Protective effect of anthocyanins on apoptosis of gastrocnemius muscle myocytes of rats after intense exercise
AbstractCurrently, in sports medicine, much attention is paid to the prevention and treatment of delayed muscle soreness syndrome (DOMS), which occurs several hours or days after unusual or intense physical activity, as well as the state of athlete overtraining. One of the main pathogenetic factors in the development of this syndrome is myocyte ultrastructural damage with apoptosis activation. Therefore, using natural antioxidants in sports nutrition for the relief of this pathology is of particular relevance.
The aim of the study was to study the effect of an anthocyanin-enriched diet on apoptosis of gastrocnemius muscle myocytes of rats after intense exercise.
Material and methods. The experiment was carried out for 4 weeks on 4 groups of male Wistar rats (12 animals in each, initial body weight ≈300 g). Animals were divided into groups of rats (groups 1 and 2), whose motor activity was limited by standard conditions for keeping animals in vivarium, and groups of physically active rats (groups 3 and 4), which received additional physical activity – treadmill training. Before the end of the experiment, the animals of groups 3 and 4 were given debilitating (until the rats refused to continue the exercise) physical activity on a treadmill. Rats of all four groups received a standard semi-synthetic diet, water ad libitum. Animals in groups 2 and 4 were additionally given blueberry and blackcurrant extract (30% anthocyanins) as part of the diet at a daily dose of 15 mg anthocyanins/kg body weight. The intensity of apoptosis of gastrocnemius muscle myocytes was studied by flow cytometry. Cells were stained with fluorochrome-conjugated annexin V and vital dye 7-aminoactinomycin. The results are presented as the percentage of intact cells and cells at different stages of apoptosis per 100 000 counted objects in each sample.
Results. The enrichment of the diet of control group rats with blueberry and black currant extract did not have a significant effect on the relative content of intact cells and the studied parameters of apoptosis of gastrocnemius muscle myocytes of rats of the 2nd group. Intense physical activity in rats of the 3rd group led to a statistically significant (p<0.05) decrease in the relative content of intact (live) cells compared with this indicator in rats of other groups (85.32±1.44 vs 90.87±0.66% – in the 1st group; 90.16±0.79% – in the 2nd group; 89.01±0.81% – in the 4th group, р<0.05). After intense physical activity in rats of the 3rd group, activation of apoptosis of gastrocnemius muscle myocytes was found, as evidenced by an increase in the relative content of objects in apoptosis compared with other groups (11.61±1.45 vs 7.88±0.60% – in the 1st group, р<0.05; 8.01±0.70% – in the 2nd group, p<0.10; 7.93±0.59% – in the 4th group р<0.05). Enrichment of the diet of exercise rats with blueberry and blackcurrant extract (4th group) had a protective effect on the intensity of the apoptosis process, the studied parameters did not differ significantly from those in rats of the control and the 2nd groups.
Conclusion. The results of the study indicate the activation of the process of apoptosis of gastrocnemius muscle myocytes of rats after intense physical activity. Enrichment of rats’ diet with anthocyanins from blueberry and black currant extracts ensures the restoration of the studied apoptosis parameters to the level of control group rats. In the control group of rats with normal physical activity, the addition of anthocyanins to the diet does not have a significant effect on the physiological process of apoptosis of gastrocnemius muscle myocytes. In this way, an evidence base for the effectiveness of the use of biologically active substances – anthocyanins – in sports nutrition for the restoration of skeletal muscles has been obtained.
Keywords:apoptosis; anthocyanins; physical activity; gastrocnemius muscle; rats
Funding. The research was carried out at the expense of a subsidy for the fulfillment of a state task within the framework of the Program for Fundamental Scientific Research of the Presidium of the Russian Academy of Sciences (subject No. FGMF-2022-0005).
Conflict of interest. The authors declare no conflict of interest.
Contribution. The concept and design of the study – Aksenov I.V., Krasutsky A.G., Trushina E.N.; data collection and statistical data processing – Trushina E.N., Mustafina O.К.; writing the text – Trushina E.N., Nikityuk D.B.; editing, approval of the final version of the article, responsibility for the integrity of all parts of the article – all authors.
For citation: Trushina E.N., Mustafina O.K., Aksenov I.V., Krasutsky A.G., Nikityuk D.B. Protective effect of anthocyanins on apoptosis of gastrocnemius muscle myocytes of rats after intense exercise. Voprosy pitaniia [Problems of Nutrition]. 2022; 91 (4): 47–53. DOI: https://doi.org/10.33029/0042-8833-2022-91-4-47-53 (in Russian)
References
1. Varga O.Yu., Ryabkov V.A. Apoptosis: concept, implementation mechanisms, significance. Ekologiya cheloveka [Human Ecology]. 2006; (7): 28–32. (in Russian)
2. Phaneuf S., Leeuwenburgh C. Apoptosis and exercise. Med Sci Sports Exerc. 2001; 33 (3): 393–6. DOI: https://doi.org/10.1097/00005768-200103000-00010
3. Podhorska-Okolow M., Sandri M., Zampieri S., Brun B., Rossini K., Carraro U. Apoptosis of myofibres and satellite cells: exercise-induced damage in skeletal muscle of the mouse. Neuropathol Appl Neurobiol. 1998; 24 (6): 518–31. DOI: https://doi.org/10.1046/j.1365-2990. 1998.00149.x
4. Kroemer G., Reed J.С. Mitochondrial control of cell death. Nat Med. 2000; 6 (5): 513–9. DOI: https://doi.org/10.1038/74994
5. Hildeman D., Mitchell Th., Kappler J., Marrack P.H. T cell apoptosis and reactive oxygen species. J Clin Invest. 2003; 111 (5): 575–81. DOI: https://doi.org/10.1172/JCI18007
6. Palmowski J., Reichel T., Boßlau T.K., Krüger K. The effect of acute running and cycling exercise on T cell apoptosis in humans: a systematic review. Scand J Immunol. 2020; 91 (2): e12834. DOI: https://doi.org/10.1111/sji.12834
7. Hotfiel T., Freiwald J., Hoppe M.W., Lutter C., Forst R., Grim C., et al. Advances in delayed-onset muscle soreness (DOMS): part I: pathogenesis and diagnostics. Sportverletz Sportschaden. 2018; 32 (4): 243–50. DOI: https://doi.org/10.1055/a-0753-1884
8. Battistelli M., Salucci S., Guescini M., Curzi D., Stocchi V., Falcieri E. Skeletal muscle cell behavior after physical agent treatments. Curr Pharm Des. 2015; 21 (25): 3665–72. DOI: https://doi.org/10.2174/1381612821666150122123412
9. Montanari S., Şahin M.A., Lee B.J. Blacker S.D., Willems M.E.T. No effects of New Zealand blackcurrant extract on physiological and performance responses in trained male cyclists undertaking repeated testing across a week period. Sports (Basel). 2020; 8 (8): 114. DOI: https://doi.org/10.3390/sports8080114
10. Braakhuis A.J., Somerville V.X., Hurst R.D. The effect of New Zealand blackcurrant on sport performance and related biomarkers: a systematic review and meta-analysis. J Int Soc Sports Nutr. 2021; 18 (1): 8. DOI: https://doi.org/10.1186/s12970-020-00398-x
11. Brandenburg J.P., Giles L.V. Blueberry supplementation reduces the blood lactate response to running in normobaric hypoxia but has no effect on performance in recreational runners. J Int Soc Sports Nutr. 2021; 18 (1): 26. DOI: https://doi.org/10.1186/s12970-021- 00423-7
12. Şahin M.A., Bilgiç P., Montanari S., Willems M.E.T. Intake duration of anthocyanin-rich New Zealand blackcurrant extract affects metabolic responses during moderate intensity walking exercise in adult males. J Diet Suppl. 2021; 18 (4): 406–17. DOI: https://doi.org/10.1080/19390211.2020.1783421
13. Kashi D.S., Shabir A., Boit M.D., Bailey S.J., Higgins M.F. The efficacy of administering fruit-derived polyphenols to improve health biomarkers, exercise performance and related physiological responses. Nutrients 2019; 11 (10): 2389. DOI: https://doi.org/10.3390/nu11102389
14. Сastañeda-Ovando A., de Lourdes Pacheco-Hernández M., Páez-Hernández M.E., Rodríguez J. A., Galán-Vidal C.A. Chemical studies of anthocyanins: a review. Food Chem. 2009; 113 (4): 859–71. DOI: https://doi.org/10.1016/j.foodchem.2008.09.001
15. Edwards M., Czank C., Woodward G.M., Cassidy A., Kay C.D. Phenolic metabolites of anthocyanins modulate mechanisms of endothelial function. J Agric Food Chem. 2015; 63 (9): 2423–31. DOI: https://doi.org/10.1021/jf5041993
16. Fairlie-Jones L., Davison K., Fromentin E., Hill A.M. The effect of anthocyanin-rich foods or extracts on vascular function in adults: a systematic review and meta-analysis of randomised controlled trials. Nutrients. 2017; 9 (8): 908. DOI: https://doi.org/10.3390/nu9080908
17. Popova A.Yu., Tutelyan V.A., Nikityuk D.B. On the new (2021) Norms of physiological requirements in energy and nutrients of various groups of the population of the Russian Federation. Voprosy pitaniia [Problems of Nutrition]. 2021; 90 (4): 6–19. DOI: https://doi.org/10.33029/0042-8833-2021-90-4-6-19 (in Russian)
18. Copetti C.L.K., Diefenthaeler F., Hansen F. Di Pietro P.F. Fruit-Derived anthocyanins: effects on cycling-induced responses and cycling performance. Antioxidants (Basel). 2022; 11 (2): 387. DOI: https://doi.org/10.3390/antiox11020387
19. Reeves P.G., Nielsen F.H., Fahey G.C. Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993; 123 (11): 1939–51. DOI: https://doi.org/10.1093/jn/123.11.1939
20. Carrizzo A., Lizio R., Di Pietro P., Ciccarelli M., Damato A., Venturini E., et al. Healthberry 865® and its related, specific, single anthocyanins exert a direct vascular action, modulating both endothelial function and oxidative stress. Antioxidants (Basel). 2021; 10 (8): 1191. DOI: https://doi.org/10.3390/antiox10081191
21. Azad M., Khaledi N., Hedayati M., Karbalaie M. Apoptotic response to acute and chronic exercises in rat skeletal muscle: eccentric & sprint interval. Life Sci. 2021; 270: 119002. DOI: https://doi.org/10.1016/j.lfs.2020.119002
22. Buchheit M., Laursen P.B. High-intensity interval training, solutions to the programming puzzle. Sports Med. 2013; 43 (5): 313–38. DOI: https://doi.org/10.1007/s40279-013-0029-x
23. Egan B., Zierath J.R. Exercise metabolism and the molecular regulation of skeletal muscle adaptation. Cell Metab. 2013; 17 (2): 162–84. DOI: https://doi.org/10.1016/j.cmet.2012.12.012
24. Salih D.A., Brunet A. FoxO transcription factors in the maintenance of cellular homeostasis during aging. Curr Opin Cell Biol. 2008; 20 (2): 126–36. DOI: https://doi.org/10.1016/j.ceb.2008.02.005
25. Cook M.D., Willems M.E. Dietary anthocyanins: a review of the exercise performance effects and related physiological responses. Int J Sport Nutr Exerc Metab. 2019; 29 (3): 322–30. DOI: https://doi.org/10.1123/ijsnem.2018-0088
26. Xu J.W., Ikeda K., Yamori Y. Upregulation of endothelial nitric oxide synthase by cyanidin-3-glucoside, a typical anthocyanin pigment. Hypertension. 2004; 44 (2): 217–22. DOI: https://doi.org/10.1161/01.HYP.0000135868.38343.c6
27. Bailey S.J., Vanhatalo A., Winyard P.G., Jones A.M. The nitrate-nitrite-nitric oxide pathway: its role in human exercise physiology. Eur J Sport Sci. 2011; 12 (4): 1–12. DOI: https://doi.org/10.1080/17461391.2011.635705