Berberine protects against diclofenac sodium-induced testicular impairment in mice by its anti-oxidant and anti-apoptotic activities

Document Type : Original Article

Authors

1 Laboratory of Physiology, Department of Zoology, Faculty of Sciences, Assiut University, Assiut, Egypt

2 Department of Cell and Histology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt

3 Department of Pathology, Al Azhar, Faculty of Medicine, Cairo, Egypt

4 Department of Medical Physiology, Faculty of Medicine, Assiut University, Assiut, Egypt

Abstract

Objective(s): This study was designed to investigate the effect of berberine (BBR) on diclofenac sodium-induced testicular impairment in mice. 
Materials and Methods: Eighteen male mice were divided randomly and equally into three groups for three weeks. One group was kept as control, the second group was injected intraperitoneally with diclofenac sodium (DS) at a dose of 10 mg/kg BW daily during the second and third weeks. The third group received daily oral administration of BBR at a dose of 50 mg/kg BW throughout the whole period of the experiment in parallel with the injection of the above-mentioned dose of DS during the second and third weeks. Plasma testosterone as well as testicular lipid peroxides (LPO), nitric oxide (NO), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were evaluated. In paraffin-embedded testicular tissues, histological examination, immuno-expression of glutathione reductase (GR), and TUNEL assay were carried out. 
Results: Testosterone levels were within the normal range in all groups. BBR decreased testicular LPO and induced SOD and GSH without marked changes in CAT and NO. The histology of testis was improved and, regularity and integrity of seminiferous tubules basement membranes, and distribution and amount of peritubular collagen fibers were normalized. BBR treated group showed few positive GR immuno-expression in spermatogenic cells and negative GR immuno-expression in interstitial cells of Leydig along with a few apoptotic spermatogenic cells. 
Conclusion: BBR is effective in protecting against DS-induced testicular dysfunction by improving oxidant/anti-oxidant balance and blocking the apoptotic cascade.

Keywords


1. Gan TJ. Diclofenac: An update on its mechanism of action and safety profile.  Curr Med Res Opin 2010; 26:1715-1731.
2. Lewis S, Aitken RJ. DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 2005; 322:33-41.
3. Agarwal A, Makker K, Sharma R. Clinical relevance of oxidative stress in male factor infertility: An update. Am J Reprod Immunol 2008; 59:2-11.
4. Adeyemi WJ, Olayaki LA. Effects of salmon calcitonin and omega-3 fatty acids on some biochemical parameters in induced knee osteoarthritic Wistar rats. Niger J Physiol Sci 2017; 32:179-188.
5. Nouri A, Heidarian E. Ameliorative effects of N-acetyl cysteine on diclofenac-induced renal injury in male rats based on serum biochemical parameters, oxidative biomarkers, and histopathological study. J Food Biochem 2019; 43:e12950.
6. El-Hadary AE, Ramadan MF. Anti-oxidant traits and protective impact of Moringa oleifera leaf extract against diclofenac sodium-induced liver toxicity in rats. J Food Biochem 2019; 43:e12704.
7. Adeyemi WJ, Omoniyi JA, Olayiwola A, Ibrahim M, Ogunyemi O, Olayaki LA. Elevated reproductive toxicity effect of diclofenac after withdrawal: Investigation of the therapeutic role of melatonin. Toxicol Rep 2019; 6:571-577.
8. Mousa AA, Elweza AE, Elbaz HT, Tahoun EA, Shoghy KM, Elsayed I. Eucalyptus globulus protects against diclofenac sodium induced hepatorenal and testicular toxicity in male rats. J Tradit Complement Med 2019; 10:521-528.
9. Mogilner JG, Lurie M, Coran AG, Nativ O, Shiloni E, Sukhotnik I. Effect of diclofenac on germ cell apoptosis following testicular ischemia-reperfusion injury in a rat. Pediatr Surg Int 2006; 22:99-105.
10. Inoue A, Muranaka S, Fujita H, Kanno T, Tamai H, Utsumi K. Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: Dependency on reactive oxygen species, Akt, Bid, cytochrome c, and caspase pathway. Free Radic Biol Med 2004; 37:1290-1299.
11. Vyas A, Purohit A, Ram H. Assessment of dose-dependent reproductive toxicity of diclofenac sodium in male rats. Drug Chem Toxicol 2019; 42:478-486.
12. Hassani-Bafrani H, Najaran H, Razi M, Rashtbari H. Berberine ameliorates experimental varicocele-induced damages at testis and sperm levels; evidences for oxidative stress and inflammation. Andrologia 2019; 51:e13179.
13. Kazaz IO, Mentese A, Demir S, Kerimoglu G, Colak F, Bodur A, et al. Berberine inhibits the ischemia-reperfusion induced testicular injury through decreasing oxidative stress. Am J Emerg Med 2019; 38:33-37.
14. Owumi SE, Aliyu‐Banjo NO, Odunola OA. Selenium attenuates diclofenac‐induced testicular and epididymal toxicity in rats. Andrologia 2020; 52:e13669.
15. Lowry OL, Rosebrough NJ, Farr AL, Randall RJ. Protein determination with the Folin phenol reaction. J Biol Chem 1951; 93:265-273.
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. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988; 141:2407-2412.
18. Beutler E, Duron O, Kelly M. Colorimetric method for determination of glutathione reduced. J Lab Clin Med 1963; 61:882.
19. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 247:3170-3175.
20. Lück H. Catalase: In: Methods of Enzymatic Analysis edited by HU. Academic Press, New York, London); 1963. p. 885-888.
21. Bancroft JD, Gamble M. Carbohydrates.  Theory and Practice of Histological Techniques. 6th ed. Churchill Livingstone, Edinburgh, London, Oxford, New York, Philadelphia, St Louis, Sydney and Toronto: Elsevier Health Sciences; 2008. p. 161-186.
22. Abd-Elkareem M, Abou Khalil NS, Sayed AEH. Cytoprotective effect of Nigella sativa seed on 4-nonylphenol-induced renal damage in the African catfish (Clarias gariepinus). Chemosphere 2020; 259:127379.
23. Sayed AEH, Abd-Elkareem M, Abou Khalil NS. Immunotoxic effects of 4-nonylphenol on Clarias gariepinus: Cytopathological changes in hepatic melanomacrophages. Aquat Toxicol 2019; 207:83-90.
24. Gaschler MM, Stockwell BR. Lipid peroxidation in cell death. Biochem Biophys Res Commun 2017; 482:419-425.
25. Huang T, Zhang G, Chong S, Liu Y, Zhang N, Fang S, et al. Effects and mechanism of diclofenac degradation in aqueous solution by US/Zn0. Ultrason Sonochem 2017; 37:676-685.
26. Kono T, Saito M, Kinoshita Y, Satoh I, Shinbori C, Satoh K. Real-time monitoring of nitric oxide and blood flow during ischemia-reperfusion in the rat testis. Mol Cell Biochem 2006; 286:139-145.
27. Sun J, Druhan LJ, Zweier JL. Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production. Arch Biochem Biophys 2010; 494:130-137.
28. Zweier JL, Chen C, Druhan LJ. S-glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling. Anti-oxid Redox Signal 2011; 15:1769–1775.
29. Nayernia K, Diaconu M, Aumüller G, Wennemuth G, Schwandt I, Kleene K, et al. Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa. Mol Reprod Dev 2004; 67:458-464.
30. Simon JP, Evan Prince S. Aqueous leaves extract of Madhuca longifolia attenuate diclofenac-induced hepatotoxicity: Impact on oxidative stress, inflammation, and cytokines. J Cell Biochem 2018; 119:6125-6135.
31. Prince SE. Diclofenac-induced renal toxicity in female Wistar albino rats is protected by the pre-treatment of aqueous leaves extract of Madhuca longifolia through suppression of inflammation, oxidative stress and cytokine formation. Biomed Pharmacother 2018; 98:45-51.
32. Simon JP, Parthasarathy M, Nithyanandham S, Katturaja R, Namachivayam A, Prince SE. Protective effect of the ethanolic and methanolic leaf extracts of Madhuca longifolia against diclofenac-induced toxicity in female Wistar albino rats. Pharmacol Rep 2019; 71:983-993.
33. Jung J, Nam Y, Sohn UD. Inhibitory effects of ECQ on indomethacin-induced gastric damage in rats. Korean J Physiol Pharmacol 2012; 16:399-404.
34. Tsunoda S, Kawano N, Miyado K, Kimura N, Fujii J. Impaired fertilizing ability of superoxide dismutase 1-deficient mouse sperm during in vitro fertilization. Biol Reprod 2012; 87:121, 121-126.
35. Saleh SR, Attia R, Ghareeb DA. The ameliorating effect of berberine-rich fraction against gossypol-induced testicular inflammation and oxidative stress. Oxid Med Cell Longev 2018; 2018:1056173.
36. Albasher G, Alkahtani S, Alarifi S. Berberine mitigates oxidative damage associated with testicular impairment following mercury chloride intoxication. J Food Biochem 2020; 44:e13385.
37. Kazaz IO, Mentese A, Demir S, Kerimoglu G, Colak F, Bodur A, et al. Berberine inhibits the ischemia-reperfusion induced testicular injury through decreasing oxidative stress. Am J Emerg Med 2020; 38:33-37.
38. Sarna LK, Wu N, Hwang S, Siow YL, O K. Berberine inhibits NADPH oxidase mediated superoxide anion production in macrophages. Can J Physiol Pharmacol 2010; 88:369-378.
39. Tian H, Kang Y, Gao H, Shi X, Fu L, Li Y, et al. Chronic infusion of berberine into the hypothalamic paraventricular nucleus attenuates hypertension and sympathoexcitation via the ROS/Erk1/2/iNOS pathway. Phytomedicine 2019; 52:216-224.
40. Liu L, Liu J, Huang Z, Yu X, Zhang X, Dou D, et al. Berberine improves endothelial function by inhibiting endoplasmic reticulum stress in the carotid arteries of spontaneously hypertensive rats. Biochem Biophys Res Commun 2015; 458:796-801.
41. Paul M, Hemshekhar M, Kemparaju K, Girish KS. Berberine mitigates high glucose-potentiated platelet aggregation and apoptosis by modulating aldose reductase and NADPH oxidase activity. Free Radic Biol Med 2019; 130:196-205.
42. Lao-ong T, Chatuphonprasert W, Nemoto N, Jarukamjorn K. Alteration of hepatic glutathione peroxidase and superoxide dismutase expression in streptozotocin-induced diabetic mice by berberine. Pharm Biol 2012; 50:1007-1012.
43. Lopes TZ, de Moraes FR, Arni RK, Rahal P, Calmon MF. Berberine associated photodynamic therapy promotes autophagy and apoptosis via ROS generation in renal carcinoma cells. Biomed Pharmacother 2020; 123:109794.
44. Nayak G, Rao A, Mullick P, Mutalik S, Kalthur SG, Adiga SK, et al. Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis. J Ethnopharmacol 2020; 259:112922.
45. Cupertino MC, Novaes RD, Santos EC, Neves AC, Silva E, Oliveira JA, et al. Differential susceptibility of germ and leydig cells to cadmium-mediated toxicity: Impact on testis structure, adiponectin levels, and steroidogenesis. Oxid Med Cell Longev 2017; 2017.
46. Motawi TK, Ahmed SA, El-Boghdady NA, Metwally NS, Nasr NN. Impact of betanin against paracetamol and diclofenac induced hepato-renal damage in rats. Biomarkers 2019:1-24.
47. Al-Bader M, Kilarkaje N. Effects of bleomycin, etoposide and cisplatin treatment on Leydig cell structure and transcription of steroidogenic enzymes in rat testis. Eur J Pharmacol. 2015; 747:150-159.
48. Lee YJ, Ahn MY, Kim HS, Kwack SJ, Park KL, Yoon S, et al. Role of phospholipase D in regulation of testicular Leydig cell hyperplasia in Sprague–Dawley rats treated with di (2-ethylhexyl) phthalate. Arch Toxicol 2011; 85:975-985.
49. Monsees TK, Franz M, Gebhardt S, Winterstein U, Schill WB, Hayatpour J. Sertoli cells as a target for reproductive hazards. Andrologia 2000; 32:239-246.
50. Altindağ F, Rağbetli MÇ. The effect of maternal treatment with diclofenac sodium and thymoquinone on testicular parameters in rat offspring. Rev Int Androl 2019.
51. Chen N, Su P, Wang M, Li Y. Ascorbic acid inhibits cadmium-induced disruption of the blood-testis barrier by regulating oxidative stress-mediated p38 MAPK pathways. Environ Sci Pollut Res 2018; 25:21713-21720.
52. Khoshvakhti H, Yurt KK, Altunkaynak BZ, Türkmen AP, Elibol E, Aydın I, et al. Effects of melatonin on diclofenac sodium treated rat kidney: A stereological and histopathological study. Ren Fail 2015; 37:1379-1383.
53. Amin M, Razi M, Sarrafzadeh-Rezaei F, Shalizar Jalali A, Najafi G. Berberine inhibits experimental varicocele-induced cell cycle arrest via regulating cyclin D1, cdk4 and p21 proteins expression in rat testicles. Andrologia 2018; 50:e12984.
54. Rashtbari H, Razi M, Hassani-Bafrani H, Najaran H. Berberine reinforces Sertoli cells niche and accelerates spermatogonial stem cells renewal in experimentally-induced varicocele condition in rats. Phytomedicine 2018; 40:68-78.
55. Orabi SH, Eldaium DA, Hassan A, El Sabagh HS, Eldaim MAA. Allicin modulates diclofenac sodium induced hepatonephro toxicity in rats via reducing oxidative stress and caspase 3 protein expression. Environ Toxicol Pharmacol 2020; 74:103306.
56.Huang J, Nguyen V, Tang X, Wei J, Lin X, Lai Z, et al. Protection from diclofenac-induced liver injury by Yulangsan polysaccharide in a mouse model. J Ethnopharmacol 2016; 193:207-213.
57. Rana C, Piplani H, Vaish V, Nehru B, Sanyal SN. Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by diclofenac and curcumin in colon cancer. Mol Cell Biochem 2015; 402:225-241.
58. Patil JB, Kim J, Jayaprakasha GK. Berberine induces apoptosis in breast cancer cells (MCF-7) through mitochondrial-dependent pathway. Eur J Pharmacol 2010; 645:70-78.
59. Yang J, Yan H, Li S, Zhang M. Berberine ameliorates MCAO induced cerebral ischemia/reperfusion injury via activation of the BDNF–TrkB–PI3K/Akt signaling pathway. Neurochem Res 2018; 43:702-710.
60. Zhao G, Yu L, Gao W, Duan W, Jiang B, Liu X, et al. Berberine protects rat heart from ischemia/reperfusion injury via activating JAK2/STAT3 signaling and attenuating endoplasmic reticulum stress. Acta Pharmacol Sin 2016; 37:354-367.
61. Sheng M, Zhou Y, Yu W, Weng Y, Xu R, Du H. Protective effect of berberine pretreatment in hepatic ischemia/reperfusion injury of rat. Transplant Proc 2015; 47:275-282.
62. Chen K, Li G, Geng F, Zhang Z, Li J, Yang M, et al. Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K–Akt signaling in diabetic rats. Apoptosis 2014; 19:946-957.