 g of salt per day) increased calcium excretion in urine and changed the serum NTX (N-terminal telopeptide) level in comparison with a low salt intake (3.9 g). However, there was no significant change in the concentration of Pyr (pyridoxine) and Dpyr (deoxypyridoline) [109]. A meta-analysis demonstrated that a high intake of Na is a factor associated with a higher risk of osteoporosis. There was no significant correlation between the amount of calcium excretion in urine and bone mineral density [110].

A low sodium diet (2 g salt/day) for 6 months decreased calcium excretion with urine in patients who consumed 3.4 g or more salt per day and reduced the concentration of P1NP (propeptide of type 1 collagen). There was no significant change in the serum NTX level.

On the other hand, a low-sodium diet (2 g salt per day) of 6-month duration in persons consuming 3.4 g or more salt per day increased the amount of excreted calcium and the serum P1NP level. The authors did not observe any changes in the serum NTX level [111].

#### **5. Microelements and Bone Mineral Density in IBD**

#### *5.1. Zinc*

Zinc (Zn) catalyses about 100 enzymes, which makes it an indispensable element for the proper functioning of the immune system as well as for protein and DNA synthesis [112]. Zinc participates in the stabilization of the mucous membrane structure and inhibits mast cells degranulation. On the other hand, zinc deficiency can lead to the release of endogenous heparin, which contributes to the development of osteoporosis. Nevertheless, the exact mechanism of zinc influence on the prevention of osteoporosis is unknown [113].

About 15% of patients with UC or CD exhibited zinc deficiency. In diarrhoea, which often affects IBD patients, Zn demand in the body increases, and its supplementation is essential [112].

The serum zinc level of postmenopausal women with osteoporosis was lower than that of the control group, suggesting the influence of Zn on bone mineralization [113]. Zinc intake correlated negatively with the excretion of Pyr and Dpyr, which are specificity and sensitivity bone resorption markers. No association with serum osteocalcin and alkaline phosphatase was observed, and more patients presented a normal serum zinc level [114].

#### *5.2. Copper*

The human body contains about 80–150 mg copper (Cu), stored mainly in the muscles, bones, brain, and liver. Cu is absorbed in the small intestine and constitutes a cofactor for many enzymes, such as lysyl oxidase, participating also in collagen synthesis [115]. Thus, copper deficiency results in anaemia, the rupture of blood vessels, fractures, and depression [116].

Although women with IBD did not consume enough copper [42], Nangliyai et al. demonstrated that the copper serum level was statistically higher in IBD patients than in the control group. In fact, BMD correlated negatively with the serum Cu level [117].

The serum Cu level was statistically lower in postmenopausal women with osteoporosis than in the group without bone disorders. The administration of calcitonin to women with osteoporosis caused an increase in the serum Cu level. Hence, the results suggested that Cu impacts the development of osteoporosis [112]. In contrast, Cu supplementation (3 or 6 mg per day) for 4 weeks did not cause a change in the level of urine osteocalcin, creatinine, and the daily excretion of Pyr and DPyr [118].

#### *5.3. Selenium*

Selenium (Se) is an antioxidant and a cofactor for numerous enzymes in the human body. Additionally, it decreases inflammation and inactivates osteoclasts, thus influencing BMD [119]. Furthermore, since deiodinase contains Se, this element determines thyroid function. The main sources of selenium are eggs, meat, and milk, but only if it was added to fodder [120].

It was observed that the serum selenium level was lower in CD patients than in healthy adults. In men with UC, the selenium concentration depended on the location of the inflammatory lesions [121].

A study showed a negative correlation between the amount of selenium intake and osteoporosis [122]. On the other hand, the serum selenoprotein level correlated positively with total BMD, and the serum selenium level with the total and femoral neck BMD in aging men from Europe. It is vital to note that the observed relationships were independent of the thyroid function [123].

The demand for selected vitamins and minerals for adults is presented in Table 2.


**Table 2.** Requirements for the selected vitamins and minerals in adults [29].

\* RDA—Recommended Dietary Allowance; \*\* AI—Adequate Intake.

#### **6. Polyphenols**

Polyphenols are bioactivity chemical compounds which occur in plants, primarily in fruits, vegetables, coffee, tea, cocoa, herbs, and spices [124]. They are antioxidants and possess anti-inflammatory properties, which may influence the course of inflammatory diseases, such as IBD [125]. Polyphenols may influence the activity of nuclear factor erythroid 2–related factor 2 (Nrf2), which protects against oxidative stress and inflammation [126]. Moreover, polyphenols affect gut microbiota composition, stimulating bifidium, and lactobacilli [127]. There only a few studies regarding the influence of selected polyphenols on the IBD course. The available research is more frequently focused on animal models.

In addition, it is crucial to acknowledge the importance of green tea polyphenols. They decrease the TNF-α level and increase COX-2 (cyclooxygenase-2) synthesis. [128].

A study based on animal models showed that green tea polyphenols increase antioxidants levels, reduce serum TNF-α and Il-6, and decrease intestinal inflammation. [129]. Rezaei et al. reported that feeding rats with honey (which contains phenols and flavonoids) and Spirulina Platensis (containing antioxidants) may protect against UC development induced by acetic acid. The supplementation of polyphenols influences, for instance, the downregulation of various inflammatory pathways [130].

Another study presented no association between the total polyphenol intake and CD or UC, but polyphenols and resveratrol may protect against the development of Crohn's disease [124]. Moreover, the 6-week supplementation of resveratrol (500 mg/day) in UC patients decreased the TNF-α level and the clinical colitis activity index score [131].

What is more, polyphenols are vital elements in the prevention of osteoporosis. The consumption of products with high amounts of polyphenols (olive oil, soy, tea, fruits, vegetables) probably protects against osteoporosis development. However, the mechanism of action is not well understood [132]. A higher intake of flavonoids was associated with a higher BMD of the spine, but no association was found with the hip BMD [133]. Hardcastle et al. reported a negative correlation between the amount of flavonoids intake and bone resorption markers [134].

#### **7. Conclusions**

The diet of IBD patients often proves to be inadequate, which affects the peak bone mass, thus leading to osteopenia, osteoporosis, and fractures. Nutrition guidelines for IBD patients should include osteoporosis prevention.

In conclusion, it is worth emphasizing that:

1. The vitamin D concentration in patients with IBD should be examined routinely, since IBD constitutes a risk factor of vitamin D deficiency. Individual doses of vitamin D are recommended.


The importance of diet in the prevention of osteoporosis in the group of patients with IBD is indisputable. Hence, it is vital to adhere to the recommendations of a clinical dietitian, as an important member of the coordinated IBD patient care team.

**Author Contributions:** Conceptualization, A.E.R. and I.K.-K.; writing—original draft preparation, A.E.R.; critical revision of the manuscript, I.K.-K., A.D., A.Z., A.M.R.; supervision, I.K.-K.; acceptance of the final version: all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to thank Translation Lab, a biomedical translation company, for revising the language and style of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Osteoporos. Int. J. Establ. Result Coop. Eur. Found. Osteoporos. Natl. Osteoporos. Found. USA* **2013**, *24*, 1285–1293. [CrossRef] [PubMed]


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Satellite Cells and Markers of Muscle Regeneration during Unloading and Reloading: E**ff**ects of Treatment with Resveratrol and Curcumin**

#### **Laura Mañas-García 1, Maria Guitart 1,2, Xavier Duran <sup>3</sup> and Esther Barreiro 1,2,\***


Received: 29 April 2020; Accepted: 18 June 2020; Published: 23 June 2020

**Abstract:** We hypothesized that treatment with pharmacological agents known to increase sirtuin-1 activity (resveratrol and curcumin) may enhance muscle regeneration. In limb muscles of mice (C57BL/6J, 10 weeks) exposed to reloading for seven days following a seven-day period of hindlimb immobilization with/without curcumin or resveratrol treatment, progenitor muscle cell numbers (FACS), satellite cell subtypes (histology), early and late muscle regeneration markers, phenotype and morphometry, sirtuin-1 activity and content, and muscle function were assessed. Treatment with either resveratrol or curcumin in immobilized muscles elicited a significant improvement in numbers of progenitor, activated, quiescent, and total counts of muscle satellite cells, compared to non-treated animals. Treatment with either resveratrol or curcumin in reloaded muscles compared to non-treated mice induced a significant improvement in the CSA of both hybrid (curcumin) and fast-twitch fibers (resveratrol), sirtuin-1 activity (curcumin), sirtuin-1 content (resveratrol), and counts of progenitor muscle cells (resveratrol). Treatment with the pharmacological agents resveratrol and curcumin enhanced the numbers of satellite cells (muscle progenitor, quiescent, activated, and total satellite cells) in the unloaded limb muscles but not in the reloaded muscles. These findings have potential clinical implications as treatment with these phenolic compounds would predominantly be indicated during disuse muscle atrophy to enhance the muscle regeneration process.

**Keywords:** muscle unloading; muscle reloading; sirtuin-1; muscle progenitor cells; activated satellite cells; quiescent satellite cells; muscle regeneration markers

#### **1. Introduction**

Disuse muscle atrophy is an important condition that is the result of progression of other chronic and acute diseases, such as cardiac and respiratory disorders; cancer; prolonged bed rest; and critical illness. Reduced physical activity leading to deconditioning is characterized by the loss of muscle mass and function in the affected muscles in patients [1–5] and in animal models [6–8]. Muscle atrophy resulting from deconditioning may also worsen disease prognosis in patients with chronic and acute diseases regardless of the underlying condition [9,10].

Several pathophysiological and biological mechanisms are involved in the loss of muscle mass and function characteristic of muscle atrophy. In this regard, a great many studies have previously demonstrated that markers of oxidative stress, inflammation, proteolysis, apoptosis, autophagy, and atrophy signaling pathways were upregulated in the atrophying muscles following periods of disuse in patients [11,12] and animal models [6–8]. Whether regenerative potential is altered in models of disuse muscle atrophy remains to be fully elucidated.

Skeletal muscles are formed as a result of the fusion of progenitor myoblasts during development. Postnatal muscle stem cells replace the muscle turnover resulting from the daily life activity of humans and animals [13]. Thus, regeneration of skeletal muscles is a tightly regulated process [13]. Muscle regeneration relies widely on the interaction between satellite cells and the microenvironment. Their numbers and subtypes may vary according to the underlying condition, such as during muscle atrophy [11,13,14], exposure to cigarette smoking [15], aging [16], and prolonged bed rest [17]. All these conditions may also take place simultaneously within the same individual and may interfere with the process of muscle regeneration in the patients.

Satellite cells are characterized by their heterogeneity, which leads to different functions within the skeletal muscle fibers. The satellite cell reservoir is composed by sublaminar cells that express paired box (Pax)-7 with no expression of myogenic factor (Myf)-5 marker [13]. Importantly, Pax-7+/Myf-5+ satellite cells are the ones that preferentially differentiate into muscle fibers, while Pax-7+/Myf-5- cells do not proliferate, representing the satellite cell reservoir of the muscles. The ability to express Myf-5 determines these two different subtypes of satellite cell populations within the skeletal muscles [13]. In line with this, Pax-7+/Myf-5- satellite cells are identified as the actual stem cells, whereas Pax-7+/Myf-5+ satellite cells are recognized as the committed myogenic progenitors [13].

Resveratrol elicits beneficial effects on tissues including skeletal muscles. It is a natural polyphenol, which is obtained from peanuts, red wine, grapes, and other plants. It is a very popular compound given its effects as a powerful antioxidant [18]. Resveratrol was also shown to improve the lifespan of different animals by attenuating oxidative stress, inflammation, and atherosclerosis [19–22]. Amelioration of injury was also shown in the gastrocnemius of rats in response to treatment with resveratrol [23]. Moreover, muscle regeneration also improved as a result of resveratrol treatment in mice [24,25].

Curcumin is another polyphenolic compound that is obtained from turmeric. Curcumin exerted beneficial effects on several tissues through different mechanisms. For instance, in mouse cells, myocardial-infarction-induced fibrosis was attenuated via sirtuin-1 activation [26]. Senescence of smooth muscle and endothelial cells also improved as a result of curcumin therapy via sirtuin-1 activity [27]. Inhibition of NF-kB elicited by curcumin was shown to attenuate muscle protein degradation in models, such as in sepsis [28,29] and during unloading in mice [30]. Moreover, muscle regeneration was also favored by treatment with the NF-kB inhibitor curcumin [31].

In models of unloading and reloading, it was shown that the acetylation statuses of the transcription factors fork-head box O (FoxO)1 and FoxO3 were relevant mediators of the process of muscle mass loss during unloading of the limb muscles in mice [6,7]. Interestingly, unloading of the gastrocnemius muscles elicited a decline in levels of histone deacetylase sirtuin-1 activity, and the activity level rose up to the control levels following a period of reloading of the hindlimb [6,7]. The beneficial effects observed in the gastrocnemius muscle in those studies were most likely due to the activity of sirtuin-1 enzyme on the transcription factors FoxO1 and FoxO3 [6,7]. Thus, it is plausible to conceive that enhancement of sirtuin-1 activity with compounds such as resveratrol and curcumin may prevent muscles from further muscle loss through attenuation of the activity of atrophy signaling. On the other hand, sirtuin-1 activity may also promote muscle repair and regeneration following disuse muscle atrophy [25].

On this basis, the current hypothesis was that treatment with pharmacological agents (resveratrol and curcumin) known to increase sirtuin-1 activity, among other functions, may enhance muscle regeneration as evaluated by the identification of biological players clearly involved in this process in the limb muscles of mice exposed to muscle reloading following a period of hindlimb unloading. Hence, the objectives were as follows, in the limb muscles of mice exposed to a seven-day period of unloading followed by another seven-day period of reloading treated with either resveratrol or curcumin: (1) progenitor muscle cell numbers (all limb muscles together) were identified using

fluorescent-activated cell sorting (FACS), (2) subtypes of satellite cells in histological preparations of gastrocnemius muscle were counted, (3) markers of early and late muscle regeneration were analyzed, (4) fiber type composition and morphometry of the gastrocnemius muscle were assessed, (5) sirtuin-1 activity and content were explored, and (6) function of the limb muscles was also explored. The experimental model employed in the current investigation has previously been well validated [6–8].

#### **2. Methods**

#### *2.1. Study Design and Animal Experiments*

Female C57BL/6J mice (10 weeks old, weight ~20 g) were obtained from Harlan Interfauna Ibérica SL (Barcelona, Spain). Female mice were used for practical reasons as previous investigations in our group had also been conducted on this type of animal [6–8,30]. Mice were kept under pathogen-free conditions in the animal house facility at Barcelona Biomedical Research Park (PRBB), with a 12/12 h light–dark cycle.

The entire study protocol is shown in Figure 1. Mice were exposed to unilateral hindlimb immobilization as previously described to reproduce a model of disuse muscle atrophy [6–8,32]. The time-points used in the current investigation have also been validated in previous studies conducted by our group [6–8]. Muscle damage was demonstrated in the limb muscles of the unloaded mice [7]. Reloading of the muscle for another 7-day period elicited an improvement in muscle damage [7].

**Figure 1.** Graphical time-line representation of all the groups and treatments administered to the mice in the study. Definition of abbreviations: PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; mL, millilitre; mg, milligram; kg, kilogram; h, hour; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin.

The left hindlimb was shaved with clippers and was enveloped using surgical tape. The hindlimb was introduced into a 1.5-mL microcentrifuge tube with cover and bottom lids removed, while maintaining the foot in a plantar-flexed position to induce the maximal atrophy of the target limb muscle [6–8,32]. As the weight of the tube was approximately 0.6 g, it did not interfere with the usual mobility of the mice. The following groups of mice were studied (n = 10/group, Figure 1): (1) non-immobilized mice, (2) 7-day-immobilized mice (7dI, left hindlimb immobilized for seven consecutive days), (3) 7dI mice treated with resveratrol (7dI + Res, intraperitoneal administration, 20 mg/kg weight/24 h) [33,34], (4) 7dI mice treated with curcumin (7dI + Cur, intraperitoneal administration, 1 mg/kg weight/24 h) from day 0 to day 7 [35], (5) 7-day-recovery mice (7dR, left hindlimb immobilized for seven consecutive days, when the plastic splint was removed and the animals were moving free in their cages, in order to evaluate muscle recovery), (6) 7dR mice treated with resveratrol (7dR + Res, intraperitoneal administration, 20 mg/kg weight/24 h) from day 7 to day 14 [33,34], and (7) 7dR mice treated with curcumin (7dR + Cur, intraperitoneal administration, 1 mg/kg weight/24 h) from day 7 to day 14 [35].

The half-life of circulating curcumin was previously established to go from 15 to 60 min in animal models and patients [36,37] and from 30 to 60 min for resveratrol [38]. The rationale to administer resveratrol or curcumin intraperitoneally was to ensure that each animal received exactly the same dose of the drug every day. Administration of resveratrol or curcumin using other routes (oral, during food or water administration) would not allow us to ensure an identical dose for each mouse. Furthermore, intraperitoneal administration avoids absorption through the gastrointestinal tract and the first barrier of the hepatic metabolism, as generally happens in oral administration [39,40]. Accordingly, intraperitoneal injection was the selected route due to the fact that it gets into the circulation faster than other routes (oral gavage), to ensure the optimal absorption of the two compounds in view of its short bioavailability in plasma, estimated as 15–60 min in mice [36–38]. In order to control for a potential injection-induced stress response, all four groups (including the non-treated controls) of animals were injected intraperitoneally.

#### *2.2. Ethics*

All animal experiments were conducted in the animal facilities at PRBB. This was a controlled study designed in accordance with the ethical regulations on animal experimentation of the European Community Directive 2010/63/EU, Spanish Legislation (*Real Decreto* 53/2013, BOE 34/11370–11421) and the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986). All animal experiments were approved by the Animal Research Committee at PRBB. Ethical approval was obtained by the Animal Research Committee (Animal Welfare Department in Catalonia, Spain, EBP-13-1485).

#### *2.3. In-Vivo Measurements in the Mice*

In all the study animals, body weight and food intake were measured at every time point, and food and water were supplied ad libitum for the entire duration of the immobilization or recovery periods. In all mice, limb strength was determined on day 0 and right at the end of each immobilization or recovery time point (as described above) using a grip strength meter (Bioseb, Vitrolles Cedex, France) following previously published methodologies [6–8,30,41] in which grip strength was also the end-point parameter in the different experimental models. Grip strength was assessed in the four limbs at the same time in all mice. In all the animals, limb strength gain was calculated as the percentage of the measurements performed at the end of the study period with respect to the same measurements obtained at baseline (grip strength at the end of the study period − grip strength on day 0)/grip strength on day 0 × 100) [6,7].

#### *2.4. Sacrifice and Sample Collection*

Mice from all the experimental groups were sacrificed after the corresponding immobilization or recovery time cohorts (7 or 14 days). Each mouse was previously inoculated intraperitoneally with 0.1 mL sodium pentobarbital (60 mg/Kg). In all cases, the pedal and blink reflexes were evaluated in order to verify total anesthetic depth. Muscles were obtained from all the animals at the time of sacrifice. For isolation of muscle cell progenitor experiments, the following muscles were obtained from all the animals at the time of sacrifice: gastrocnemius, tibialis anterior (TA), extensor digitorum longus (EDL) and quadriceps femoris (QF, entire muscles in all cases). All study muscles from the hindlimb were pooled together by groups of two for each experimental condition: non-immobilized, 7dI and 7dR, with/without treatment with either resveratrol or curcumin. For immunofluorescence experiments, gastrocnemius muscle was obtained at the time of sacrifice in each experimental cohort and was embedded in optimum cutting temperature (OCT, Sakura Finetek, Torrance, CA, USA). For stem cell progenitor isolation experiments, the muscle samples were preserved in cold Dulbecco's Modified Eagle's Medium (DMEM) to be immediately processed as described below. For identification of several muscle regeneration markers, gastrocnemius muscle samples were snap-frozen in liquid nitrogen to be thereafter stored frozen at −80 ◦C to be further used.

#### *2.5. Tissue Embedding*

The gastrocnemius muscles of all study groups were fixed in 4% paraformaldehyde solution, pH 6.9 (EMD Millipore corporation, Billerica, MA, USA) and were embedded progressively with increasing concentrations of sucrose. They were subsequently embedded in tissue-tek OCT compound to be snap-frozen in 2-methyl-butane immersed in liquid nitrogen as previously described [42]. Ten-μm frozen sections were cut using a cryostat microtome (Leica CM3050S, Leica Biosystems, Wetzlar, Germany) at −20 ◦C and were mounted on glass slides.

#### *2.6. Biological Analyses*

*Muscle fiber type and morphometry.* Slow- and fast-twitch muscle fibers were identified using immunofluorescence procedures with anti-myosin heavy chain (MyHC) I and anti-MyHC II antibodies, respectively. Muscle cross-sections were air-dried for thirty minutes and were rinsed with phosphate-buffered saline (PBS) for another fifteen minutes. PBS was used to rinse sections in the different incubation steps. After rising, the sections were put in cold methanol for six more minutes. The sections were then boiled using a pressure cooker in 10 mM citrate buffer (pH 6.0) for twenty minutes and were then cooled down at room temperature for two hours. Subsequently, sections were incubated with mouse IgG blocking reagent (MOM, Vector Laboratories, Burlingame, CA, USA) for one hour, and in blocking solution (3% bovine serum albumin (BSA), 10% goat serum and 0.5% triton in PBS) for another hour. Afterwards, they were incubated overnight with the mouse monoclonal anti-MyHC I antibody (ab11083, Abcam) and anti-MyHC II antibody (ab51263, Abcam) prepared in blocking solution at 4 ◦C. Following incubation with the primary antibody and after rising with PBS, the sections were incubated with the corresponding secondary antibody and 4',6-diamino-2-fenilindol (DAPI), which specifically stained deoxyribonucleic acid (DNA) allowing identification of all nuclei for one hour at room temperature: Alexa Fluor® 488 AffiniPure goat anti-mouse IgG, Fcγ Subclass 1 Specific (Jackson Immunoresearch, West Grove, USA), which was also prepared in the blocking solution. The sections were mounted using 70% glycerol in 30% PBS. Myofibers positively stained with the anti-MyHC type I antibody or anti-MyHC type II were fluorescein isothiocyanate (FITC)-stained in green in two consecutives muscle cross-sections. The cross-sectional area (CSA), mean least diameter, and proportions of type I and type II, were assessed using a fluorescence microscope (x20 objective, Nikon Eclipse Ni, Nikon, Tokyo, Japan) coupled with an image-digitizing camera (Zyla 4.2 sCMOS camera, Andor, Belfast, UK) and the Image J software (National Institute of Health, available at http://rsb.info.nih.gov/ij/). In each muscle cross-section, at least 100 fibers were measured and counted,

separately, from all study groups of mice. Fibers that were stained simultaneously for both anti-type I and anti-type II primary antibodies were identified as the hybrid fibers. They were all counted in the histological preparations of all the study groups.

*Satellite cell identification using immunofluorescence microscopy*. Immunofluorescence staining was used to detect satellite cells in both quiescent and activated states using specific antibodies (see below). Briefly, muscle cross-sections were air-dried for 30 min and were rinsed with PBS for another 15 min. PBS was used to rinse the sections among the different incubation steps. After rinsing, the sections were put in cold methanol for six more minutes. Then, the sections were boiled using a hot bath in 0.1 M citrate buffer (pH 6.0) for 12 min and were then blocked with 10% goat serum in PBS for two hours.

Subsequently, sections were incubated with MOM for 30 min. Afterwards, they were incubated overnight with a mixture of two antibodies: mouse monoclonal anti-Pax-7 antibody (Developmental Studies Hybridoma Bank, Iowa, IA, USA) and rabbit polyclonal anti-Myf-5 antibody (Aviva Systems Biology, San Diego, CA, USA), prepared in an antibody solution (1% goat serum dissolved in PBS, at 4 ◦C. Anti-Pax-7 antibody alone was used to detect quiescent satellite cells, while the mixture of anti-Pax-7 and anti-Myf-5 antibodies detected committed satellite cells [13]. The addition of quiescent and committed satellite cells corresponded to the total number of satellite cells. Following incubation with the primary antibodies and after rinsing with PBS, the sections were incubated at room temperature with the corresponding secondary antibodies for one hour: Alexa Fluor® 488 AffiniPure goat anti-mouse IgG, Fcγ Subclass 1 Specific and Alexa Fluor® plus 555 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Waltham, USA) also prepared in an antibody solution. Finally, the sections were mounted using the fluorescent mounting medium DAPI G-Fluoromount medium (Southern Biotech, Birmingham, AL, USA), which specifically marks DNA (allowing identification of all nuclei) in the muscle sections. A fluorescence microscope (×40 objective, Nikon Eclipse Ni, Nikon, Tokyo, Japan) coupled with a digitizing camera was used to identify and count the number of satellite cells (10 fields) in each study sample. Results were expressed as Pax-7+/Myf-5- (quiescent) satellite cells, Pax-7+/Myf-5+ (activated) satellite cells or the addition of both (as total satellite cells) to the total number of counted myonuclei in the 10 fields. Additionally, negative control experiments were carried out by omission of the primary antibodies and incubation of the muscle samples only with secondary antibody, to confirm the specificity of each antibody.

*Sirtuin-1 activity.* Briefly, frozen muscle samples from the gastrocnemius muscle of all study animals were homogenized in a buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 100 mM NaF, 10 mM Na pyrophosphate, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton-X, and no protease inhibitors. Homogenates were centrifuged in a 4 mL buffer containing 30% sucrose, 10 mM Tris HCl (pH 7.5), 10 mM NaCl, and 3 mM MgCl2 at 4 ◦C and at 1300 *g* for 10 min. The pellets were washed with cold 10 mM Tris-HCl (pH 7.5) and 10 mM NaCl, to be subsequently centrifuged at 4 ◦C and at 1300 *g* for 10 min. The resultant pellets, which contained the nuclei, were resuspended in 200 μL of extraction buffer containing 50 mM HEPES potassium hydroxide (HEPES KOH, pH 7.5), 420 mM NaCl, 0.5 mM EDTA, 0.1 mM Ethylene glycol tetraacetic acid (EGTA) and 10% glycerol. The nuclei were subsequently sonicated in 15-s cycles. Afterwards, the sonicated nuclei remained on ice for 30 min. Following centrifugation at 4 ◦C and at 12,000 *g* for 10 min, the supernatants (crude nuclear extracts) were stored at −80 ◦C until further use [43,44]. Sirtuin-1 activity was evaluated using 50 μg of crude nuclear extracts from the gastrocnemius muscle of all experimental mice.

*Satellite cell isolation using fluorescence-activated cell sorting (FACS).* A schematic representation of these methodologies is shown in Figure 2. Immediately after dissection, muscles were processed following a modified version of a previously described protocol [45]. Pools of gastrocnemius, TA, EDL and QF muscles were first minced with scissors and secondly with a razor blade (Electron Microscopy Science, Hatfield, PA, USA). The minced muscles were collected in a 50-mL tube containing 40 mL of cold DMEM to be subsequently washed to remove fat tissue. Enzymatic digestion with a DMEM media containing 2.5 U/mL collagenase (Serva Electrophoresis, Heidelberg, Germany) and 2.5 U/mL

dispase (Sigma Aldrich, Sant Louis, MO, USA) was immediately performed at 37 ◦C in an agitation bath for 10 min.

**Figure 2.** Representative flowchart corresponding to the isolation protocol of the satellite cells using FACS analyses in the mouse limb muscles. Each specific step is described in a box and the flow of the entire protocol is indicated by the dark thick arrows. Definition of abbreviations: QF, quadriceps femoris; TA, tibialis anterior; EDL, extensor digitorum longus; FACS, fluorescence-activated cell sorting; DAPI, 4',6-diamino-2-fenilindol; α7, alpha-7 integrin.

The digestion procedure was repeated four times and the digested muscle solution was then filtered through a 100-μm mesh filter (Corning, New York, NY, USA). Immediately afterwards, the digestion was stopped by adding 2 volumes of 10% fetal bovine serum (FBS) in PBS, and the muscle solution was filtered through a 70-μm mesh filter (Corning). The filtered solution was centrifuged at 300× *g* for 5 min. The pellet was kept and the supernatant was centrifuged again in order to recover the maximum amount of cells. Finally, the two cell pellets were combined in a fresh tube to be re-suspended in FACS buffer ((1 mL PBS solution containing 2.5% goat serum (Sigma Aldrich)). The number of cells was counted using a Neubauer chamber.

Prior to incubation with antibodies, the cells were washed in 20-mL cold DMEM and centrifuged at 1700 rpm for 10 min to recover the cell pellet. Cells were resuspended in 1 <sup>×</sup> 106 cell/100 <sup>μ</sup>L FACS-specific buffer. All the cells were incubated with antibodies used to specifically identify the satellite cells: Phycoerythrin (PE)-conjugated anti-alpha-7 integrin (Ablab, Vancouver, Canada), a heterodimeric integral membrane protein critical for the modulation of cell–matrix interactions, and allophycocyanin (APC)-conjugated anti-CD34 (BD Pharmigen, San Jose, CA, USA), as a cell surface sialomucin (a mucopolysaccharide molecule containing sialic acid) with reported anti-adhesive, motile, and pre-proliferative properties for 30 min. Additionally, the cells from all study groups of mice were incubated at the same time (30 min) with specific antibodies to exclude other cell type populations that might also have been present in the muscle extracts, namely endothelial cells, leukocytes and

hematopoietic cells: PE-cyanine7 (Cy7)-conjugated anti-CD31 (Biolegend, San Diego, CA, USA), a marker of endothelial cells, both anti-CD11b (Biolegend) and anti-CD45 (Biolegend) as markers of leukocytes, and anti-Sca-1 (Biolegend), a marker of hematopoietic stem cells. The excluded cell populations were named negative-lineage (Lin (−)).

Subsequently, all the study cells were incubated with DAPI in order to exclude the dead cells (cells DAPI+ exclusively) five minutes prior to the start of FACS analyses (FACS Aria II SORP, BD Biosciences, San Jose, CA, USA). Once DAPI+ cells and other cell types (endothelial, leukocytes, and hematopoietic) were excluded, the cells identified using FACS were small and of a round shape, indicating a relatively low complexity. Cells stained for PE-conjugated anti-alpha-7 integrin and APC-conjugated anti-CD34 were sorted and were named alpha-7 integrin+/CD34<sup>+</sup> muscle progenitors. Muscle progenitor cells were expressed as the percentage of alpha-7 integrin+/CD34<sup>+</sup> progenitors to the total grams of tissue.

*Ribonucleic acid (RNA) extraction.* Total RNA was first isolated from the gastrocnemius muscle of mice using Trizol reagent following the manufacturer's protocol (Life Technologies, Carlsbad, CA, USA). Total RNA concentrations were determined spectrophotometrically using a NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA).

*Procedures of messenger (mRNA) reverse transcription (RT).* A single RT was performed from which all the target genes of the study were analyzed. First-stranded complementary deoxyribonucleic acid (cDNA) was generated from mRNA using oligo(dT)12–18 primers and the Super-Script III reverse transcriptase following the manufacturer's instructions (Life Technologies).

*Quantitative real-time* polymerase chain reaction *amplification (qRT-PCR).* TaqMan-based qPCR reactions were performed using the ABI PRISM 7900HT Sequence Detector System (Life Technologies, Carlsbad, CA, USA) together with commercially available gene expression assays. The probes corresponding to the following genes involved in muscle regeneration were tested: marker of cell proliferation Ki67 *(Ki67*) (Mm01278617\_m1, Life Technologies). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (*Gapdh*) (Mm99999915\_g1, Life Technologies) served as the endogenous control for the mRNA gene [46,47]. Reactions were run in duplicate, and mRNA data were collected and subsequently analyzed using the Sequence Detection System relative quantification software, version 2.4 (Applied BioSystems), in which the comparative CT method (2-ΔΔCT) for relative quantification was employed [48].

*Immunoblotting of 1D electrophoresis.* Protein levels of the different molecular markers analyzed in the study were explored by means of immunoblotting procedures as previously described [6,7,30]. Briefly, frozen muscle samples from the gastrocnemius muscle of all mouse experimental groups were homogenized in a buffer containing 50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM Na pyrophosphate, 5 mM EDTA, 0.5% Triton-X, 2 micrograms/mL leupeptin, 100 micrograms/mL phenylmethanesulfonyl fluoride (PMSF), 2 micrograms/mL aprotinin and 10 micrograms/mL pepstatin A. The entire procedure was conducted at 4 ◦C. Protein levels in crude homogenates were spectrophotometrically determined with the Bradford method using triplicates in each case and BSA as the standard (Bio-Rad protein reagent, Bio-Rad Inc., Hercules, CA, USA). The final protein concentration in each sample was calculated from at least two Bradford measurements that were almost identical. Equal amounts of total protein (ranging from 5 to 60 micrograms, depending on the antigen and antibody) from crude muscle homogenates were always loaded onto the gels, as well as identical sample volumes/lanes. For the purpose of comparison among the different groups of experimental and control rodents, muscle sample specimens were always run together and kept in the same order. Two independent sets of immunoblots were conducted and four fresh 10-well mini-gels were always simultaneously loaded for each of the antigens. Experiments were confirmed at least twice for all the antigens analyzed in the investigation.

Proteins were then separated by electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes, blocked with BSA and incubated overnight with selective primary antibodies. Protein levels of sirtuin-1 and myogenic markers Pax-7, myoD and myogein were identified in the gastrocnemius using specific primary antibodies: NAD-dependent protein deacetylase sirtuin-1

(anti-sirtuin-1 antibody, ProteinTech Group Inc., Rosemont, IL, USA), MyoD (anti-MyoD antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), myogenin (anti-myogenin antibody, Santa Cruz Biotechnology), Pax-7 (anti-Pax-7 antibody; Abcam, Cambridge, UK) and GAPDH (anti-GAPDH antibody, Santa Cruz Biotechnology). Antigens from all samples were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and a chemiluminescence kit. For each of the antigens, samples from the different groups were always detected in the same picture under identical exposure times. The specificity of the different antibodies was confirmed by omission of the primary antibody and incubation of the membranes only with secondary antibodies.

PVDF membranes were scanned with the Alliance Q9 Advanced (Uvitec Cambridge, England, UK). Optical densities of specific bands were quantified using the ImageJ software (National Institute of Health, available at http://rsb.info.nih.gov/ij/). Final optical densities obtained in each specific group of mice corresponded to the mean values of the different samples (lanes) of each of the study antigens. In order to validate equal protein loading among the various lanes, the glycolytic enzyme GAPDH was used as the protein loading control in all the immunoblots [46,47].

#### *2.7. Statistical Analysis*

The Shapiro–Wilk test was used to test normality of the study variables. The results are presented as mean values (standard deviation). The variables of food intake and percentage changes in total body weight, limb strength, and muscle structure are represented in Tables 1 and 2. The biological variables are represented in the figures. For each specific treatment (either resveratrol or curcumin), two-way analysis of variance (ANOVA) was performed using STATA (Software for Statistics and Data Science, StataCorp LLC, College Station, TX, USA) independently. For all the study variables, the following effects were analyzed: immobilization/recovery, treatment with either resveratrol or curcumin, and interaction between treatment and condition. Moreover, potential differences between two specific experimental groups were analyzed using post-hoc analysis contrast of marginal linear predictions: (1) comparisons between non-immobilized and immobilized mice, (2) comparisons between recovery and immobilized mice, (3) comparisons between treated and non-treated animals during the unloading period, and (4) comparisons between the treated and non-treated rodents during the recovery period. *p* ≤ 0.05 was established as the level of significance.

#### **3. Results**

#### *3.1. Physiological Characteristics of the Study Animals*

*Non-immobilized versus immobilized conditions.* Compared with non-immobilized animals, in immobilized mice, total body and gastrocnemius weight and limb strength gain were significantly reduced, while food intake was not modified (Table 1).



presented(standarddeviation).recovery;g,grams.significancerepresented *p* and \*\*\*, *p* < 0.001 between 7dI animals and non-immobilized mice; §, *p* < 0.05 and §§, *p* < 0.01 between the 7dI and 7dR groups of animals. The effect of immobilization and treatment and interactioneffectsarealsoindicatedasactual*p*valuesforeachvariable;*p*<0.05and*p*<0.001arepresentedinbold.

*Reloading versus unloading conditions*. Compared with unloaded animals, in recovery mice, total body and gastrocnemius weight and limb strength gain significantly increased, while food intake was not modified (Table 1).

*Unloading with either resveratrol or curcumin versus unloading*. Compared to non-treated unloaded mice, in the gastrocnemius of 7dI + Res and 7dI + Cur mice, food intake, total body and gastrocnemius weight, and limb strength gain did not significantly differ (Table 1).

*Reloading with either resveratrol or curcumin versus reloading*. Compared to non-treated reloaded animals, in the gastrocnemius of 7dR + Res and 7dI + Cur mice, food intake, total body and gastrocnemius weight, and limb strength gain did not significantly differ (Table 1).

#### *3.2. Structural Phenotypic Characteristics*

*Non-immobilized versus immobilized conditions.* Compared with non-immobilized animals, in the gastrocnemius of immobilized mice, the cross-sectional area of both type I and type II muscle fibers significantly decreased, while the proportions of hybrid fibers increased (Table 2).

*Reloading versus unloading conditions*. Compared to non-treated unloading animals, in the gastrocnemius of the reloading mice, the CSA of type II fibers significantly increased (Table 2). Fiber type proportions of both slow- and fast-twitch and hybrid fibers did not significantly differ between 7dI and 7dR mice (Table 2). The CSA of slow-twitch and hybrid fibers did not differ between these two groups of animals (Table 2).

*Unloading with either resveratrol or curcumin versus unloading*. Compared to non-treated unloaded mice, in the gastrocnemius of 7dI + Res or 7dI + Cur mice, no significant differences were detected in either cross-sectional area or muscle fiber type proportions (Table 2).

*Reloading with either resveratrol or curcumin versus reloading*. Compared to non-treated reloaded animals, in the gastrocnemius of 7dR + Cur mice, the cross-sectional area of the hybrid fibers increased, as almost did the CSA of fast-twitch fibers in the 7dR + Res animals, while no significant differences were observed in the other parameters (Table 2).



54

+ Res versus 7dR. The effect of

immobilization

 and treatment and interaction effects are also indicated as actual *p* values for each variable; *p* < 0.05 and *p* < 0.01 are presented in bold.

*Nutrients* **2020**, *12*, 1870

#### *3.3. Sirtuin-1 Content and Activity*

*Non-immobilized versus immobilized conditions.* Compared with non-immobilized animals, in the gastrocnemius of immobilized mice, sirtuin-1 activity did not differ, while sirtuin-1 protein levels significantly decreased (Figure 3A–C, respectively).

**Figure 3.** (**A**) Mean values and standard deviation of sirtuin-1 activity levels in the gastrocnemius muscle of the different study groups of mice, as measured by fluorescence in arbitrary units (a.u.). Definition of abbreviations: a.u., arbitrary units; I, immobilization; R, recovery; Res, resveratrol; Cur, Curcumin. *p* = 0.116: statistical significance between 7dR + Cur versus 7dR. The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**B**) Mean values and standard deviation of sirtuin-1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*, *p* < 0.05 between 7dI animals and non-immobilized mice; §, *p* < 0.05 between the 7dI and 7dR groups of animals; #, *p* < 0.05 for any group of pharmacologically treated mice (resveratrol or curcumin) compared to their respective group (7dI or 7dR mice). The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**C**) Representative immunoblots of sirtuin-1 and GAPDH proteins in the gastrocnemius muscle of all study groups of mice. Arrowheads indicate the corresponding analyzed band. Definition of abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; kDa, kilodalton; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin.

*Reloading versus unloading conditions*. Compared to non-treated unloading animals, in the muscles of the reloading mice, sirtuin-1 activity levels did not differ, while those of sirtuin-1 protein levels significantly increased (Figure 3A–C, respectively).

*Unloading with either resveratrol or curcumin versus unloading*. Compared to non-treated unloaded mice, in the gastrocnemius of 7dI + Res or 7dI + Cur mice, no significant differences were detected in either sirtuin-1 protein content or activity (Figure 3A–C, respectively).

*Reloading with either resveratrol or curcumin versus reloading*. Compared to non-treated reloaded animals, in the gastrocnemius of 7dR + Cur mice a significant rise in sirtuin-1 activity was almost (*p* = 0.116) observed, while sirtuin-1 protein levels showed a significant increase in 7dR + Res animals (Figure 3A–C, respectively).

#### *3.4. Satellite Cell Counts*

*Non-immobilized versus immobilized conditions.* A significant decline in α7-integrin +/CD34<sup>+</sup> cells was detected in the limb muscles of the unloaded mice compared to non-immobilized animals (Figure 4A). Moreover, activated (Pax-7+ and Myf-5+) satellite cells increased in the gastrocnemius of the immobilized mice compared to the non-immobilized mice, while quiescent/regenerative potential (Pax-7+ and Myf-5-) cells decreased, and total numbers of satellite cells did not significantly differ in muscles between the two experimental groups (Figure 4B–D and Figure 5A, respectively).

*Reloading versus unloading conditions*. A significant increase in α7-integrin+/CD34<sup>+</sup> cells was seen in the limb muscles of the reloading mice compared to immobilized animals (Figure 4A). Activated (Pax-7+ and Myf-5+) satellite cell numbers did not differ in the gastrocnemius of the recovery mice compared to unloaded muscles, while quiescent/regenerative potential (Pax-7+ and Myf-5-) cells and total numbers of satellite cells increased (Figure 4B–D and Figure 5A,B, respectively).

*Unloading with either resveratrol or curcumin versus unloading*. A significant increase in α7-integrin+/CD34<sup>+</sup> cells was observed in the limb muscles of the unloading mice treated with either resveratrol or curcumin compared to immobilized animals (Figure 4A). A significant rise in activated (Pax-7+ and Myf-5+) satellite cells, quiescent/regenerative potential (Pax-7+ and Myf-5-) cells, and in total satellite cell numbers was observed in the gastrocnemius of the immobilized mice treated with either resveratrol or curcumin compared to immobilized animals (Figure 4B–D and Figure 5A, respectively).

*Reloading with either resveratrol or curcumin versus reloading*. A significant increase in α7-integrin+/ CD34<sup>+</sup> cells was observed in the limb muscles of the reloading mice treated with resveratrol compared to recovery animals, whereas curcumin elicited no significant modifications (Figure 4A). No significant differences in activated (Pax-7+ and Myf-5+) satellite cells, quiescent/regenerative potential (Pax-7+ and Myf-5-) cells, or total satellite cell numbers were detected in the gastrocnemius between recovery mice treated with either resveratrol or curcumin and non-treated recovery animals (Figure 4B–D and Figure 5B, respectively).

#### **Figure 4.** *Cont.*

**Figure 4.** (**A**) Mean values and standard deviation of the percentage of satellite cells (alpha-7 integrin+/CD34+) measured by FACS analyses in the limb muscles of the different study groups of mice. Definition of abbreviations: α7, alpha-7 integrin; g, grams; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*, *p* < 0.05 between the 7dI animals and non-immobilized mice; §, *p* < 0.05 between the 7dI and 7dR groups of animals; #, *p* < 0.05 for any group of pharmacologically treated mice (resveratrol or curcumin) compared to their respective group (7dI or 7dR mice). The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**B**) Mean values and standard deviation of the percentage of activated satellite cell counts as identified by the number of Pax-7/Myf-5-positive cells in the gastrocnemius muscle of the different study groups of mice. *Definition of abbreviations*: Pax-7, paired box-7; Myf-5, myogenic factor 5; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*\*, *p* < 0.01 between 7dI animals and non-immobilized mice; #, *p* < 0.05 for any group of pharmacologically treated mice (resveratrol or curcumin) compared to their respective group (7dI or 7dR mice). The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**C**) Mean values and standard deviation of the percentage of quiescent satellite cell counts as identified by the number of Pax7-positive cells (Myf-5-negative) in the gastrocnemius muscle of the different study groups of mice. Definition of abbreviations: Pax-7, paired box-7; Myf-5, myogenic factor 5; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*, *p* < 0.05 between 7dI animals and non-immobilized mice; §, *p* < 0.05 between the 7dI and 7dR groups of animals; #, *p* < 0.05 for any group of pharmacologically treated mice (resveratrol or curcumin) compared to their respective group (7dI or 7dR mice). The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**D**) Mean values and standard deviation of the percentage of total satellite cell counts as identified by the number of quiescent and activated satellite cells in the gastrocnemius muscle of the different study groups of mice. Definition of abbreviations: I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: §, *p* < 0.05 between the 7dI and 7dR groups of animals; #, *p* < 0.05 and ##, *p* < 0.01 for any group of pharmacologically treated mice (resveratrol or curcumin) compared to their respective group (7dI or 7dR mice). The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable.

**Figure 5.** (**A**) Representative images of immunofluorescence staining of DAPI (left panels), Pax-7 (middle-left panels), Myf-5 (middle panels) and cells positively stained for both Pax-7 and Myf-5 markers (middle-right panels), and negative control (right panels) in the gastrocnemius muscle of the non-immobilized mice and immobilized mice with and without treatment (7dI, 7dI + Res and 7dI + Cur groups of mice). Thin arrows indicate Pax-7-positive cells, and arrowheads indicate double-stained nuclei for both Pax-7- and Myf-5-positive cells (activated satellite cells). Definition of abbreviations: Pax-7, paired box-7; Myf-5, myogenic factor 5; DAPI, 4',6-diamino-2-fenilindol; I, immobilization; Res, resveratrol; Cur, curcumin. (**B**) Representative images of immunofluorescence staining of DAPI (left panels), Pax-7 (middle-left panels), Myf-5 ((middle panels) and cells positively stained for both Pax-7 and Myf-5 markers (middle-right panels) and negative control (right panels) in the gastrocnemius muscle of the recovery mice with and without treatment (7dR, 7dR + Res and 7dR + Cur groups of mice). Thin arrows indicate Pax-7-positive cells, and arrowheads indicate double-stained nuclei for both Pax-7- and Myf-5-positive cells (activated satellite cells). Definition of abbreviations: Pax-7, paired box-7; Myf-5, myogenic factor 5; DAPI, 4',6-diamino-2-fenilindol; R, recovery; Res, resveratrol; Cur, curcumin.

#### *3.5. Myogenic Markers of Muscle Regeneration*

*Non-immobilized versus immobilized conditions.* In the gastrocnemius muscle of immobilized rodents compared to non-immobilized mice, gene expression of *Ki67* did not differ (Figure 6A). In the gastrocnemius muscle of immobilized mice compared to non-immobilized animals, protein expression levels of Pax-7 significantly improved, those of myogenin did not differ, while those of MyoD increased (Figure 6B–E, respectively).

*Reloading versus unloading conditions*. In the gastrocnemius muscle of recovery mice compared to immobilized animals, gene expression of *Ki67* increased (Figure 6A). In the gastrocnemius muscle of recovery mice compared to immobilized animals, protein expression of Pax-7 significantly declined, myogenin increased, and MyoD significantly decreased (Figure 6B–E, respectively).

*Unloading with either resveratrol or curcumin versus unloading*. In the gastrocnemius of unloading mice treated with either resveratrol or curcumin compared to non-treated unloaded muscles, no differences were observed in *Ki67* expression levels (Figure 6A). In the gastrocnemius of unloading mice treated with either resveratrol or curcumin compared to non-treated unloaded muscles, protein expression of Pax-7, myogenin and MyoD did not differ (Figure 6B–E, respectively).

*Reloading with either resveratrol or curcumin versus reloading.* In the gastrocnemius of reloading mice treated with curcumin or resveratrol, compared to non-treated reloaded muscles, gene expression of *Ki67* did not differ (Figure 6A). In the gastrocnemius of reloading mice treated with either resveratrol or curcumin compared to non-treated reloaded muscles, protein expression of Pax-7, myogenin and MyoD did not differ (Figure 6B–E, respectively).

**Figure 6.** *Cont.*

**Figure 6.** (**A**) Mean values and standard deviation of gene expression of *Ki67* in the gastrocnemius muscle of the different study groups of mice. Definition of abbreviations: Ki67, cell proliferation Ki-67; mRNA, messenger ribonucleic acid; a.u., arbitrary units; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: §, *p* < 0.05 between the 7dI and 7dR groups of animals. The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**B**) Mean values and standard deviation of Pax-7 protein content, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; Pax-7, paired box-7; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*, *p* < 0.05 between the 7dI animals and non-immobilized mice; §, *p* < 0.05 between the 7dI and 7dR groups of animals. The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**C**) Mean values and standard deviation of myogenin protein content, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: §, *p* < 0.05 between the 7dI and 7dR groups of animals. The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**D**) Mean values and standard deviation of MyoD protein content, as measured by optical densities in arbitrary units (OD, a.u.). Definition of abbreviations: OD, optical densities; a.u., arbitrary units; MyoD, myogenic differentiation 1; I, immobilization; R, recovery; Res, resveratrol; Cur, curcumin. Statistical significance is represented as follows: \*, *p* < 0.05 between 7dI animals and non-immobilized mice; §, *p* < 0.05 between the 7dI and 7dR groups of animals. The effect of immobilization and treatment and interaction effects are also indicated as actual *p* values for each variable. (**E**) Representative immunoblots of Pax-7, MyoD, myogenin and GAPDH proteins in the gastrocnemius muscle of all study groups of mice. Arrowheads indicate the corresponding analyzed band. Definition of abbreviations: Pax-7, paired box-7; MyoD, myogenic differentiation 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; kDa, kilodalton; I, immobilization; R, recovery; Res, resveratrol, Cur, curcumin.

#### **4. Discussion**

The main findings of the current investigation were that in immobilized mice compared to non-immobilized mice total body and gastrocnemius weight, limb strength gain, CSA of both slow- and fast-twitch fibers, sirtuin-1 protein levels, numbers of muscle progenitor cells (α7-integrin+/CD34+), and numbers of quiescent (Pax-7+ and Myf-5-) satellite cells significantly decreased, while proportions of hybrid fibers, activated (Pax-7+ and Myf-5+) satellite cell counts, and both Pax-7 and MyoD protein levels significantly increased.

In the muscles of the reloading animals compared to unloaded mice, total body and gastrocnemius weight, limb strength gain, CSA of fast-twitch fibers, sirtuin-1 protein levels, numbers of α7-integrin+/CD34<sup>+</sup> satellite cells, numbers of quiescent (Pax-7+ and Myf-5-) satellite cells, total counts of satellite cells, *Ki67* levels (gene expression), and myogenin protein levels significantly improved.

In muscles of the immobilized mice treated with either resveratrol or curcumin compared to non-treated immobilized animals, counts of satellite α7-integrin+/CD34<sup>+</sup> cells, activated (Pax-7+ and Myf-5+) satellite cells, quiescent (Pax-7+ and Myf-5-) satellite cells, and the total counts of satellite cells improved.

In muscles of the reloaded animals treated with either resveratrol or curcumin compared to non-treated reloaded mice, the CSA of both hybrid (only in curcumin-treated mice) and fast-twitch fibers (only in resveratrol-treated animals), sirtuin-1 activity levels (only in curcumin-treated animals), sirtuin-1 protein levels (only in resveratrol-treated mice), and counts of α7-integrin+/CD34<sup>+</sup> satellite cells (only in the resveratrol-treated mice) significantly increased.

Unloading of the limb muscles induced a significant decline in the numbers of muscle progenitor cells and quiescent satellite cells, while a rise in activated satellite cell counts was observed. These are interesting findings that put research forward on the impact of a seven-day period of unloading on the initial steps of the muscle regeneration process.

In different experimental models, treatment with resveratrol has been shown to improve muscle mass and phenotype as a result of sirtuin-1 activity [49] and attenuation of apoptosis [25]. Increased sirtuin-1 during reloading of previously unloaded muscles also induced an improvement in muscle mass and function [6]. In the current study, CSA of fast-twitch myofibers also improved in response to treatment with resveratrol during reloading of hindlimb muscles. These findings are in line with previous results obtained in our group (unpublished observations). On the other hand, curcumin administration during reloading also elicited an improvement in muscle phenotype and function through attenuation of apoptosis and proteolysis (ubiquitin–proteasome system) in experimental models [30,35]. Furthermore, NF-kB-dependent muscle wasting was also alleviated as a result of curcumin treatment in septic rats [28,29,50].

In the present study, treatment of immobilized mice with either resveratrol or curcumin induced a significant improvement in the numbers of muscle progenitor cells, quiescent, activated and total satellite cell counts in the limb muscles compared to non-treated immobilized animals. In line with this, it was previously shown [51] that sirtuin-1 maintained satellite cells in a stem-like state. This probably accounted for the rise in the numbers of quiescent and total satellite cells seen in the muscles of the unloaded mice treated with either curcumin or resveratrol. Furthermore, in trained elderly subjects treated with resveratrol, satellite cell numbers also increased in their limb muscles [52]. Collectively, these findings suggest that resveratrol and curcumin favor muscle regenerative potential following unloading.

Importantly, treatment with resveratrol was also shown to favor the process of muscle regeneration through several mechanisms. In fact, similar levels of muscle progenitor cells to those encountered in the current investigation were also reported in a former study [25]. Interestingly, a modest enhancement of myogenic precursor cell proliferation was seen in resveratrol-treated muscles following reloading [25]. Furthermore, recovery of muscle mass and of the size of fast-twitch fibers in the rat hindlimb muscles was also mediated by the action of proapoptotic proteins including cleaved caspase-3 [25]. Another mechanism whereby resveratrol may favor muscle regeneration was its ability to attenuate

muscle damage (contusion model) in mice [24]. Furthermore, local and systemic markers of muscle regeneration were attenuated in response to resveratrol treatment in mice [24].

Specifically, muscle regenerative markers and recovery of normal tissue architecture was favored by the systemic administration of curcumin in mice in response to freeze injury [31]. The beneficial effects seen in muscles were due to blocking of NF-kB activity [31]. In a previous study from our group [30], curcumin also elicited an improvement in muscle phenotype and function via attenuation of NF-kB activity (lower levels of acetylation) in mice exposed to the same experimental conditions as in the present study. Taken together, these findings suggest that NF-kB is a probably a major regulator of the muscle regeneration process detected in the hindlimb muscles of mice in the present investigation.

Protein levels of early markers of muscle regeneration (Pax-7 and MyoD) significantly increased in the gastrocnemius of the immobilized mice compared to non-immobilized animals. These findings are in accordance with previous reports [8,17,53] and suggest that the process of muscle regeneration has been triggered. On the other hand, protein levels of the early markers Pax-7 and MyoD significantly declined in the gastrocnemius of the reloaded muscles, while those of the late marker myogenin significantly rose. These results are also consistent with previous reports in which late markers of muscle regeneration were upregulated during recovery in limb muscles [8,17,53].

Importantly, treatment with either curcumin or resveratrol in the reloading periods elicited an increase in sirtuin-1 activity and sirtuin-1 protein levels, while it did not induce any significant modifications in levels of any of the analyzed markers of muscle regeneration, cells or myogenic markers (ki67, pax-7, myoD, and myogenin) in the gastrocnemius of any of the reloaded animals. In keeping with this, a potential role of sirtuin-1 activity was established as a negative regulator of early myogenic markers (proliferation of muscle precursor cells) of muscle regeneration [54]. Another plausible explanation relies on the fact that the process of muscle regeneration would be almost entirely complete during the reloading phase, and treatment with any of the phenolic compounds would not be able to exert additional beneficial effects. Thus, treatment with either resveratrol or curcumin did not elicit any significant modification of the markers of muscle regeneration beyond the physiological effects elicited by the experimental conditions of unloading or reloading.

In line with this, treatment with resveratrol during recovery for two weeks of aged rats exposed to tail suspension also induced moderate therapeutic benefits as identified by muscle mass recovery and increased size of the fast-twitch fibers of limb muscles, probably resulting from the attenuation of apoptosis [25]. Another study [55] also demonstrated that the beneficial effects induced by resveratrol on the limb muscles of type I diabetic mice was not dependent on sirtuin-1 activity. In that investigation, mitochondrial membrane potential was restored in muscle fibers without the interference of sirtuin-1 activity in the mouse muscles [55].

#### **5. Conclusions**

Unloading of the limb muscles triggered a program of muscle regeneration characterized by the activation of satellite cells and the upregulation of early myogenic factors. Treatment with the pharmacological agents resveratrol and curcumin enhanced the numbers of the identified subtypes of satellite cells (muscle progenitor, quiescent, activated, and total satellite cells) in the unloaded limb muscles but not in the reloaded muscles. These findings have potential clinical implications as treatment with these phenolic compounds would predominantly be indicated during disuse muscle atrophy to enhance the muscle regeneration process. Treatment with either curcumin or resveratrol would not elicit as many beneficial effects during reloading.

**Author Contributions:** L.M.-G.: animal experiments, molecular biology, data analyses, results preparation including graphical and tabular representation, and manuscript draft writing; M.G.: animal experiments, molecular biology, data analyses; X.D.: statistical analyses of all the study results; E.B.: study design, data analyses and interpretation, results preparation, and manuscript writing—final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** Instituto de Salud Carlos III: FIS 18/00075 (FEDER), Instituto de Salud Carlos III: CIBERES 2019. Sociedad Española de Neumología y Cirugía Torácica: SEPAR 2018.

**Acknowledgments:** Laura Mañas-García was a recipient of a predoctoral fellowship from the Department of Experimental and Health Sciences of Pompeu Fabra University (DCEXS-UPF). We confirm that we have read the journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The current research has been supported by Instituto de Salud Carlos-III, contract grant numbers, CIBERES, FIS 18/00075 (FEDER), Spanish Ministry of Science and Innovation, Spanish Respiratory Society (SEPAR), contract grant numbers, SEPAR 2018.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Editorial Support:** None to declare.

#### **References**


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