Effects of High-Intensity Interval Training and High-Fat Diet on Hepatic Oxidative Stress and Inflammatory Markers in Male C57BL/6 Mice

Sabet, Pooyan; Esfarjani, Fahimeh; Marandi, Mohammad

doi:10.22089/spj.2025.17788.2357

Effects of High-Intensity Interval Training and High-Fat Diet on Hepatic Oxidative Stress and Inflammatory Markers in Male C57BL/6 Mice

Document Type : Research Paper

Authors

pooyan sabet ¹

Fahimeh Esfarjani ²

mohammad marandi ³

¹ Ph.D.Student, Department of ExercisePhysiology,Faculty of Sport Sciences,University of Isfahan ,Isfahan, Iran

² Associate Professor in Exercise Physiology, Faculty of Sport Sciences,University of Isfahan ,Isfahan ,Iran

³ Professor in Exercise Physiology, Faculty of Sport Sciences,University of Isfahan, Isfahan ,Iran

10.22089/spj.2025.17788.2357

Abstract

Background and Purpose
The liver is a vital organ with essential roles in metabolism, energy storage, and detoxification. Oxidative stress and inflammation are critical factors that can severely compromise liver health. Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the body's capacity to neutralize them with its antioxidant defenses. This imbalance can activate various transcription factors, which in turn regulate the expression of genes involved in inflammatory pathways. Inflammatory markers, which are part of the body's response to injury and infection, are closely linked to liver tissue damage.Key genes involved in these processes include HMOX1 (heme oxygenase 1), NFE2L2 (nuclear factor erythroid 2–related factor 2, or Nrf2), and NOS2 (inducible nitric oxide synthase). HMOX1 is a protective enzyme that counteracts oxidative damage and helps control inflammation. NFE2L2 is a master regulator of the antioxidant response and also helps reduce inflammation. The expression of NOS2 is induced by inflammatory stimuli like cytokines and microbial products, leading to nitric oxide production during inflammation. The dysregulated expression of these genes is a common feature of liver diseases, highlighting their role in pathogenesis and progression.High-fat diets (HFD) are known to exacerbate oxidative stress and activate inflammatory pathways, contributing to conditions such as non-alcoholic fatty liver disease (NAFLD), fibrosis, cirrhosis, hepatocellular carcinoma, diabetes, and cardiovascular disorders, although the precise mechanisms are not fully understood. Conversely, high-intensity interval training (HIIT) has gained recognition for its cardiovascular and metabolic benefits, including the ability to reduce oxidative stress by improving oxygen consumption and enhancing antioxidant defenses.This study investigates the combined effects of HIIT and a high-fat diet on hepatic oxidative stress and the expression of inflammatory genes in C57BL/6J mice.
Materials and Methods
Of course. Here is the native and polished version of your text:Forty male C57BL/6 mice (approximately one month old, weighing 14 ± 1 g) were housed under controlled conditions, including a 12-hour light/dark cycle, 50-60% humidity, and a temperature of 24°C, with free access to food and water. The mice were randomly divided into four experimental groups for a 10-week period: 1) normal diet (50% carbohydrate, 30% protein, 20% fat) without exercise; 2) normal diet with HIIT; 3) high-fat diet (60% fat, 20% protein, 20% carbohydrate) without exercise; and 4) high-fat diet combined with HIIT.The HIIT protocol was performed on a treadmill. Following a one-week acclimation period with incremental speeds, the formal training began. The protocol consisted of five 40-minute sessions per week, each including a 3-minute warm-up, 35 minutes of interval running, and a 2-minute cooldown. The training intensity started at 15 m/min and was increased by 2 m/min every two weeks, reaching a final speed of 23 m/min in the last week.To evaluate molecular outcomes, mRNA expression levels of the HMOX1, NFE2L2, and NOS2 genes were quantified using real-time PCR, while NOS2 protein expression was analyzed by Western blotting. All data were analyzed using ANOVA, with a significance threshold of P < 0.05.
Results
After 10 weeks, mice fed a high-fat diet demonstrated significant metabolic and hepatic alterations compared to those on a normal diet. We observed pronounced increases in fasting blood glucose (73.34%, P=0.0001), liver weight (22.67%, P=0.0001), and liver enzymes, with AST rising by 101% and ALT by 58.46% (P=0.0001 for both).At the molecular level, the high-fat diet markedly upregulated hepatic gene expression of HMOX1 and NOS2 by 173.62% (P=0.0001) and 125.30% (P=0.0003), respectively. Conversely, NFE2L2 gene expression was significantly suppressed, decreasing by 50.52% (P=0.0008). This change was consistent with a 130.52% increase in NOS2 protein expression (P=0.0002).The addition of exercise to the high-fat diet regimen elicited a significant restorative effect. Compared to the sedentary high-fat diet group, the exercise group showed increased expression of NFE2L2 (21.66%, P=0.0008), HMOX1 (7.63%, P=0.0001), and the NOS2 gene (13.90%, P=0.0003), along with a 12.32% rise in NOS2 protein levels (P=0.0002).
Conclusion
High-intensity interval training (HIIT) demonstrates a capacity to mitigate hepatic oxidative stress and inflammation, independent of weight loss. The underlying mechanisms involve HIIT's role in enhancing hepatic lipid and carbohydrate metabolism, which reduces fat accumulation and improves insulin sensitivity. Furthermore, HIIT suppresses hepatic lipogenesis and adipose tissue lipolysis, while modulating key signaling pathways such as TLR4/NF-κB, leading to decreased production of pro-inflammatory cytokines like TNF-α and IL-6. These collective actions reduce inflammatory cytokine production and limit hepatocyte necrosis, thereby promoting overall liver health. As a result, integrating HIIT with a balanced diet presents a promising non-pharmacological strategy to counter high-fat diet-induced hepatic dysfunction. These findings highlight the essential synergy between nutrition and physical activity in preventing chronic diseases and point toward valuable directions for future research.
Article Message
A high-fat diet promotes hepatic oxidative stress and inflammation, leading to tissue damage and functional impairment. High-intensity interval training (HIIT) represents an effective strategy to counteract these adverse effects.
Ethical Considerations
This study was conducted in strict accordance with ethical guidelines and received approval from the Research Ethics Committee of Isfahan University (Approval code : IR.UI.REC.1403.151). No funding was received from public, commercial, or nonprofit sources.
Authors’ Contributions
All authors equally contributed to the study’s design, execution, and manuscript preparation.
Conflict of Interest
No conflicts of interest are declared.
Acknowledgments
This work is based on a dissertation submitted in partial fulfillment of the requirements for a Professional Doctorate in Sport Physiology at Isfahan University. The authors extend their sincere gratitude to all those who contributed to the successful completion of this research.

Keywords

High-Intensity Interval Training

High-Fat Diet

Oxidative Stress

Inflammatory Markers

Subjects

sport metabolism

1. Kalra A, Yetiskul E, Wehrle CJ, Tuma F. Physiology, liver. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. Available at: https://pubmed.ncbi.nlm.nih.gov/30571059/

2. Singh AK, Rana HK, Pandey AK. The oxidative stress: causes, free radicals, targets, mechanisms, affected organs, effects, indicators. Antioxidants Effects in Health. 2022:33-42. https://doi.org/10.1016/B978-0-12-819096-8.00012-4

3. Jomova K, Raptova R, Alomar SY, Alwasel SH, Nepovimova E, Kuca K, Valko M. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Archives of Toxicology. 2023;97(10):2499-574. https://doi.org/10.1007/s00204-023-03562-9

4. Husain S, Hillmann K, Hengst K, Englert H. Effects of a lifestyle intervention on the biomarkers of oxidative stress in non-communicable diseases: a systematic review. Frontiers in Aging. 2023;4:1085511. http://doi.org/10.3389/fragi.2023.1085511

5. Hu P, Liu Y, Li S, Zhao Y, Gu H, Zong Q, et al. Lactoferrin relieves Deoxynivalenol-induced oxidative stress and inflammatory response by modulating the Nrf2/MAPK pathways in the liver. Journal of Agricultural and Food Chemistry. 2023;71(21):8182-91. https://doi.org/10.1021/acs.jafc.3c01035

6. Fanaei H, Mard SA, Sarkaki A, Goudarzi G, Khorsandi L. Gallic acid protects the liver against NAFLD induced by dust exposure and high-fat diet through inhibiting oxidative stress and repressing the inflammatory signaling pathways NF-kβ/TNF-α/IL-6 in Wistar rats. Avicenna Journal of Phytomedicine. 2021;11(5):527. https://doi.org/10.3389/fphar.2024.1515172

7. Tsopmejio ISN, Yuan J, Diao Z, Fan W, Wei J, Zhao C, et al. Auricularia polytricha and Flammulina velutipes reduce liver injury in DSS-induced Inflammatory Bowel Disease by improving inflammation, oxidative stress, and apoptosis through the regulation of TLR4/NF-κB signaling pathways. The Journal of Nutritional Biochemistry. 2023;111:109190. https://doi.org/10.1016/j.fbio.2021.101426

8. Zhang C-h, Xiao Q, Sheng J-q, Liu T-t, Cao Y-q, Xue Y-n, et al. Gegen Qinlian Decoction abates nonalcoholic steatohepatitis associated liver injuries via anti-oxidative stress and anti-inflammatory response involved inhibition of toll-like receptor 4 signaling pathways. Biomedicine & Pharmacotherapy. 2020;126:110076. https://doi.org/10.3389/fcimb.2023.1066053

9. Dang Y, Ma C, Chen K, Chen Y, Jiang M, Hu K, et al. The effects of a high-fat diet on inflammatory bowel disease. Biomolecules. 2023;13(6):905. https://doi.org/10.3390/biom13060905

10. Yoo W, Zieba JK, Foegeding NJ, Torres TP, Shelton CD, Shealy NG, et al. High-fat diet–induced colonocyte dysfunction escalates microbiota-derived trimethylamine N-oxide. Science. 2021;373(6556):813-8. https://doi.org/10.1126/science.aba3683

11. El-Emam SZ, Soubh AA, Al-Mokaddem AK, Abo El-Ella DM. Geraniol activates Nrf-2/HO-1 signaling pathway mediating protection against oxidative stress-induced apoptosis in hepatic ischemia-reperfusion injury. Naunyn-Schmiedeberg's archives of Pharmacology. 2020;3.58-93:1849. https://doi.org/10.1007/s00210-020-01887-1

12. Huang J, Shen X-D, Yue S, Zhu J, Gao F, Zhai Y, et al. Adoptive transfer of heme oxygenase-1 (HO-1)-modified macrophages rescues the nuclear factor erythroid 2-related factor (Nrf2) antiinflammatory phenotype in liver ischemia/reperfusion injury. Molecular Medicine. 2014;20:448-55. https://doi.org/10.2119/molmed.2014.00103

13. Zeng X-F, Varady KA, Wang X-D, Targher G, Byrne CD, Tayyem R, et al. The role of dietary modification in the prevention and management of metabolic dysfunction-associated fatty liver disease: An international multidisciplinary expert consensus. Metabolism. 2024;161:156028. https://doi.org/10.1016/J.metabol.2024

14. Jamioł-Milc D, Gudan A, Kaźmierczak-Siedlecka K, Hołowko-Ziółek J, Maciejewska-Markiewicz D, Janda-Milczarek K, Stachowska E. Nutritional support for liver diseases. Nutrients. 2023;15:3640(16). https://doi.org/10.3390/nu15163640

15. Younossi ZM, Corey KE, Lim JK. AGA clinical practice update on lifestyle modification using diet and exercise to achieve weight loss in the management of nonalcoholic fatty liver disease: expert review. Gastroenterology. 2021;160(3):912-8. https://doi.org/10.1053/j.gastro.2020.11.051

16. You Y, Li W, Liu J, Li X, Fu Y, Ma X. Bibliometric review to explore emerging high-intensity interval training in health promotion: a new century picture. Frontiers in Public Health. 2021;9:697633. https://doi.org/10.3389/fpubh.2021.697633

17. Wu Z-J, Wang Z-Y, Gao H-E, Zhou X-F, Li F-H. Impact of high-intensity interval training on cardiorespiratory fitness, body composition, physical fitness, and metabolic parameters in older adults: A meta-analysis of randomized controlled trials. Experimental Gerontology. 2021;150:111345. https://doi.org/10.1016/j.exger.2021.111345

18. Martland R, Mondelli V, Gaughran F, Stubbs B. Can high-intensity interval training improve physical and mental health outcomes? A meta-review of 33 systematic reviews across the lifespan. Journal of Sports Sciences. 2020;38(4):430-69. https://doi.org/10.1093/schbul/sbaa029.707

19. Abdollahi M, Marandi SM, Ghaedi K, Safaeinejad Z, Kazeminasab F, Shirkhani S, et al. Insulin-related liver pathways and the therapeutic effects of aerobic training, green coffee, and chlorogenic acid supplementation in prediabetic mice. Oxidative Medicine and Cellular Longevity. 2022;2. https://doi.org/10.4093/dmj.2022.0265

20. Tan BL, Norhaizan ME. Effect of high-fat diets on oxidative stress, cellular inflammatory response and cognitive function. Nutrients. 2019;11(11):2579. https://doi.org/10.3390/nu11112579

21. Sears B, Perry M. The role of fatty acids in insulin resistance. Lipids in health and disease. 2015;14(1):121.doi:10.1186/s12944-015-0123-1

22. Lian C-Y, Zhai Z-Z, Li Z-F, Wang L. High fat diet-triggered non-alcoholic fatty liver disease: A review of proposed mechanisms. Chemico-Biological Interactions. 2020;330:109199. https://doi.org/10.1016/j.cbi.2020.109199

23. Vancells Lujan P, Vinas Esmel E, Sacanella Meseguer E. Overview of non-alcoholic fatty liver disease (NAFLD) and the role of sugary food consumption and other dietary components in its development. Nutrients. 2021;13(5):1442. https://doi.org/10.3390/nu13051442

24. Consoli V, Sorrenti V, Grosso S, Vanella L. Heme oxygenase-1 signaling and redox homeostasis in physiopathological conditions. Biomolecules. 2021;11(4):589. https://doi.org/10.3390/biom11040589

25. Campbell NK, Fitzgerald HK, Dunne A. Regulation of inflammation by the antioxidant haem oxygenase 1. Nature Reviews Immunology. 2021;21(7):411-25. https://doi.org/10.1038/s41577-020-00491-x

26. Ryter SW. Heme oxygenase-1: an anti-inflammatory effector in cardiovascular, lung, and related metabolic disorders. Antioxidants. 2022;11(3):555. https://doi.org/10.3390/antiox11030555

27. Minhas R, Bansal Y, Bansal G. Inducible nitric oxide synthase inhibitors: a comprehensive update. Medicinal Research Reviews. 2020;40(3):823-55. https://doi.org/10.1002/med.21636

28. Cinelli MA, Do HT, Miley GP, Silverman RB. Inducible nitric oxide synthase: Regulation, structure, and inhibition. Medicinal Research Reviews. 2020;40(1):158-89. https://doi.org/10.1002/med.215599

29. Basu P, Averitt DL, Maier C, Basu A. The effects of nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) activation in preclinical models of peripheral neuropathic pain. Antioxidants. 2022;11(2):430. https://doi.org/10.3390/antiox11020430

30. Francisqueti-Ferron FV, Ferron AJT, Garcia JL, Silva CCVdA, Costa MR, Gregolin CS, et al. Basic concepts on the role of nuclear factor erythroid-derived 2-like 2 (Nrf2) in age-related diseases. International Journal of Molecular Sciences. 2019;20(13):3208. https://doi.org/10.3390/ijms20133208

31. Seminotti B, Grings M, Tucci P, Leipnitz G, Saso L. Nuclear factor erythroid-2-related factor 2 signaling in the neuropathophysiology of inherited metabolic disorders. Frontiers in Cellular Neuroscience. 2021;15:785057 . https://doi.org/10.3389/fphar.2023.1264842

32. Smolková K, Mikó E, Kovács T, Leguina-Ruzzi A, Sipos A, Bai P. Nuclear factor erythroid 2-related factor 2 in regulating cancer metabolism. Antioxidants & Redox Signaling. 2020;33(13):966-97. https://doi.org/10.1089/ars.2020.8024

33. Ma Y, Wu Z, Gao M, Loor J. Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro. Journal of Dairy Science. 2018;101(6):5329-44. https://doi.org/10.31168/jds.2017-14128

34. Atakan MM, Li Y, Koşar ŞN, Turnagöl HH, Yan X. Evidence-based effects of high-intensity interval training on exercise capacity and health: a review with historical perspective. International Journal of Environmental Research and Public Health. 2021;18(13):7201. https://doi.org/10.3390/ijerph18137201

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