The Effect of Resveratrol Supplementation and Aerobic Exercise on Serum Levels of Activin A and TGF-β in Rats with Non-Alcoholic Fatty Liver Disease

Background & Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) is one of the main causes of abnormal liver function, and the prevalence of this disease is reported to be around 20% in the world (1). NAFLD is often associated with obesity, dyslipidemia, and insulin resistance in metabolic syndrome (2). NAFLD includes the accumulation of triglycerides, necrosis, and apoptosis in fat cells, which are associated with inflammatory responses in the liver. In total, these cases increase the possibility of liver cirrhosis (4). The large family of transforming growth factor beta (TGF-β) together with TGF-β1 and activin-A is a part of bone morphogenic proteins and is considered a multipurpose cytokine in a wide range of tissues and cells. These factors play a role in cell differentiation, cell number homeostasis and tissue repair, inflammation, cell proliferation, and apoptosis (5). Activin-A increases the expression of collagen and TGF-β1. It causes mitochondrial β-oxidation and downregulation of fatty acid synthase activity. In addition, activin-A inhibits proliferation and stimulates apoptosis of liver cells, which helps complete liver regeneration; Hepatocyte proliferation and liver regeneration are controlled by activin-A (7). Today, for the management of NAFLD, lifestyle modification techniques through changes in diet and physical activity are considered. Physical activity, including aerobic, resistance, and interval, reduces NAFLD disease by regulating intrahepatic fat status, reducing oxidative stress and inflammation, apoptosis, and autophagy process (8). There are various medicinal agents for the treatment of various diseases. The combination of using herbal medicines and engaging in physical activity has attracted the attention of researchers. Among these substances is resveratrol, which is found in purple and red plants, especially in the skin of red grapes and the berry family (9). Resveratrol has antioxidant, anti-cancer, and anti-inflammatory uses, and was first extracted from an Asian plant called Polygonum capsidatum (10). Resveratrol has inherent protective properties to prevent the spread of disease. Its anti-inflammatory effect has been determined by reducing pro-inflammatory cytokines and preventing the production of nitric oxide in macrophages. Reservatol also regulates lipid metabolism and prevents lipid oxidation and platelet aggregation (11). Methods: In this experimental study, 56 old male Wistar rats with an age of 40-50 weeks and an average weight of 250-300 grams were selected. At first, the rats were divided into two groups: control and NAFLD patients. The rats in the control group were fed a standard diet (including 12% fat, 57% carbohydrates, 28% protein, and 3% other items) for 6 weeks. To induce NAFLD, rats in the experimental group were subjected to a high-fat diet (including 22% fat, 50% carbohydrate, 24% protein, and 4% other items) for 6 weeks. Then, the rats of the patient group with NAFLD were divided into 7 groups, and each group included 7 rats. Seven groups include (1) patient group, (2) group receiving saline, (3) group receiving resveratrol supplement, (4) continuous training group, (5) intermittent training group, (6) continuous training-resveratrol group, and (7) intermittent training-resveratrol group. The rats in the training group and the supplement+training group were familiarized with the treadmill for 5 minutes at a speed of 8-10 m/min with zero inclines for one week during 5 sessions. The main continuous training program was carried out for 8 weeks and 5 sessions per week, in the first week, the speed was 15 meters per minute and the time was 5 minutes. Every week, the speed increased by 1-2 meters per minute and the time increased by 1-2 minutes. In the eighth week, the speed reached 20 meters per minute and the time was 60 minutes (12). The interval training program started in the first week with a speed of 10 meters per minute and a time of 10-15 minutes with a zero-degree slope. In the following weeks, training consisted of 10 minutes daily (2:30 x 4) at a speed of 50 m/min and a 10-degree incline with periods of 2:30 minutes (more than 90% of maximal oxygen consumption), separated by 3-minute rest periods. The number of sessions was 5 sessions per week (13). In the supplement and supplement+training groups, they were injected intraperitoneally with a dose of 20 mg per kilogram of body weight. After completing the research period, all rats were anesthetized by intraperitoneal injection of ketamine and xylazine 48 hours after the last training session. Liver samples were kept at -80 C. Serum activin-A and TGF-β levels were measured using a kit and ELISA method. The Shapiro-Wilk test was used to determine the normality of data distribution. If the data were normal, one-way variance analysis and Tukey's post hoc statistical tests were used. The significance level for all calculations was considered p≥0.05. All statistical operations were performed using SPSS version 20 software. Results: Data analysis shows that there is a significant difference in the mean and standard deviation of TGF-β and activin-A serum levels between different groups (P=0.0001). It was also observed that aerobic training and red grape extract (resveratrol) have a significant effect on the serum levels of TGF-β and activin-A in elderly rats with NAFLD in different groups (P=0.0001). Conclusion: The findings of the present research showed that the lowest serum levels of activin-A were observed in the resveratrol+continuous training group and the highest levels were observed in the patient and saline groups. Fatty liver increased activin-A levels in the patient group, and aerobic training and red grape extract (resveratrol) significantly decreased serum activin-A levels in elderly rats with NAFLD in the resveratrol+continuous training group; Of course, in the supplement group, continuous training and interval training+resveratrol also had a decreasing trend, but it did not reach a significant level. NAFLD appears to result from an imbalance between hepatic fatty acid synthesis and oxidation (5). Our findings suggest that NAFLD should be added to the list of liver disorders that could be activin-A-mediated mechanisms. Previously, activin-A was reported to inhibit liver growth by inhibiting hepatic proliferation and inducing apoptosis, as well as by inducing extracellular matrix production during liver fibrosis (14). The results of the present study showed that the levels of TGF-β increased in the high-fat food consumption group; while training and resveratrol supplementation decreased the level of TGF-β in the experimental groups. Studies have shown that the TGF-β1/Smad signaling pathway may play an important role in activating HSCs and regulating the production, degradation, and accumulation of ECM proteins (19). Therefore, inhibiting the TGF-β/Smad signaling pathway can be an attractive therapeutic target for the prevention of liver fibrosis (20). Finally, the findings of the current research showed that the serum levels of TGF-β and activin-A increased in rats with NFALD. The reason for the increased levels of cytokines may be related to the cirrhosis process itself. In previous research, it has been shown that poor liver function leads to cirrhosis secondarily and disrupts the metabolism of TGF-β and causes the accumulation of this multifunctional cytokine in the body.