Introduction
Oxidative stress and apoptosis due to hydrogen peroxide poisoning have a major effect on the physiological and pathophysiological mechanisms of the lung. Regular exercise seems to reduce cellular apoptosis and create protective adaptations against poisoning by reducing free radicals and catecholamine autoxidation [8-10]. The change in the activity of antioxidant enzymes is related to the intensity, duration and type of aerobic exercises. There is also great interest in studying the effect of natural antioxidants in improving athletic performance, recovery and immune system [13]. In this regard, the use of Tribulus terrestris extract can be one of the ways to strengthen the antioxidant defense system [14]. Comprehensive information on the simultaneous use of aerobic exercise and alcoholic extract of Tribulus terrestris is not available. Therefore, this study aims to evaluate the effect of 8 weeks of aerobic exercise and Tribulus terrestris extract supplementation on changes in cytochrome C oxidase, Adenosine Triphosphate (ATP), Malondialdehyde (MDA) and Prooxidants-Antioxidant Balance (PAB), Bax, Bcl-2, and Caspase-3 in lung tissue of male rats poisoned with hydrogen peroxide.
Methods
In this experimental study, 49 male rats aged 10-12 weeks were randomly divided into seven groups: Control, Poisoned, Poisoning+ Exercise, Poisoning+ supplementation 1 (Tribulus terrestris 5 mg/kg), Poisoning+ supplementation 2 (Tribulus terrestris 10 mg/kg), Poisoning+ Exercise+ supplementation 1, Poisoning+ Exercise+ supplementation 2. The aerobic exercise program was for eight weeks (5 days a week) and included running on a treadmill at a speed of 20 meters per minute, 60 minutes for each session. Alcoholic extract of Tribulus terrestris was administered to the target animals at doses of 5 and 10 mg/kg by gavage for eight weeks. The amount of MDA was assessed by ELISA method using special kits (Minneapolis, USA) [19]. TMB cation solution was used to evaluate the PAB due to its electrochemical and optical properties [20]. To quantify the concentration of cytochrome C oxidase, the ELISA kits from COX Company, USA was used, and to measure intracellular ATP, the ELISA kits (KA1661) from Promega Company, USA was used [21,22]. Finally, immunohistochemistry was used to evaluate the expression of Bax, Bcl-2, and Caspase 3 in lung tissue. To determine the concentration of variables, the images were analyzed in ImageJ version 49.1 software and described in an ordinal scale [23]. Independent t-test was used to compare the variables between the two groups. The study hypotheses were analyzed using two-way analysis of variance and Least Significant Difference (LSD) post hoc test, considering a significance level of P<0.05.
Results
Induction of oxidative stress by H2O2 caused a significant decrease in ATP and Bcl-2 levels and a significant increase in MDA, cytochrome C, PAB, Bax, and Caspase 3 levels compared to the control group. A summary of the effects of aerobic exercise alone, Tribulus terrestris extract alone and their combined effects on oxidative stress and lung apoptosis markers are presented in Table 1.




Discussion
It seems that 8 weeks of aerobic exercise can reduce the effects of oxidative stress and apoptosis caused by hydrogen peroxide poisoning. The androgenic effect of Tribulus terrestris extract increases pulmonary protection in rats by altering the expression levels of proteins in the apoptotic signaling pathway. However, the effects of Tribulus terrestris extract may be dose-dependent in some cases. Although the combination of aerobic exercise with Tribulus terrestris extract supplementation has better results in controlling oxidative stress enzymes and pulmonary apoptotic factors, for better results, longer duration and and higher doses should be used. Limitations of this study included the inability to control animal appetite, the use of Wistar rats, possible physiological changes in the laboratory environment, the effect of ketamine on the evaluated indicators, lack of a placebo group, and gavage of animals which can influence the results of the study.
Ethical Considerations
Compliance with ethical guidelines
This article has been approved by the Research Assistant of Islamic Azad University, Central Tehran Branch (Code: IR.IAU.PS.REC.1398.322).
Funding
This article was extracted from PhD. dissertation of Mr. Ali Rasouli Foshazadeh in Faculty of Physical Education and Sports Sciences of Islamic Azad University, Central Tehran Branch.
Authors' contributions
All authors have participated equally in the design, implementation and writing of sections of the present study.
Conflicts of interest
The authors declared no conflict of interest.
Acknowledgements
The authors would like to thank all participants as well as the Shahid Beheshti Medical Sciences Laboratory for their cooperation and support.
 

1. Ornatowski W, Lu Q, Yegambaram M, Garcia AE, Zemskov EA, Maltepe E, et al. Complex interplay between autophagy and oxidative stress in the development of pulmonary disease. Redox Biology. 2020; 36:101679. [DOI:10.1016/j.redox.2020.101679] [PMID] [PMCID]
2. Gallelli CA, Calcagnini S, Romano A, Koczwara JB, Ceglia Md, Dante D, et al. Modulation of the oxidative stress and lipid peroxidation by endocannabinoids and their lipid analogues. Antioxidants. 2018; 7(7):93. [DOI:10.3390/antiox7070093] [PMID] [PMCID]
3. Ghazizadeh H, Saberi-Karimian M, Aghasizadeh M, Sahebi R, Ghazavi H, Khedmatgozar H, et al. Pro-oxidant-antioxidant balance (PAB) as a prognostic index in assessing the cardiovascular risk factors: A narrative review. Obesity Medicine. 2020; 19:100272. [DOI:10.1016/j.obmed.2020.100272]
4. Manning AA, Zhao L, Zhu Z, Xiao H, Redington CG, Ding VA, et al. IL-39 acts as a friend to pancreatic cancer. Medical Oncology. 2018; 36(1):12. [DOI:10.1007/s12032-018-1236-y] [PMID]
5. Sun Y, Cui D, Zhang Z, Zhang T, Shi J, Jin H, et al. Attenuated oxidative stress following acute exhaustive swimming exercise was accompanied with modified gene expression profiles of apoptosis in the skeletal muscle of mice. Oxidative Medicine and Cellular Longevity. 2016; 2016:8381242. [DOI:10.1155/2016/8381242] [PMID] [PMCID]
6. Shlyonsky V, Boom A, Mies F. Hydrogen peroxide and sodium transport in the lung and kidney. BioMed Research International. 2016; 2016:1-7. [DOI:10.1155/2016/9512807] [PMID] [PMCID]
7. de Sousa CV, Sales MM, Rosa TS, Lewis JE, de Andrade RV, Simões HG. The Antioxidant Effect of Exercise: A systematic review and meta-analysis. Sports Medicine. 2017; 47(2):277-93. [DOI:10.1007/s40279-016-0566-1] [PMID]
8. Gar C, Rottenkolber M, Haenelt M, Potzel AL, Kern-Matschilles S, Then C, et al. Altered metabolic and hormonal responses to moderate exercise in overweight/obesity. Metabolism. 2020; 107:154219. [DOI:10.1016/j.metabol.2020.154219] [PMID]
9. Rosety-Rodríguez M, Camacho A, Rosety MA, Fornieles G, Diaz AJ, Rosety I, et al. A short-term training program reduced oxidative damage in elderly diabetic rats. Revista de Investigación Clínica. 2013; 65(4):331-5. [PMID]
10. Mathot E, Liberman K, Cao Dinh H, Njemini R, Bautmans I. Systematic review on the effects of physical exercise on cellular immunosenescence-related markers-An update. Experimental Gerontology. 2021; 149:111318. [DOI:10.1016/j.exger.2021.111318] [PMID]
11. Zhang X, Wang L, Lu H, Zong Z, Chen Z, Li Y, et al. Preservation of hydrogen peroxide-induced oxidative damage in HepG-2 cells by rice protein hydrolysates pretreated with electron beams. Scientific Reports. 2020; 10(1):8415. [DOI:10.1038/s41598-020-64814-7] [PMID] [PMCID]
12. Hossini s. [Effects of Aerobic Exercise and Curcumin on Apoptosis: A review (Persian)]. Pars Journal of Medical Sciences. 2018; 16(2):58-66. [DOI:10.52547/jmj.16.2.58]
13. Mason SA, Trewin AJ, Parker L, Wadley GD. Antioxidant supplements and endurance exercise: Current evidence and mechanistic insights. Redox Biology. 2020; 35:101471. [DOI:10.1016/j.redox.2020.101471] [PMID] [PMCID]
14. Amin A, Lotfy M, Shafiullah M, Adeghate E. The Protective Effect of Tribulus terrestris in Diabetes. Annals of the New York Academy of Sciences. 2006; 1084:391-401. [DOI:10.1196/annals.1372.005] [PMID]
15. Kilany O, Abdou R, El-Beltagy M, Mohammad H. Protective effects of tribulus terrestris against gentamicin mediated nephrotoxicity, oxidative damage and apoptosis in male rats. Egyptian Academic Journal of Biological Sciences. 2020; 12:41-58. [DOI:10.21608/EAJBSZ.2020.85736]
16. Haghshenas R, Jafari M, Ravasi A, Kordi M, Gilani N, Shariatzadeh M, et al. The effect of eight weeks endurance training and high-fat diet on appetite-regulating hormones in rat plasma. Iranian Journal of Basic Medical Sciences. 2014; 17(4):237-43. [PMID] [PMCID]
17. Darash K, Ghanbarzadeh M, Nikbakht M. Effect of simultaneous eight-week exercise and crocin usage on the oxidation and anti-oxidation indices of male rats’ testicles subjected to apoptosis. Thrita. 2019; 8(1):e90438. [DOI:10.5812/thrita.90438]
18. Hosseini E. [The effect of tribulus terrestris extract on hepatic complications due to the gelofen consumption in adult female rats (Persian)]. Journal of Advanced Biomedical Sciences. 2016; 6(2):155-61. [Link]
19. Abbasnezhad M, Jafari M, Asgari A, Hajihoseini R, Hajigholamali M, Salehi M, et al . [The study regarding effect of paraoxon on oxidative stress (Persian)]. Journal of Mazandaran University of Medical Sciences. 2009; 19(73):16-26. [Link]
20. Tavana S, Amini S, Hakhamaneshi MS, Andalibi P, Hajir MS, Ardalan A, et al. Prooxidant-antioxidant balance in patients with phenylketonuria and its correlation to biochemical and hematological parameters. Journal of Pediatric Endocrinology and Metabolism. 2016; 29(6):675-80. [DOI:10.1515/jpem-2015-0398] [PMID]
21. Javidtabrizi N, Bashiri J, Narimanirad M. [Effect of 12 weeks of treadmill aerobic training on cytochrome c and caspase-9 gene expression in cardiac muscle of male rats (Persian)]. Qom University of Medical Sciences Journal. 2017; 11(6):1-9. [Link]
22. Bangsbo J, Krustrup P, González-Alonso J, Saltin B. ATP production and efficiency of human skeletal muscle during intense exercise: Effect of previous exercise. American Journal of Physiology-Endocrinology and Metabolism. 2001; 280(6):E956-64. [DOI:10.1152/ajpendo.2001.280.6.E956] [PMID]
23. Yadegari M, Riahy S, Mirdar S, Hamidian G. [Interactive effects of reducing exercise intensity and Adiantum capillus veneris extract on remodeling and modulation of pulmonary apoptotic indices in the rats exposed to the hypoxia (Persian)]. Scientific Journal of Kurdistan University of Medical Sciences. 2018; 23(2):81-91. [DOI:10.29252/sjku.23.2.81]
24. Wendakoon CN, Calderon P, Gagnon D, editors. Evaluation of selected medicinal plants extracted in different ethanol concentrations for antibacterial activity against human pathogens. Journal of Medicinally Active Plants. 2012; 1(2):59-68. [DOI:10.7275/R5GH9FV2]
25. Akbari M, Shahidi F, Rajabi H, Kashef M, Mazaheri Z. [The simultaneous effect of six weeks forced swimming and crocin supplementation on the expression of 3-cardiomyocyte genesase 3 in male rats infected with hydrogen peroxide (Persian)]. Razi Journal of Medical Sciences. 2018; 25(9):26-37. [Link]
26. Seyfi A, Shahidi F, Salehpor M. The interactive effect of crocin supplementation on the alteration of malondialdehyde and cardiomyocyte catalase in male rats poisoned with hydrogen peroxide.Qom University of Medical Sciences Journal. 2019; 13(8):5-13. [DOI:10.29252/qums.13.8.5]
27. Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biology. 2017; 11:613-9. [DOI:10.1016/j.redox.2016.12.035] [PMID] [PMCID]
28. Houghton CR, Hawkins RA, Williamson DH, Krebs HA. The effects of physical training on the metabolic response to short-term severe exercise in the rat. Biochemical Journal. 1971; 124(5):57P. [DOI:10.1042/bj1240057Pa] [PMID] [PMCID]
29. Delfani N, Peeri M, Matin Homaee H. [Effect of aerobic exercise and hydroalcoholic extract of tribulus terrestris on mitochondrial oxidative stress markers in heart tissue of rats poisoned with hydrogen peroxide (Persian)]. Complementary Medicine Journal. 2021; 11(1):30-43. [DOI:10.32598/cmja.11.1.995.1]
30. Ghanbari-Niaki A, Kraemer RR, Abednazari H. [Time-course alterations of plasma and soleus agouti-related peptide and relationship to ATP, glycogen, cortisol, and insulin concentrations following treadmill training programs in male rats (Persian)]. Hormone and Metabolic Research. 2011; 43(2):112-6. [DOI:10.1055/s-0030-1267998] [PMID]
31. Shokohirad N, Bagherpour T, Nemati N, Hojati V. [The effect of pumpkin seed and endurance training on oxidative stress factors and DNA damage (Persian)]. Daneshvar Medicine: Basic and Clinical Research Journal. 2020; 28(3):28-41. [Link]
32. Shetab-Boushehri S, Samavati-Sharif M, Ravasi A, Kordi M, Javadi E, Minaii B. Effect of oral iron supplementation and endurance training on cytochrome C oxidase activity in rat soleus muscle. International Journal of Pharmacy and Pharmaceutical Sciences. 2010; 2(2):33-5. [Link]
33. Naseri L, Akbari Bazm M, Khazaei M. [A review on therapeutic effects of tribulus terrestris (Persian)]. Journal of Medicinal Plants. 2019; 4:1-22. [DOI:10.29252/jmp.4.72.1]
34. Fakourian A, Matinhomaee H, Azarbayjani MA, Farzanegi P. Effect of aerobic training and l-carnitine consumption on some oxidative stress factors in diabetic rat kidney. Armaghan-e-Danesh. 2019; 24(3):293-305. [Link]
35. Roh HT, So WY. The effects of aerobic exercise training on oxidant-antioxidant balance, neurotrophic factor levels, and blood-brain barrier function in obese and non-obese men. Journal of Sport and Health Science. 2017; 6(4):447-53. [DOI:10.1016/j.jshs.2016.07.006] [PMID] [PMCID]
36. Shams Z, Azarbayjani MA, peeri M, Matin Homaee H. [The effect of aerobic training and vitamin don gpx concentration and PABin lung tissue of rats exposed to hydrogen peroxide (Persian)]. Razi Journal of Medical Sciences. 2020; 26(12):156-66. [Link]
37. Ghajari H, Hosseini SA, Farsi S. The effect of endurance training along with cadmium consumption on Bcl-2 and bax gene expressions in heart tissue of rats. Annals of Military and Health Sciences Research. 2019; 17(1):e86795. [DOI:10.5812/amh.86795]
38. Mehri A, Hosseinpourdelaware S, Azizi M, Azarbaijani M A, Farzangi P. [The effect of aerobic training and resveratrol on some regulatory and executive factors of cardiomyocytes apoptosis in STZ-diabetic male rats (Persian)]. Medical Sciences. 2020; 30(1):59-66. [DOI:10.29252/iau.30.1.59]
39. Kazemi A, Mirza-zade E. The effect of endurance training on tumor tissue levels of caspase-3 and caspase-9 in mice with breast cancer. Iranian Journal of Breast Diseases. 2018; 11(3):32-43. [DOI:10.30699/acadpub.ijbd..11.3.32]
40. Li F, Shi W, Zhao EY, Geng X, Li X, Peng C, et al. Enhanced apoptosis from early physical exercise rehabilitation following ischemic stroke. Journal of Neuroscience Research. 2017; 95(4):1017-24. [DOI:10.1002/jnr.23890] [PMID]
41. Sadighi A, Abdi A, Azarbayjani MA, Barari A. [Effect of aerobic exercise on some factors of cardiac apoptosis in male rats (Persian)]. Feyz. 2019; 23(5):495-502. [Link]
42. Mardani Z, Hosseini SA, Matinhomaee H, Rahmati-Ahmadabad S. Effect of endurance training with coriander seed consumption on caspase-3 and cytochrome-c in the heart tissue of H2O2-poisoned rats. Modern Care Journal. 2020; 17(2):e100003. [DOI:10.5812/modernc.100003]
43. Reshma PL, Binu P, Anupama N, Vineetha RC, Abhilash S, Nair RH, et al. Pretreatment of tribulus terrestris l. causes anti-ischemic cardioprotection through mapk mediated anti-apoptotic pathway in rat. Biomedicine & Pharmacotherapy. 2019; 111:1342-52. [DOI:10.1016/j.biopha.2019.01.033] [PMID]
44. Yin L, Wang Q, Wang X, Song LN. Effects of tribulus terrestris saponins on exercise performance in overtraining rats and the underlying mechanisms. Canadian Journal of Physiology and Pharmacology. 2016; 94(11):1193-201. [DOI:10.1139/cjpp-2016-0086] [PMID]