The Effect of Intensive Aerobic Training with Omega-3 Supplementation on Level of Pro-Inflammatory Cytokines in Liver Tissue of Obese Mice

Anbari, Kosar; Taheri Kalani, Abdolhossein; Omidi, Mahnaz

doi:10.22089/spj.2025.18512.2402

The Effect of Intensive Aerobic Training with Omega-3 Supplementation on Level of Pro-Inflammatory Cytokines in Liver Tissue of Obese Mice

Document Type : Research Paper

Authors

Kosar Anbari

Abdolhossein Taheri Kalani

Mahnaz Omidi

Department of Sport Sciences, Faculty of Humanities, Il.C., Islamic Azad University, Ilam, Iran

10.22089/spj.2025.18512.2402

Abstract

Background and Purpose
An imbalance between energy intake and expenditure leads to overweight and obesity, which is closely associated with excessive fat accumulation in adipose tissue. Adipocytes, the primary cells in adipose tissue, possess the capability to produce and secrete pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). These cytokines play a pivotal role in the pathogenesis of various liver diseases, including non-alcoholic fatty liver disease (NAFLD), by promoting chronic low-grade inflammation and metabolic dysfunction. Accumulating evidence suggests that structured exercise training can significantly improve liver cell function, potentially through mechanisms involving enhanced mitochondrial biogenesis, improved insulin sensitivity, and reduced oxidative stress, although the precise pathways remain incompletely elucidated. Nutrition emerges as another critical modulator of inflammatory processes, with diets enriched in omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), demonstrating robust positive effects on human health, including prevention of inflammation and cardiometabolic disorders. Chronic inflammation constitutes a central driver in the development of metabolic disorders such as obesity, type 2 diabetes, and NAFLD, underscoring the necessity to explore combined exercise and dietary interventions capable of improving overall health by modulating key inflammatory markers. Despite extensive research on individual interventions, available evidence reveals a notable gap regarding the synergistic or potentially interfering effects of concurrent exercise training and ω-3 supplementation. Therefore, the present study was designed to investigate the effect of intensive aerobic training combined with ω-3 supplementation on the protein levels of pro-inflammatory cytokines IL-6 and TNF-α in the liver tissue of obese mice, providing insights into their combined therapeutic potential for obesity-related liver inflammation.
Materials and Methods
This experimental study involved 30 male mice aged 12-14 weeks, randomly divided into five groups (n=6 per group): normal diet (ND), high-fat diet (HFD), HFD+ω-3 supplementation, HFD+Training (HFD+T), and HFD+ω-3+Training (HFD+ω-3+T). All animals were housed under strictly controlled environmental conditions, including an average temperature of 22±4°C, relative humidity of 40-50%, and a standardized 12:12-hour light/dark cycle, with unrestricted access to water and group-specific food. The entire protocol spanned 16 weeks, comprising two weeks of familiarization, eight weeks of obesity induction, and six weeks of intensive aerobic training intervention with or without ω-3 supplementation. Mice in the ND group received a standard chow diet throughout the study, formulated with 15% fat, 25% protein, and 60% carbohydrates to maintain normal body composition. Following the initial two-week acclimation period on standard diet, obesity was induced in the intervention groups (HFD, HFD+ω-3, HFD+T, HFD+ω-3+T) using a custom high-fat diet (HFD) sourced from the Royan Institute of Isfahan. This HFD composition included 45% fat, 35% carbohydrates, and 20% protein (in kcal), delivering 4.60 kcal/g of energy, which effectively promoted rapid weight gain and metabolic alterations characteristic of obesity. By the conclusion of the eight-week induction phase, the average body weight of obese mice reached 32.89 g, confirming successful model establishment. Subsequently, the HFD was discontinued across all intervention groups, reverting them to the standard diet to isolate the effects of the subsequent interventions. The intensive aerobic training protocol was administered over six weeks, consisting of five sessions per week. Each session involved progressive treadmill running progressing from 10 to 50 minutes at an intensity of 70-75% of maximal oxygen consumption (VO₂max), with speeds ranging from 25-30 meters per minute and a 15% incline to simulate high-intensity aerobic demand. Groups receiving ω-3 supplementation (HFD+ω-3 and HFD+ω-3+T) were administered 500 mg/kg body mass of EPA and DHA daily via oral gavage, ensuring consistent dosing. At study termination, liver tissues were harvested, and protein levels of IL-6 and TNF-α were quantified using the Bradford assay for total protein normalization and enzyme-linked immunosorbent assay (ELISA) for cytokine detection, following standard laboratory protocols.
Results
Statistical analysis revealed significant differences in body mass across the experimental groups (p<0.05). Tukey's post hoc test indicated that at the onset of the intervention phase, body mass in the HFD, HFD+T, HFD+ω-3, and HFD+ω-3+T groups was markedly higher than in the ND group (all comparisons p=0.0001), validating the obesity induction. At the intervention's conclusion, body mass remained significantly elevated in the HFD group (p=0.0001), HFD+T group (p=0.029), and HFD+ω-3 group (p=0.031) relative to ND, reflecting persistent obesity effects; however, no significant difference emerged between HFD+ω-3+T and ND (p=0.067), suggesting effective mitigation by the combined intervention. Regarding inflammatory markers, data analysis demonstrated a significant elevation in hepatic IL-6 and TNF-α protein levels in the HFD+T (p=0.0001), HFD+ω-3 (p=0.0001), and HFD+ω-3+T (p=0.012) groups compared to ND, consistent with obesity-induced inflammation. Critically, all intervention groups exhibited substantial reductions versus HFD (p=0.0001). Furthermore, IL-6 and TNF-α levels were significantly lower in HFD+ω-3 (p=0.042) and HFD+ω-3+T (p=0.0001) compared to HFD+T alone, although the difference between HFD+ω-3 and HFD+ω-3+T was not statistically significant (p=0.054), indicating additive rather than fully synergistic suppression in cytokine reduction.
Conclusion
The findings confirm that HFD markedly elevates IL-6 and TNF-α protein levels in liver tissue relative to ND, exacerbating pro-inflammatory states. The mechanisms underlying HFD-induced increases in these markers remain incompletely understood but are hypothesized to involve adipose tissue expansion and ectopic fat deposition in the liver, leading to hepatocyte damage through inflammatory cytokine release and reactive oxygen species (ROS) generation. In contrast, intensive aerobic training, ω-3 supplementation, and their combination effectively attenuated IL-6 and TNF-α levels. Notably, ω-3 supplementation outperformed intensive aerobic training alone in cytokine reduction, highlighting its potent anti-inflammatory properties. Regular aerobic exercise mitigates liver inflammation in models of nonalcoholic fatty liver disease by decreasing macrophage infiltration, elevating antioxidant enzyme expression (e.g., superoxide dismutase, catalase), and normalizing ROS homeostasis. ω-3 fatty acids exert superior anti-inflammatory effects by serving as precursors for specialized pro-resolving mediators such as resolvins, protectins, and maresins, which actively resolve inflammation at sites of injury. These PUFAs competitively inhibit pro-inflammatory ω-6 fatty acid derivatives—including arachidonic acid metabolites like prostaglandins, leukotrienes, and lipoxins—produced via cyclooxygenase and lipoxygenase pathways. The study's culminating observation—a synergistic interaction between intensive aerobic exercise and ω-3 supplementation—manifested in enhanced suppression of IL-6 and TNF-α protein expression in obese mice liver tissue, suggesting complementary mechanisms that amplify anti-inflammatory outcomes beyond individual therapies.
Article Message
The combination of intensive aerobic exercise and ω-3 supplementation demonstrates a clear synergistic effect in modulating inflammatory markers within liver tissue of obese models. Considering chronic inflammation's foundational role in fatty liver disease pathogenesis and broader metabolic disorders, these findings advocate exercise and targeted nutritional interventions as efficacious strategies for disease management, extending beyond inflammation control to metabolic restoration. Nevertheless, additional mechanistic studies are warranted to delineate precise signaling pathways and translate these benefits to clinical populations.
Ethical Considerations
The ethics review board of Islamic Azad University, Ilam branch, approved the present study with the code of IR.IAU.ILAM.REC.1402.063.
Authors’ Contributions
Conceptualization: Abdolhossein Taheri Kalani, Kosar Anbari
Data Collection: Kosar Anbari, Mahnaz Omidi
Data Analysis: Abdolhossein Taheri Kalani, Mahnaz Omidi
Manuscript Writing: Mahnaz Omidi, Kosar Anbari
Review and Editing: Abdolhossein Taheri Kalani
Literature Review: Abdolhossein Taheri Kalani, Mahnaz Omidi, Kosar Anbari
Project Manager: Abdolhossein Taheri Kalani
Any Other Contribution: Avin Stem Gen Bio Health Inc. performed experimental analysis.
Conflict of Interest
The authors declare no conflicts of interest regarding the publication of this study.
Acknowledgments
The authors thank Avin Stem Gen Bio Health Inc. for technical support performing.

Keywords

Intensive Aerobic Training

Omega-3

High-Fat Diet

Supplementation

Inflammation

Subjects

sport nutrition

1. Chooi YC, Ding C, Magkos F. The epidemiology of obesity. Metabolism. 2019;92:6-10. http://doi.org/10.1016/j.metabol.2018.09.005

2. Duan Y, Pan X, Luo J, Xiao X, Li J, Bestman PL, Luo M. Association of inflammatory cytokines with non-alcoholic fatty liver disease. Front Immunol. 2022;13:880298. http://doi.org/10.3389/fimmu.2022.880298

3. Zheng H, Sechi LA, Navarese EP, Casu G, Vidili G. Metabolic dysfunction-associated steatotic liver disease and cardiovascular risk: a comprehensive review. Cardiovasc Diabetol. 2024;23(1):346. http://doi.org/10.1186/s12933-024-02434-5

4. Masetto Antunes M, Godoy G, Masi LN, Curi R, Barbosa Bazotte R. Prefrontal cortex and hippocampus inflammation in mice fed high-carbohydrate or high-fat diets. J Med Food. 2022;25(1):110-3. http://doi.org/10.1089/jmf.2021.0026

5. Engin A. Nonalcoholic fatty liver disease and staging of hepatic fibrosis. Adv Exp Med Biol. 2024;1460:539-574. http://doi.org/10.1007/978-3-031-63657-8_18

6. Auguet T, Bertran L, Binetti J, Aguilar C, Martı́nez S, Sabench F, et al. Relationship between IL-8 circulating levels and TLR2 hepatic expression in women with morbid obesity and nonalcoholic steatohepatitis. Int J Mol Sci. 2020;21(11):4189. http://doi.org/10.3390/ijms21114189

7. Ponziani FR, Bhoori S, Castelli C, Putignani L, Rivoltini L, Del Chierico F, et al. Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in nonalcoholic fatty liver disease. Hepatology. 2019;69(1):107-20. http://doi.org/10.1002/hep.30036

8. Cheng R, Wang L, Le S, Yang Y, Zhao C, Zhang X, et al. A randomized controlled trial for response of microbiome network to exercise and diet intervention in patients with nonalcoholic fatty liver disease. Nat Commun. 2022;13(1):2555. http://doi.org/10.1038/s41467-022-29968-0

9. Thyfault JP, Bergouignan A. Exercise and metabolic health: beyond skeletal muscle. Diabetologia. 2020;63(8):1464-74. http://doi.org/10.1007/s00125-020-05177-6

10. Wang H, Cheng R, Xie L, Hu F. Comparative efficacy of exercise training modes on systemic metabolic health in adults with overweight and obesity: a network meta-analysis of randomized controlled trials. Front Endocrinol. 2024;14:1294362. http://doi.org/10.3389/fendo.2023.1294362

11. Nalcakan GR. The effects of sprint interval vs. continuous endurance training on physiological and metabolic adaptations in young healthy adults. J Hum Kinet. 2014;44:97-109. http://doi.org/10.2478/hukin-2014-0115

12. Kalaki-Jouybari F, Shanaki M, Delfan M, Gorgani-Firouzjae S, Khakdan S. High-intensity interval training (HIIT) alleviated NAFLD feature via miR-122 induction in liver of high-fat high-fructose diet induced diabetic rats. Arch Physiol Biochem. 2020;126(3):242-9. http://doi.org/10.1080/13813455.2018.1510968

13. Javadi Kia M, Barjaste Yazdi A, Khajei R, Hosein Abadi M R. Effect of high-intensity interval training and portulaca oleracea extract on c-reactive protein and tumor necrosis factor-α in rats with nonalcoholic fatty liver disease. North Khorasan Univ Med Sci. 2023;15(2):13-21. http://doi.org/10.32592/nkums.15.2.13 [In Persian].

14. Khaleghzadeh H, Afzalpour ME, Ahmadi MM, Nematy M, Sardar MA. Effect of high intensity interval training along with Oligopin supplementation on some inflammatory indices and liver enzymes in obese male Wistar rats with non-alcoholic fatty liver disease. Obes Med. 2020;17:100177. http://doi.org/10.1016/j.obmed.2019.100177

15. Ghasemi M, Abdi A, Abbassi Daloii A. The effect of combining aerobic exercise and royal jelly on the expression of pro-inflammatory cytokines in liver tissue of obese rats. Iranian Journal of Diabetes and Metabolism. 2023;22(5):282-293. [In Persian].

16. Kawanishi N, Yano H, Mizokami T, Takahashi M, Oyanagi E, Suzuki K. Exercise training attenuates hepatic inflammation, fibrosis and macrophage infiltration during diet induced-obesity in mice. Brain Behav Immun. 2012;26(6):931-41. http://doi.org/10.1016/j.bbi.2012.04.006

17. Chacińska M, Zabielski P, Książek M, Szałaj P, Jarząbek K, Kojta I, Chabowski A, Błachnio-Zabielska AU. The impact of omega-3 fatty acids supplementation on insulin resistance and content of adipocytokines and biologically active lipids in adipose tissue of high-fat diet fed rats. Nutrients. 2019;11(4):835. http://doi.org/10.3390/nu11040835

18. Gerling CJ, Mukai K, Chabowski A, Heigenhauser GJF, Holloway GP, Spriet L, et al. Incorporation of omega-3 fatty acids into human skeletal muscle sarcolemma and mitochondrial membranes following 12 weeks of fish oil supplementation. Front Physiol. 2019;10:348. http://doi.org/10.3389/fphys.2019.00348

19. Jeong HY, Moon YS, Cho KK. ω-6 and ω-3 polyunsaturated fatty acids: inflammation, obesity and foods of animal resources. Food Sci Anim Resour. 2024;44(5):988-1010. http://doi.org/10. 5851/kosfa.2024.e65

20. Torabi Palat Kaleh G, Abdi A, Abbassi Daloii A. The effect of aerobic exercise and omega-3 on inflammatory and oxidative stress in the heart tissue of elderly HFD rats. J Isfahan Med Sch. 2023;41(716):277-86. http://doi.org/10.48305/jims.v41.i716.0277. [In Persian].

21. Malekshahi Moghadam A, Saedisomeolia A, Djalali M, Djazayery A, Pooya S, Sojoudi F. Efficacy of omega-3 fatty acid supplementation on serum levels of tumour necrosis factor-alpha, C-reactive protein and interleukin-2 in type 2 diabetes mellitus patients. Singapore Med J. 2012;53(9):615-9.

22. Aliasghari F, Eftekhari M, Babaei Beigi M, Hasanzadeh J, Mazooji N. The effect of conjugated linoleic acids and omega-3 fatty acids supplementation on some inflammatory and oxidative stress markers in atherosclerosis. Iranian J Nutr Sci Food Technol. 2013;7(4):35-44. [In Persian].

23. Jalili R, Somi MH, Hosseinifard H, Salehnia F, Ghojazadeh M, Makhdami N, et al. The evaluation of effective drugs for the treatment of non-alcoholic fatty liver disease: a systematic review and network meta-analysis. Adv Pharm Bull. 2020;10(4):542-55. http://doi.org/10.34172/apb.2020.065.

24. Daneshyar S, Mirakhori Z, Forutan Z. The effect of high-fat diet and continuous endurance training on expression of TFEB and E2F1 transcription factors in visceral adipose tissue of mice. Feyz. 2023;27(6):646-56. http://doi.org/10.48307/FMSJ.2022.26.6.646 [In Persian].

25. Nemati N, Bagherpour T, Mokheilefi M. Effect of aerobic training and cinnamon on expression of resistin gene in adipose tissue of male rats fed with a high-fat diet. Jundishapur J Health Sci. 2023;16(1):e135644. http://doi.org/10.5812/jjhs-135644

26. Pereira MG, Ferreira JC, Bueno CR Jr, Mattos KC, Rosa KT, Irigoyen MC, et al. Exercise training reduces cardiac angiotensin II levels and prevents cardiac dysfunction in a genetic model of sympathetic hyperactivity-induced heart failure in mice. Eur J Appl Physiol. 2009;105(6):843-50. http://doi.org/10.1007/s00421-008-0967-4

27. Bardova K, Funda J, Pohl R, Cajka T, Hensler M, Kuda O, et al. Additive effects of omega-3 fatty acids and thiazolidinediones in mice fed a high-fat diet: Triacylglycerol/fatty acid cycling in adipose tissue. Nutrients. 2020;12(12):3737. http://doi.org/10.3390/nu12123737

28. Feng D, Zou J, Su D, Mai H, Zhang S, Li P, et al. Curcumin prevents high-fat diet-induced hepatic steatosis in ApoE^-/- mice by improving intestinal barrier function and reducing endotoxin and liver TLR4/NF-κB inflammation. Nutr Metab. 2019;16:79. http://doi.org/10.1186/s12986-019-0410-3.

29. Dey T, Ghosh A, Mishra S, Pal PK, Chattopadhyay A, Pattari SK, et al. Attenuation of arsenic induced high fat diet exacerbated oxidative stress mediated hepatic and cardiac injuries in male Wistar rats by piperine involved antioxidative mechanisms. Food Chem Toxicol. 2020;142:111477. http://doi.org/10.1016/j.fct.2020.111477

30. Jeong JH, et al. The effects of either resveratrol or exercise on macrophage infiltration and switching from M1 to M2 in high fat diet mice. J Exe Nut Biochem. 2015;19(2):65. http://doi.org/10.5717/jenb.2015.15060203

31. Ishihara T, Yoshida M, Arita M. Omega-3 fatty acid-derived mediators that control inflammation and tissue homeostasis. Int Immunol. 2019;31:559-567. http://doi.org/10.1093/intimm/dxz001

32. Veras ASC, Gomes RL, Almeida Tavares ME, Giometti IC, Cardoso APMM, da Costa Aguiar Alves B, et al. Supplementation of polyunsaturated fatty acids (PUFAs) and aerobic exercise improve functioning, morphology, and redox balance in prostate obese rats. Sci Rep. 2021;11(1):6282. http://doi.org/10.1038/s41598-021-85337-9

33. Montazer S, Gholami M, Azarbayjani MA, Abed Natanzi H. Effects of aerobic training and omega-3 supplementation on the levels of CRP, MDA and lipid profile in overweight and obese women. J Bas Res Med Sci. 2021;8(4):60-70. Available at: http://jbrms.medilam.ac.ir/article-1-581-en.html