Modulatory Effect of Limosilactobacillus fermentum grx08 on the Anti-Oxidative Stress Capacity of Liver, Heart, and Kidney in High-Fat Diet Rats

Abstract

To explore the modulating effect of Limosilactobacillus fermentum (L. fermentum) grx08 on anti-oxidative stress in the liver, heart, and kidney of high-fat diet in rats, a low-fat diet as a control and a high-fat diet was used to induce oxidative stress injury in rats. L. fermentum grx08 and its heat-inactivated bacteria were used to intervene. The results showed that the high-fat diet had caused oxidative stress injury in the liver, heart, and kidney of rats. L. fermentum grx08 significantly reduced the serum levels of liver, heart, and kidney injury markers (ALT, AST, LDH, CK-MB, UA, and Crea), while restoring the balance of lipid metabolism in the liver. It also enhanced the activity of antioxidant enzymes such as GSH-Px in the liver, heart, and kidney, scavenging NO radicals and reducing the content of MDA, a product of lipid peroxidation, which can regulate the anti-oxidative stress capacity of the liver, heart, and kidney to varying degrees. Among them, L. fermentum grx08 showed better modulating effect on kidney anti-oxidative stress, followed by liver, and the weakest modulating effect on heart. At the same time, L. fermentum grx08 heat-inactivated bacteria also had a partial modulatory effect as well as a similar effect profile to that of live bacteria.

1. Introduction

Modern dietary changes and excessive intake of high-fat and high-energy diets have led to disturbances in fat, protein, and carbohydrate metabolism in the body. Excess energy gradually accumulates in the body in the form of fat, which leads to impaired mitochondrial function and increased oxidative injury in cells [1]. The liver plays a crucial role in maintaining the metabolic balance of the body, and the metabolic disorders associated with obesity are first manifested in the liver. Excessive deposition of fat in hepatocytes leads to the development of oxidative stress injury in the liver [2]. At the same time, the disruption of glucolipid metabolism caused by a chronic high-fat diet can further lead to abnormal heart and vascular function, and the resulting lipotoxicity can also increase kidney oxidative stress levels and decrease antioxidant defense levels, leading to kidney injury [3]. In the metabolic syndrome caused by a high-fat diet, in addition to liver injury, heart and kidney injury have been found. A clinical correlation between non-alcoholic fatty liver disease (NAFLD) and heart and kidney injury has also been found [4].

Probiotics can enhance the anti-oxidative stress capacity of the liver either by themselves or by modulating intestinal microorganisms [5]. Limosilactobacillus rhamnosus LV108 can alleviate NAFLD in high-fat diet rats by regulating lipid metabolism and apoptosis [6]. L. fermentum DALI02 has been found to alleviate lipid peroxidation and improve lipid metabolism in vivo, thereby reducing oxidative stress and inflammation. They are both able to reduce lipid peroxidation products in the liver and enhance the activity of antioxidant enzymes [7]. In addition, probiotics have also been found to have a degree of protective effect on kidney function impairment in hyperuricemia, enhancing the anti-oxidative stress capacity of the kidney [8]. In addition, Limosilactobacillus rhamnosus GG has been found to be able to play a role in the prevention and treatment of cardiovascular disease through various mechanisms such as anti-inflammation, anti-oxidative stress, regulation of intestinal flora, and protection of the intestinal barrier [9,10]. Although the modulatory effects of probiotics on the anti-oxidative stress capacity of liver, heart, and kidney have been demonstrated, fewer studies have explored the effects of probiotics in the same model, and there is also a lack of comparative studies on the effects of probiotics on the anti-oxidative stress of the liver, heart, and kidney.

In previous studies, L. fermentum grx08 has been found to have strong antioxidant capacity in vitro, and its adjunctive hypolipidemic effect has also been confirmed in animal experiments [11]. We hypothesized that L. fermentum grx08 may have the potential to modulate the anti-oxidative stress capacity of the liver, heart, and kidney in high-fat diet rats. The aim of this work was to investigate the effect and extent of the effect of L. fermentum grx08 on the anti-oxidative stress capacity of the liver, heart, and kidney in high-fat diet rats. Therefore, we established a high-fat diet rat model to investigate the modulating effect of L. fermentum grx08 on the anti-oxidative stress capacity of the liver, heart, and kidney, and compared its modulating effect.

2. Materials and Methods

2.1. Microorganism and Preparation of Oral Samples

L. fermentum grx08 was cultured in sterile MRS broth (Hopebio., Yancheng, China) from the 3% inoculum with 18 h incubation at 37 °C (DNP-9022 incubator, Shanghai Jinghong, Shanghai, China). Cell pellets were harvested at 5000× g for 10 min (H1750 centrifuge, Changsha Xiangyi, Changsha, China), resuspended with saline (repeated three times) and adjusted the concentration of the bacterium to 1 × 10⁹ CFU/mL (plate count), which is the sample of L. fermentum grx08 suspension. L. fermentum grx08 suspension was heat inactivated at 80 °C for 20 min (TW12 water bath, JULABO, Seelbach, Germany), which was the sample of L. fermentum grx08 heat inactivated suspension.

2.2. Animals and Treatment

Thirty healthy male Wistar rats were purchased from Comparative Medical Center of Yangzhou University, Jiangsu, China. The rats were 5 weeks old and weighed about 145 g at the start of the experiment. All animals were housed under a 12-h light/12-h dark cycle in a controlled room with a temperature of 23 ± 3 °C and a humidity of 50% ± 10%. The animals were acclimated to their new circumstances for one week. All rats were allowed free access to food and water. All 5 rats were placed in a cage and only one cage of rats (Normal control group (NC)) was fed a low-fat diet (LFD: Flour 20%, rice flour 10%, corn 20%, drum skin 26%, soy material 20%, fish meal 2%, bone meal 2%), and other rats were fed a high-fat diet (HFD: 10% lard, 10% egg powder, 1% cholesterol and 0.2% bile salts, and 78.8% LFD) for 4 weeks. High-fat diet rats were randomly divided into 4 groups (n = 5): model control group (MC), positive control group (PC), L. fermentum grx08 suspension treatment group (Grx08), and L. fermentum grx08 heat inactivated suspension treatment group (Grx08H). In the next 4 weeks, all rats received the following treatments by lavage: NC and MC: physiological saline (1 mL/100 g), PC: VC solution (1 mL/100 g, 10 mg/mL), Grx08: L. fermentum grx08 suspension (1 mL/100 g, 1 × 10⁹ CFU/mL), and Grx08H: L. fermentum grx08 heat inactivated suspension (1 mL/100 g, 1 × 10⁹ CFU/mL). At the end of the 8th week, rats underwent 12 h of fasting prior to being anaesthetized and dissected. All rats were euthanized at the anestrus period following anesthesia under 1% sodium pentobarbital.

2.3. Body Weight and Organ Index

The body weight of rats was measured once a week and fasting weight was measured before execution. After autopsy, the liver, heart, and kidney were weighed, and the organ index was calculated.

Organ index = organ weight (g)/body weight (100 g).

2.4. Blood Samples Handling

Blood was obtained from each group on the last day of the 8th week. Serum was incubated at 37 °C for 30 min and then centrifuged at 3000× g for 15 min (H1750 centrifuge, Changsha Xiangyi, Changsha, China), the supernatant was then taken and stored at −20 °C.

2.5. Organ Samples Handling

Part of the liver/heart/kidney was put in cold physiologic saline immediately and tissue homogenate was prepared (10%, w/v). The hypothalamus homogenates were centrifuged at 4000× g for 15 min at 4 °C (H1750 centrifuge, Changsha Xiangyi, Changsha, China), and the supernatant was collected and stored at −20 °C until further analyzed.

Rat liver, heart, and kidney tissues were trimmed to 0.5 cm × 0.5 cm × 0.5 cm size, fixed in 4% paraformaldehyde, and stored at 4 °C.

2.6. Indicator Testing

Liver total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), uric acid (UA), and creatinine (Crea) (Ningbo Meikang, Ningbo, China) were measured by a Model 7020 fully automated biochemical analyzer (Hitachi, Tokyo, Japan); HE staining, nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured according to the method provided in the assay kit (Nanjing Jiancheng, Nanjing, China).

2.7. Statistical Methods

Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA, USA). The results are presented as the means ± standard deviation, and comparisons between different groups were assessed by analysis of variance (ANOVA) with Tukey post hoc test (one-way ANOVA-Tukey). Values of p < 0.05 were considered statistically significant. The graphics were created using GraphPad Prism 9 (San Diego, CA, USA).

3. Result

3.1. Effect of L. fermentum grx08 on Body Weight and Organ Index of Rats

There was no significant difference in the initial body weight of rats (p > 0.05). Compared with the group NC, the final body weight and liver index of rats in the group MC were significantly increased (p < 0.05), and then were significantly reduced after L. fermentum grx08 and its heat-sterilized bacteria intervention (p < 0.05). In contrast, there was no significant difference (p > 0.05) in the heart and kidney indices of rats in all intervention groups, and the high-fat diet did not cause significant changes in heart and kidney indices of rats. L. fermentum grx08 could alleviate the obesity induced by a high-fat diet as well as the increase of the liver index (

Table 1. Body weight and organ index of rats.

	Initial Weight (g)	Final Weight (g)	Liver	Heart	Kidney
NC	145.00 ± 7.97	352.80 ± 12.07 *	3.53 ± 0.04 *	0.29 ± 0.02	0.67 ± 0.03
MC	146.50 ± 12.26	415.25 ± 17.73	4.54 ± 0.05	0.28 ± 0.02	0.59 ± 0.03
PC	147.00 ± 8.52	375.50 ± 12.07 *	4.49 ± 0.05	0.32 ± 0.02	0.62 ± 0.06
Grx08	143.20 ± 4.32	358.80 ± 22.99 *	3.89 ± 0.03 *	0.28 ± 0.01	0.57 ± 0.04
Grx08H	145.40 ± 5.41	384.60 ± 37.42	4.19 ± 0.03 *	0.32 ± 0.03	0.59 ± 0.05

Same column comparison, * indicates significant difference compared to the model group (p < 0.05), n = 5.

).

3.2. Effect of L. fermentum grx08 on Organ Oxidative Stress Injuries

Compared with the group NC, the serum AST, ALT, LDH, CK-MB, UA, and Crea levels of rats in the group MC were significantly increased (p < 0.05), and the high-fat diet caused injury to the liver, heart, and kidney of rats. After L. fermentum grx08 intervention, serum AST, ALT, LDH, CK-MB, UA, and Crea were significantly decreased in rats compared with the group MC (p < 0.05). L. fermentum grx08 heat-inactivated bacteria had the same effect but had no significant effect on Crea (p > 0.05). L. fermentum grx08 had the potential to enhance the resistance to the anti-oxidative stress capacity of the liver, heart, and kidney in rats (Figure 1).

3.3. Modulatory Effect of L. fermentum grx08 on Anti-Oxidative Stress of Rat Liver

Compared with the group NC, TC, TG, and LDL-C contents were significantly increased (p < 0.05) and HDL-C contents were significantly decreased (p < 0.05) in the liver of rats in the group MC. After intervention with L. fermentum grx08 and its heat-inactivated bacteria, TC, TG, and LDL-C contents were significantly reduced (p < 0.05) and HDL-C contents were significantly increased (p < 0.05) in the rat liver. The group PC had no significant effect on lipid accumulation in the liver (p > 0.05) (Figure 2a).

Compared with the group NC, the liver NO and MDA contents were significantly increased (p < 0.05) and GSH-Px contents were significantly decreased (p < 0.05) in the group MC. L. fermentum grx08 significantly reduced NO and MDA levels (p < 0.05), while significantly increasing GSH-Px activity (p < 0.05). Its heat-inactivated bacteria only significantly reduced MDA levels (Figure 2b).

Compared with the group NC, the hepatocyte structure of rat liver in the group MC was severely damaged, the cell gap was unclear, fat deposition was obvious, and a large number of hepatocyte fat vacuoles were visible. After the intervention of L. fermentum grx08 and its heat-inactivated bacteria, compared with the group MC, the hepatocyte structure of rats was more intact, with clearer boundaries and fewer fat vacuoles, which was closer to that of the group NC. Fat vacuoles of hepatocytes were still visible in the group PC (Figure 2c).

3.4. Modulatory Effect of L. fermentum grx08 on Anti-Oxidative Stress of Rat Heart

Compared with the group NC, there were no significant changes in NO content and SOD activity, a significant increase in MDA content (p < 0.05) and a significant decrease in GSH-Px activity (p < 0.05) in the hearts of rats in the group MC. Both L. fermentum grx08 and its heat-inactivated bacteria significantly reduced NO content (p < 0.05) and increased GSH-Px activity (p < 0.05) compared to the group MC, with no significant effect on MDA content (p > 0.05). The group PC had no significant effect on the anti-oxidative stress capacity of the heart (p > 0.05) (Figure 3a). The case-study observation did not reveal significant fat accumulation and lesions in the heart tissue of the group MC, as well as significant changes in the intervention of L. fermentum grx08 and its heat-inactivated bacteria (Figure 3b).

3.5. Modulatory Effect of L. fermentum grx08 on Anti-Oxidative Stress of Rat Kidney

Compared with the group NC, the NO and MDA contents in the kidney of rats in the group MC were significantly increased (p < 0.05), and the SOD and GSH-Px activities were significantly decreased (p < 0.05). Both L. fermentum grx08 and its heat-inactivated bacteria significantly reduced the content of NO and MDA in rat kidney (p < 0.05) and significantly increased the activity of GSH-Px (p < 0.05), and L. fermentum grx08 also significantly increased the activity of SOD (p < 0.05). Of particular note, the levels of NO and MDA as well as the activities of SOD and GSH-Px in the kidney after L. fermentum grx08 intervention were restored to the level of the group NC. In addition, there was no significant effect of the group PC on both MDA and SOD in the kidney (p > 0.05) (Figure 4a). The case study observation did not reveal any significant fat accumulation and lesions in kidney tissue of the group MC, as well as significant changes in the intervention of L. fermentum grx08 and its heat-inactivated bacteria (Figure 4b).

4. Discussion

A high-fat, high-energy diet results in numerous health problems. In addition to causing fatty liver, injury to the heart and kidney cannot be ignored. We used a high-fat diet to induce oxidative stress injury in the rat liver, heart, and kidney, and intervened with L. fermentum grx08 to investigate the modulating effect of L. fermentum grx08 on the anti-oxidative stress capacity of the liver, heart, and kidney in rats.

The changes in organ indices were mainly influenced by the accumulation of organ lipids. The liver is the main organ of lipid metabolism and has a regulatory role in the homeostasis of lipid metabolism in the body. Imbalance of lipid metabolism leads to the accumulation of fat in the liver first [2], which makes the liver index increase. In contrast, injury to the heart and kidney is caused by disorders of lipid metabolism [3,4] and does not result in significant accumulation of lipids, so the cardiac and renal indices did not change significantly in this study. The results of the case sections of the liver, heart, and kidney showed the same situation, with significant fat vacuoles only in the liver sections. L. fermentum grx08 greatly reduced fat vacuoles and the rat liver index, and alleviated liver lipid accumulation. Further measurements of lipid levels in the rat liver also revealed that the high-fat diet caused lipid accumulation in the liver, and L. fermentum grx08 was able to significantly reduce lipid accumulation in the liver and restore the balance of lipid metabolism in the liver.

A number of markers in the serum can more accurately reflect the extent of organ injury. Serum ALT and AST are important indicators of the extent of liver injury. When hepatocytes are damaged, cell membrane permeability increases and these two aminotransferases enter the circulation, leading to an increase in their serum concentrations [12]. LDH is the catalase for the final step of the glycolytic process, and the disruption of the cardiomyocyte membrane or abnormal permeability causes the release of LDH from the circulation [13]. CK-MB is mainly found in the myocardium and has a high sensitivity and specificity for heart injury, and is an important indicator for the diagnosis of heart injury [14].UA is derived from the breakdown of purine compounds in the body and dietary intake, and elevated serum UA is closely related to kidney injury [15]. Crea is also an important indicator for the evaluation of kidney injury, which results in a decrease in the glomerular filtration rate and, consequently, an increase in serum Crea [16]. In this study, the high-fat diet caused significant increases in ALT, AST, LDH, CK-MB, UA, and Crea in the group MC (p < 0.05), suggesting that although lipids only accumulated significantly in the liver, rats showed injury to the liver, heart, and kidney, which was significantly reduced after L. fermentum grx08 intervention (p < 0.05). It tentatively suggested that L. fermentum grx08 could enhance the anti-oxidative stress capacity of the liver, heart, and kidney in rats.

A long-term high-fat diet also causes the body to produce large amounts of free radicals through enzyme systems or non-enzyme systems. Free radicals attack unsaturated fatty acids in biological membranes and trigger lipid peroxidation, forming lipid peroxides, which deplete antioxidant substances in the body and lead to cell necrosis and apoptosis [17]. NO radicals are the main free radicals in the body [18]. MDA is the product of lipid peroxidation by free radicals, and its content reflects the degree of lipid peroxidation in the body and indirectly reflects the severity of free radical attack on cells [19]. SOD is the most important enzyme for scavenging free radicals in the body, and GSH-Px is an important peroxide-degrading enzyme that plays an important role in protecting cellular organelles from oxidative injury. Under normal conditions, antioxidants such as SOD and GSH-Px work together in the body to effectively scavenge free radicals in the body and maintain redox homeostasis [20]. We found that the high-fat diet caused different degrees of elevated NO and MDA and decreased GSH-Px in the liver, heart, and kidney, which indicated different degrees of oxidative stress injury. L. fermentum grx08 was able to modulate the anti-oxidative stress capacity of the liver, heart, and kidney to different degrees. L. fermentum grx08 had better modulating effects on the anti-oxidative stress capacity of kidney, enhancing the levels of both antioxidant enzymes SOD and GSH-Px, scavenging NO radicals and reducing lipid peroxidation products MDA, and all of them were restored to levels in the group NC. L. fermentum grx08 had the second highest modulatory effect on the anti-oxidative stress capacity of the liver and the weakest modulatory effect on the heart. We also found lower levels of NO and MDA in the kidney in comparison, which may also be the reason why the kidney was the first to recover after the L. fermentum grx08 intervention. In addition, we found that L. fermentum grx08 heat-inactivated bacteria also had some regulatory effects, and the regulatory effects on the liver, heart, and kidney were consistent with those of live bacteria.

5. Conclusions

In this study, we explored the regulatory effects of L. fermentum grx08 on oxidative stress injury in the liver, heart, and kidney of high-fat diet rats and compared its regulatory capacity. It was found that L. fermentum grx08 could regulate the balance of liver lipid metabolism, increase the activity of some antioxidant enzymes, scavenge free radicals, and reduce lipid oxidation products in the liver, heart, and kidney. It could also regulate the anti-oxidative stress capacity of the liver, heart, and kidney in varying degrees, especially increasing the anti-oxidative stress capacity of the kidney.