**4. Discussion**

Excessive fat accumulation in hypertrophic adipose tissue associated with obesity is responsible for oxidative stress, chronic inflammation, and dysregulated adipokine secretion [25]. It is believed that the therapeutic potential of natural dietary compounds against obesity and obesity-related disorders should focus on improving the fat function in pathogenic hypertrophic adipocytes by reducing oxidative stress, alleviating inflammation, and regulating underproduction or overproduction of clinically relevant adipocyte factors. However, most bioactive compounds or extracts strongly a ffect preadipocytes, their viability, proliferation, and di fferentiation into mature fat cells, without any significant e ffects on the pathological status of hypertrophic adipocytes. Therefore, in this work, the influence of the *Sambucus nigra* fruit extract on mature fully di fferentiated insulin-resistant 3T3-L1 adipocytes was investigated.

In our study, we found no reduction in cell viability and lipid content in hypertrophic 3T3-L1 adipocytes after exposure to EDB extract. However, as a result of the treatment, the intracellular ROS generation was significantly down-regulated and probably, oxidative stress accompanying excessive fat accumulation was also importantly reduced. Oxidative stress induced by enhanced lipid content is reported to be involved in the pathogenesis of obesity-related comorbidities including insulin resistance and diabetes, cardiovascular complications, and cancer [26]. It was found that ROS are intensively generated in visceral adipose tissue by adipocytes during the metabolism of excess nutrients and also by macrophages, which accumulate in adipose tissue in obesity state. The increased release of fatty acids from overproduced fat accumulated in adipose tissue, activate NADPH oxidases (NOX) and induce or aggravate ROS production. Other factors that also contribute oxidative stress to obesity include hyperleptinemia, low antioxidant defense, or chronic inflammation [27]. Our results showed that EDB extract could reduce ROS generation by lowering the expression of NOX4, the major NOX isoform in adipocytes. Treatment of hypertrophied 3T3-L1 adipocytes with EDB extract caused a significant decreasing in *NOX4* mRNA expression. Furthermore, up-regulation of mRNA expression of antioxidant enzymes, like SOD and GPx, could also contribute to enhancing adipocyte antioxidant defense e fficiency. Numerous studies have shown the high antioxidant capacity of *Sambucus nigra* fruit [6,10]. However, the antioxidant e ffects of EDB on adipocytes have not ye<sup>t</sup> been reported in the literature. In the present study, we demonstrated that introduction of EDB extract to the culture of hypertrophic adipocytes resulted in decreased ROS generation in cells. The antioxidant action of EDB extract in adipocytes may be a potential protective mechanism against obesity-associated pathological risk factors, including insulin resistance and chronic inflammation.

Additionally, EDB extract treatment modulated the leptin and adiponectin gene expression and protein secretion in hypertrophic 3T3-L1 adipocytes. Leptin and adiponectin are adipocytokines, which influence energy homeostasis, glucose and lipid metabolism, cardiovascular function, and immune response [28]. Leptin is primarily secreted by fully di fferentiated adipocytes, and its crucial role is to regulate energy intake and expenditure through controlling appetite and glucose metabolism. Reflecting the increased amount of adipose tissue, obese individuals often have elevated leptin concentration and the simultaneous apparent loss of e fficacy of leptin, which is a result of leptin resistance, the state that leads to uncontrolled food intake, pro-inflammatory state, diabetes mellitus, and other obesity-related complications [29]. In contrast to leptin, adiponectin is down-regulated in obesity, and the circulating adiponectin levels are inversely correlated with body fat amount. Adiponectin enhances energy metabolism and fatty acid oxidation, promotes insulin sensitivity, improves glucose tolerance, and exerts anti-inflammatory e ffects [28]. Low serum adiponectin and high serum leptin levels are considered as risk factors for developing type 2 diabetes (T2DM), obesity, dyslipidemia, hypertension, and cardiovascular diseases. In this study, a remarkable decrease in leptin expression and secretion was observed in response to EDB extract treatment of hypertrophied 3T3-L1 adipocytes, which may help counteract the leptin resistance state. Whereas, adiponectin mRNA expression and protein secretion in treated adipocytes were significantly increased. The e ffect of EDB extract on adiponectin production may indicate anti-inflammatory potential and insulin-sensitizing activity of *Sambucus nigra* fruit.

The association of visceral obesity with T2DM is a long-recognized phenomenon. The primary determinant of this correlation is the fact that central obesity is the critical factor in the emergence of insulin resistance. The insulin-resistant state results in defective insulin-stimulated glucose uptake and consequently in hyperglycemia, elevated circulating free fatty acids level, abnormal fat accumulation, and dysregulation of hepatic glucose production, that, in combination with a paucity of insulin secretion by pancreatic β-cells, leads to T2DM [30]. These metabolic abnormalities may arise from impairment in insulin signaling pathways and subsequent defect in translocation of insulin-responsive glucose transporter protein (GLUT-4) and in adipose tissue, also from down-regulation of *GLUT-4* gene [31].

The e ffects of EDB extract on glucose uptake and *GLUT-4* expression were evaluated in this study. Experiments were performed both with mature 3T3-L1 adipocytes sensitive to insulin and adipocytes treated with TNFα to induce an inflammatory status and insulin resistance. Analysis revealed that EDB extract stimulated the 2-NBDG uptake in both types of adipocytes and up-regulated mRNA expression of *GLUT-4* in insulin-resistant cells, suggesting insulin-like and insulin-sensitizing activities of the extract. The signaling pathways involved in the development of these activities will be further examined in future studies. This is the first study assessing the e ffects of EDB extract on glucose uptake in 3T3-L1 cells. Although several recent reports have suggested the anti-diabetic and hypoglycemic properties of elderberry, it has been found that EDB methanolic extracts markedly stimulate glucose uptake in liver HepG2 cells and also exert inhibitory e ffect towards carbohydrate hydrolyzing enzyme [32]. Furthermore, EDB extracts, EDB anthocyanins, mainly cyanidin-3-glucoside and cyanidin-3-sambubioside, procyanidins, and their metabolites were found to enhance glucose uptake in human skeletal muscle cells [33]. Whereas, EDB lipophilic and polar extracts were reported to modulate glucose metabolism or lower insulin secretion contributing to the mitigation of insulin resistance in T2DM rats [13].

Anti-obesity and anti-diabetic activity of EDB extract could be related to the inhibition of dietary fat and sugar absorption from the intestinal tract. There is some evidence that polyphenols from berry fruits, such as strawberry, raspberry, blueberry, bilberry, black and red currant, lingonberry, red and green gooseberry, cranberry, and chokeberry, contribute to the inhibition of digestive enzymes involved in the hydrolysis of dietary lipids and carbohydrates [34]. Based on our research, the *Sambucus nigra* fruit may be included in the class of berries considered as e ffective inhibitors of α-amylase, α-glucosidase, and pancreatic lipase activity.

Obesity is known to be accompanied by metaflammation—low-grade chronic inflammation condition triggered by excess nutrients in metabolic cells [35]. An attribute of obesity-related inflammation is enhanced infiltration of macrophages into expanding adipose tissue, activation of specialized immune cells, and secretion of proinflammatory cytokines such as TNFα, IL-6, and MCP-1 leading to an unresolved inflammatory response, which a ffects normal metabolism and insulin action [35]. Inhibition of obesity-induced inflammation could, thus, be a therapeutic intervention against adipose tissue dysfunction and related co-morbidities. In recent years, the use of anti-inflammatory nutrients provided through diet as a potential approach against obesity has been extensively studied [36,37].

In the present study, we evaluated anti-inflammatory e ffects of EDB extract in LPS-stimulated RAW 264.7 macrophages. Activated macrophages produce cytokines such as TNFα, IL-1β, and IL-6 as well as pro-inflammatory mediators, such as NO and PGE2 [38]. IL-6 and TNFα are potent proinflammatory cytokines, which play a central role in inflammatory response and are characterized by a broad spectrum of functions with various e ffects in adipose tissue. TNFα substantially influences lipid metabolism and adipocytes apoptosis. It can disrupt insulin signaling pathway promoting insulin resistance and adipocytes dysfunction [39]. TNFα has, thus, been believed to be the crucial mediator in the detrimental paracrine loop between adipocytes and macrophages [40]. IL-6 has a pivotal role in acute phase reactions. It also influences hormonal balance and energy homeostasis and may a ffect the increase of free fatty acids level. Circulating levels of IL-6 and TNFα are elevated in obese individuals and patients with insulin resistance [41]. In general, the regulation of TNFα and IL-6 secretion is considered to be a potent treatment strategy for inflammation-associated diseases [42].

The research presented in this work suggests that EDB extract dose-dependently down-regulates mRNA expression and protein production of TNFα and IL-6 in activated RAW 264.7 macrophages and therefore alleviates the cellular inflammatory response induced by LPS. In addition to TNFα and IL-6, EDB extract significantly reduced the production of inflammatory mediators—PGE2 and NO. Increased level of PGE2 is observed in obese adipose tissue due to remarkable up-regulation of COX-2—the key enzyme in eicosanoid metabolism, of which expression is induced in inflammation state [43]. It has been suggested that COX-2-mediated inflammation in visceral fat is responsible for insulin resistance and fatty liver development in high-fat-induced obese rats [44]. The same study revealed that COX-2 inhibition significantly reversed adipocyte hypertrophy, macrophage infiltration, and decreased markers of adipocyte di fferentiation. Nitric oxide formed by iNOS is a short-lived vasodilator that acts as an important regulator of physical homeostasis, while its overproduction has been closely correlated with the pathological conditions including septic shock, osteoporosis and rheumatoid arthritis, insulin resistance, and inflammation [45]. In the present study, EDB extract was found to suppress PGE2 and NO production via down-regulation of *COX-2* and *iNOS* expression. These findings indicate that inhibition of PGE2 and NO generation is one of the anti-inflammatory mechanisms of the extract. Several recent studies have shown the anti-inflammatory potential of EDB fruit preparations. In our previous study, we demonstrated the anti-inflammatory potential of gastrointestinally digested EDB extract following intestinal absorption in a co-culture model of intestinal epithelial Caco-2 cells and LPS-stimulated RAW 264.7 macrophages [7]. The analyzed extract down-regulated the expression of genes (IL-1β, IL-6, TNFα, COX-2) involving in the inflammatory pathway in a range comparable to that of budesonide. This study demonstrated adequate bioavailability and intestinal permeability of EDB compounds that are probably su fficient to evoke systemic anti-inflammatory e ffects [7]. Moreover, there is increasing evidence that the EDB bioactives can penetrate the blood–brain barrier and modulate the immune response induced in di fferent types of brain injuries, including ischemic stroke. It has been found that EDB extract and its phenolic components significantly inhibit activation of microglia, considered to be resident macrophages responsible for the initial immune response to brain injuries. Treatment of activated microglial bv-2 cells with EBD extract led to diminishing ROS and NO generation, and as a consequence, attenuating the neuroinflammatory process [16].

Results of the study, as discussed above, indicate that *Sambucus nigra* fruit extract may o ffer substantial preventive and therapeutic potential for the treatment of obesity and obesity-related disorders, accompanied by oxidative stress, inflammationm and insulin resistance. Moreover, the extract can inhibit digestive enzyme activity, and consequently, significantly reduce the intestinal absorption of dietary lipids and carbohydrates, which is an e ffective strategy for the prevention and treatment of obesity and metabolic comorbidities.

Considering the findings of *in vitro* studies, we can postulate a nutraceutical application of the *Sambucus nigra* fruit extract. The scientific community focuses grea<sup>t</sup> attention on introducing nutraceuticals into the daily diet to prevent the occurrence of the pathological conditions, to delay or avoid the need for drug treatment and to support pharmacological therapy. Nutraceuticals as pharmafoods should be evaluated in the clinical aspects regarding safety, side effects, bioavailability, beneficial health effects, mechanisms of action and efficacy, and any possible interactions between food and drugs assumed together with them [46,47]. Thus, the developing of clinical studies will be of significant importance for clinically justified promotion of the *Sambucus nigra* fruit extract as a safe nutraceutical with the capacity of prevention or treatment of obesity and obesity-related immune-metabolic disorders.

**Author Contributions:** Conceptualization, J.Z.-W. and A.O.; methodology, J.Z.-W., A.O., M.O. and K.K.; formal analysis, A.O. and R.D.; investigation, J.Z.-W. and M.O.; resources, R.D.; writing—original draft preparation, J.Z.-W.; writing—review and editing, A.O.; project administration, A.O.; funding acquisition, A.O.

**Funding:** This research was funded by THE NATIONAL SCIENCE CENTRE, POLAND, gran<sup>t</sup> number 2015/19/B/NZ9/01054.

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