INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is becoming one of the most common chronic liver diseases worldwide, but there is currently no treatment approved by the U.S. Food and Drug Administration. Previous studies have shown that telomeres and telomerase play a role in the initiation and progression of NAFLD (1). The association between telomerase mutations and the progression of chronic liver disease to cirrhosis may be due to decreased telomerase activity, resulting in impaired maintenance of telomere length (3). In advanced liver injury, telomere function is impaired, and mutations in telomerase complexes (including hTERT and hTERC) have been observed in patients with liver cirrhosis (4). TRF2-interacting protein (TRF2IP) is associated with TRF1 (7). It has been reported that the rate of telomere shortening is affected by psychological stress and exercise training (8).
Evidence suggests that antioxidants and anti-inflammatories can also slow the rate of telomere shortening during aging (13). Resveratrol may be effective in patients with liver disorders (15).  It could also potentially prevent and improve NAFLD (16,17).
However, whether resveratrol consistently shows effects on NAFLD in vivo and in randomized clinical trials, as well as its precise mechanism of action, are critical questions that require confirmation and further research for its development.
Regarding the effect of physical exercise on NAFLD, research showed that aerobic training improved liver structural damage, pathological changes, and fat deposition in NAFLD rats (18,19).
Few studies have investigated the effects of exercise and resveratrol on factors that influence telomere changes. Although exercise and certain foods appear to increase telomere length, the mechanisms by which these factors occur are unknown.
Understanding the effects of aerobic exercise and supplements, such as resveratrol, on telomeres could provide a better understanding of the cellular mechanisms that affect diseases caused by NAFLD and be useful in preventing and developing diseases in these conditions.
Therefore, the present study aimed to investigate the effect of aerobic training and resveratrol on the expression of TRF1, TRF2, and TERT in liver tissue in rats with NAFLD.
METHODS
To conduct the present experimental study, 35 eight-week-old male Wistar rats were selected as samples and transferred to the research center. After the rats were transferred to the laboratory, induced NAFLD, and familiarized with exercise on a treadmill for rodents, they were randomly divided into two groups: NAFLD patients and healthy controls.
The rats in the patient group were further divided into four experimental subgroups: NAFLD: non-alcoholic fatty liver, NAFLDT: non-alcoholic fatty liver-exercise, NAFLDRSV: non-alcoholic fatty liver-resveratrol, and NAFLDTRSV: non-alcoholic fatty liver-exercise-resveratrol. The rats in the exercise group performed an eight-week (five days a week) aerobic exercise program, while the other rats did not participate in any exercise program (21).
For resveratrol supplementation, 1 g of resveratrol powder (Nutrabio, USA, with pharmacological grade and purity of 99.87%) was prepared as a stock at a concentration of 10 mg/mL (22).
Moreover, 48 hours after the last training session, and 12 to 14 hours of fasting, all animals were anesthetized with an intraperitoneal injection of a combination of ketamine (30-50 mg/kg body weight) and xylazine (3-5 mg/kg body weight).
Immediately after isolation and washing with saline, the tissue was immediately placed in tubes containing RNA later to prevent RNA degradation, transferred to liquid nitrogen, and then stored in a refrigerator at -80°C until measurement.
Finally, descriptive statistics (mean and standard deviation) and inferential statistics (Shapiro-Wilk, Levine, one-way analysis of variance, and Tukey's post hoc test) were used to statistically analyze the data using SPSS statistical software version 26 at a significance level of P≤0.05.
RESULTS
Data analysis indicated that there was a significant increase in the rate of changes in TRF1 expression in the NAFLD groups (P=0.0001) compared to the control group. Moreover, a significant decrease was observed in the NAFLDT (P=0.042) and NAFLDTRSV (P=0.0001) groups compared to NAFLD.
The results further demonstrated that a significant decrease in the amount of changes in TRF2 expression was observed in the NAFLD groups (P=0.0001) compared to CN. A significant increase was also observed in the NAFLDT (p=0.030), NAFLDRSV (p=0.020), and NAFLDTRSV (p=0.0001) groups compared to NAFLD; and NAFLDTRSV compared to the NAFLDT (p=0.029) and NAFLDRSV (p=0.042) groups.
The results also showed that there was a significant increase in the rate of changes in TERT expression in the NAFLD (P=0.0001), NAFLDT (P=0.0001), and NAFLDRSV (P=0.0001) groups compared to the control group. A significant decrease was observed in the NAFLDT (P=0.040), NAFLDRSV (P=0.049), and NAFLDTRSV (P=0.0001) groups compared to NAFLD; and in NAFLDTRSV compared to the NAFLDT (P=0.047) and NAFLDRSV (P=0.038) groups.
CONCLUSION
Considering the roles of TRF1, TRF2, and TERT in cellular function, it appears that altering the levels of these markers following physical activity and resveratrol supplementation can partially prevent the development of various liver diseases associated with NAFLD.
Ethical Considerations
Compliance with ethical guidelines
This study obtained ethical approval from the Faculty of Medical Sciences, Islamic Azad University, Sari Branch, Iran, with the ethics code IR.IAU.SARI.REC.1404.155.
Funding
There was no funding support.
Authors' Contributions
All authors participated in the design, implementation, data analysis, and writing of all parts of the research.
Conflict of Interest
The authors declare that there is no conflict of interest.
Acknowledgments
This work was extracted from the master's thesis in the field of exercise physiology, Islamic Azad University, Qaemshahr Branch, Iran. The authors would like to express their sincere gratitude to all those who contributed to the conduct of this research.