Rapamycin protects testes against germ cell apoptosis and oxidative stress induced by testicular ischemia-reperfusion

Document Type: Original Article


1 Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences

2 Experimental Medicine Research Center, Tehran University of Medical Sciences

3 Department of Pharmacology, Faculty of Pharmacy, Urmia University of Medical Sciences

4 Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

5 Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran

6 Department of Pharmacology, Faculty of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

7 Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

8 Department of Pathology, Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran

9 Department of Anatomy and Reproductive Biology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

10 Department of Pathology, Urmia University of Medical Sciences, Urmia, Iran


Objective(s):Rapamycin is an immunosuppressant compound with a broad spectrum of pharmaco-logical activities. In recent years, it has been used successfully to decrease ischemia-reperfusion injury in several organ systems. The purpose of the present study was to examine the effect of rapamycin on testicular ischemia-reperfusion injury.
Materials and Methods: Seventy-two adult male Wistar rats were divided into six groups: control (group1), sham-operated (Group2), T/D + DMSO as vehicle group (group3), and groups 4–6; respectively received 0.5, 1, and 1.5 mgkg-1 of rapamycin , IP 30 min before detorsion. Ischemia was achieved by twisting the right testis 720o clockwise for 1 hr. The right testis of 6 animals from each group were excised 4 hr after detorsion for the measurement of lipid peroxidation, caspase-3, and antioxidant enzyme activities. Histopathological changes and germ cell apoptosis were determined by measuring mean of seminiferous tubules diameters (MSTD) and TUNEL test in right testis of 6 animals per group, 24 hr after detorsion.
Results: Testicular T/D caused increases in the apoptosis, malondialdehyde (MDA), and caspase-3 levels and decreases in the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in ipsilateral testis (P<0.001). The rats treated with rapamycin had significant decreases in the MDA and caspase-3 levels and significant increases in the SOD, CAT and GPx activities in ipsilateral testis compared with the T/D group (P<0.001); germ cell apoptosis was decreased, and MSTD was improved.
Conclusion: Rapamycin administration during testicular torsion decreased ischemia/reperfusion (I/R) cellular damage.


1.   Visser AJ, Heyns CF. Testicular function after torsion of the spermatic cord. BJU Int 2003; 92:200-203.

2.   Filho DW, Torres MA, Bordin AL, Crezcynski-Pasa TB, Boveris A. Spermatic cord torsion, reactive oxygen and nitrogen species and ischemia–reperfusion injury. Mol Aspects Med 2004; 25:199-210.

3.   Miller JL. Sirolimus approved with renal transplant indication. Am J Health Syst Pharm 1999; 56:2177-2178.

4.   Calap-Quintana P, Soriano S, Llorens JV, Al-Ramahi I, Botas J, Moltó MD, et al. TORC1 inhibition by rapamycin promotes antioxidant defences in a drosophila model of friedreich’s ataxia. PloS One 2015; 10:e0132376.

5.   Wang Y, Liu Y, Lu J, Zhang P, Wang Y, Xu Y, et al. Rapamycin inhibits FBXW7 loss-induced epithelial–mesenchymal transition and cancer stem cell-like characteristics in colorectal cancer cells. Biochem Biophys Res Commun 2013; 434:352-356.

6.   Long S, Rieck M, Sanda S, Bollyky JB, Samuels P, Goland R, et al. Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function. Diabetes 2012; 61:2340-2348.

7.   Chong ZZ, Shang YC, Zhang L, Wang S, Maiese K. Mammalian target of rapamycin: hitting the bull's-eye for neurological disorders. Oxid Med Cell Longev 2010; 3:374-391.

8.   Chong ZZ, Maiese K. Mammalian target of rapamycin signaling in diabetic cardiovascular disease. Cardiovasc Diabetol 2012; 11:45.

9.   Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002; 110:163-175.

10. Payabvash S, Kiumehr S, Tavangar SM, Dehpour AR. Ethyl pyruvate reduces germ cell–specific apoptosis and oxidative stress in rat model of testicular torsion/detorsion. J Pediatr Surg 2008; 43:705-712.

11. Turner T, Tung K, Tomomasa H, Wilson LW. Acute testicular ischemia results in germ cell-specific apoptosis in the rat. Biol Reprod 1997; 57:1267-1274.

12. Koji T, Hishikawa Y, Ando H, Nakanishi Y, Kobayashi N. Expression of Fas and Fas ligand in normal and ischemia-reperfusion testes: involvement of the Fas system in the induction of germ cell apoptosis in the damaged mouse testis. Biol Reprod 2001; 64:946-954.

13. Yazdani I, Ghazi‐Khansari M, Saeedi Saravi SS, Nobakht M, Majdani R, Rezayat SM, et al. Nortriptyline protects testes against germ cell apoptosis and oxidative stress induced by testicular ischaemia/reperfusion. Andrologia 2016; 49:2.

14. Zhang WH, Wang H, Wang X, Narayanan MV, Stavrovskaya IG, Kristal BS, et al. Nortriptyline protects mitochondria and reduces cerebral ischemia/hypoxia injury. Stroke 2008; 39:455-462.

15. Motaghinejad M, Karimian M, Motaghinejad O, Shabab B, Yazdani I, Fatima S. Protective effects of various dosage of Curcumin against morphine induced apoptosis and oxidative stress in rat isolated hippocampus. Pharmacol Rep 2015; 67:230-235.

16. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95:351-358.

17. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105:121-126.

18. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70:158-169.

19. Paoletti F, Mocali A. Determination of superoxide dismutase activity by purely chemical system based on NAD (P) H Oxidation. Methods Enzymol 1990; 186:209-220.

20. Namura S, Zhu J, Fink K, Endres M, Srinivasan A, Tomaselli KJ, et al. Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci 1998; 18:3659-3668.

21. Cosentino MJ, Nishida M, Rabinowitz R, Cockett AT. Histopathology of prepubertal rat testes subjected to various durations of spermatic cord torsion. J Androl 1986; 7:23-31.

22. Turner TT, Bang HJ, Lysiak JL. The molecular pathology of experimental testicular torsion suggests adjunct therapy to surgical repair. J Urol 2004; 172:2574-2578.

23. Lysiak JJ, Nguyen QA, Kirby JL, Turner TT. Ischemia-reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression. Biol Reprod 2003; 69:202-210.

24. Shimizu S, Saito M, Kinoshita Y, Shomori K, Satoh I, Satoh K. Ischemic preconditioning and post-conditioning to decrease testicular torsion-detorsion injury. J Urol 2009; 182:1637-1643.

25. Barlas M, Hatiboğlu C. The effect of nitric oxide in testicular ischemia-reperfusion injury. Int Urol Nephrol 2002; 34:81-86.

26. Beheshtian A, Salmasi AH, Payabvash S, Kiumehr S, Nezami BG, Rahimpour S, et al. Role of endogenous cannabinoids in ischemia/reperfusion injury following testicular torsion in rats. Int J Urol 2008; 15:449-454.

27. Savaş Ç, Dindar H, Aras T, Yücesan S. Pentoxifylline improves blood flow to both testes in testicular torsion. Int Urol Nephrol 2002; 33:81-85.

28. Yíldíz H, Durmus AS, Şimşek H, Yaman M. Protective effect of sildenafil citrate on contralateral testis injury after unilateral testicular torsion/detorsion. Clinics 2011; 66:137-142.

29. Singh AK, Singh S, Garg G, Rizvi SI. Rapamycin alleviates oxidative stress induced damage in rat erythrocytes. Biochem Cell Biol 2016; 94:471-479.

30. Podbielski J, Schoenberg L. Use of sirolimus in kidney transplantation. Prog Transplant 2001; 11:29-32.

31. Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 2005; 16:525-537.

32. Kamada Y, Sekito T, Ohsumi Y. Autophagy in yeast: ATOR-mediated response to nutrient starvation. Germany: TOR, Springer; 2004. P. 73-84.

33. Hosoi H, Dilling MB, Liu LN, Danks MK, Shikata T, Sekulic A, et al. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol Pharmacol 1998; 54:815-824.

34. Koehl GE, Andrassy J, Guba M, Richter S, Kroemer A, Scherer MN, et al. Rapamycin protects allografts from rejection while simultaneously attacking tumors in immunosuppressed mice. Transplantation 2004; 77:1319-1326.

35. Fang B, Xiao H. Rapamycin alleviates cisplatin-induced ototoxicity in vivo. Biochem Biophys Res Commun 2014; 448:443-447.

36. Inman SR, Davis NA, Olson KM, Lukaszek VA, McKinley MR, Seminerio JL. Rapamycin preserves renal function compared with cyclosporine A after ischemia/reperfusion injury. Urology 2003; 62:750-754.

37. Lieberthal W, Fuhro R, Andry CC, Rennke H, Abernathy VE, Koh JS, et al. Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells. Am J Physiol Renal Physiol 2001; 281:F693-F706.

38. Lui SL, Chan KW, Tsang R, Yung S, Lai KN, Chan TM. Effect of rapamycin on renal ischemia‐reperfusion injury in mice. Transpl Int 2006; 19:834-839.

39. Matsuda T, Yamaguchi Y, Matsumura F, Akizuki E, Okabe K, Liang J, et al. Immunosuppressants decrease neutrophil chemoattractant and attenuate ischemia/reperfusion injury of the liver in rats. J Trauma 1998; 44:475-484.

40. Puglisi RN, Strande L, Santos M, Schulte G, Hewitt CW, Whalen TV. Beneficial effects of cyclosporine and rapamycin in small bowel ischemic injury. J Surg Res 1996; 65:115-118.

41. Serr F, Lauer H, Armann B, Ludwig S, Thiery J, Fiedler M, et al. Sirolimus improves early microcirculation, but impairs regeneration after pancreatic ischemia‐reperfusion injury. Am J Transplant 2007; 7:48-56.

42. Das A, Salloum FN, Durrant D, Ockaili R, Kukreja RC. Rapamycin protects against myocardial ischemia–reperfusion injury through JAK2–STAT3 signaling pathway. J Mol Cell Cardiol 2012; 53:858-869.

43. Khan SA, Salloum F, Das A, Xi L, Vetrovec GW, Kukreja RC. Rapamycin confers preconditioning-like protection against ischemia–reperfusion injury in isolated mouse heart and cardiomyocytes. J Mol Cell Cardiol 2006; 41:256-264.

44. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011; 147:728-741.

45. Gross GJ, Peart JN. KATP channels and myocardial preconditioning: an update. Am J Physiol Heart Circ Physiol 2003; 285:H921-H930.

46. Liu Y, Sato T, Seharaseyon J, Szewczyk A, O'rourke B, Marban E. Mitochondrial ATP‐dependent potassium channels: viable candidate effectors of ischemic preconditioninga. Ann N Y Acad Sci 1999; 874:27-37.

47. O’Rourke B. Evidence for mitochondrial K+ channels and their role in cardioprotection. Circ Res 2004; 94:420-432.

48. Forbes RA, Steenbergen C, Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res 2001; 88:802-809.

49. Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, et al. Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 2000; 87:460-466.

50. Oldenburg O, Cohen MV, Downey JM. Mitochondrial K(ATP) channels in preconditioning. J Mol Cell Cardiol 2003; 35:569-575.

51. Dzeja PP, Bast P, Ozcan C, Valverde A, Holmuhamedov EL, Van Wylen DG, et al. Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection. Am J Physiol Heart Circ Physiol 2003; 284:H1048-H1056.

52. Narayan P, Mentzer RM, Lasley RD. Adenosine A1 receptor activation reduces reactive oxygen species and attenuates stunning in ventricular myocytes. J Mol Cell Cardiol 2001; 33:121-129.

53. O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D,               et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006; 66:1500-1508.

54. Shi Y, Yan H, Frost P, Gera J, Lichtenstein A. Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade. Mol Cancer Ther 2005; 4:1533-1540.

55. Desai BN, Myers BR, Schreiber SL. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc Natl Acad Sci U S A 2002; 99:4319-4324.

56. Sasaki N, Sato T, Ohler A, O’Rourke B, Marbán E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 2000; 101:439-445.