Resveratrol Ameliorates Microcystin-LR-Induced Testis Germ Cell Apoptosis in Rats via SIRT1 Signaling Pathway Activation

Abstract

Microcystin-leucine arginine (MC-LR), a cyclic heptapeptide produced by cyanobacteria, is a strong reproductive toxin. Studies performed in rat Sertoli cells and Chinese hamster ovary cells have demonstrated typical apoptosis after MC-LR exposure. However, little is known on how to protect against the reproductive toxicity induced by MC-LR. The present study aimed to explore the possible molecular mechanism underlying the anti-apoptosis and protective effects of resveratrol (RES) on the co-culture of Sertoli–germ cells and rat testes. The results demonstrated that MC-LR treatment inhibited the proliferation of Sertoli–germ cells and induced apoptosis. Furthermore, sirtuin 1 (SIRT1) and Bcl-2 were inhibited, while p53 and Ku70 acetylation, Bax expression, and cleaved caspase-3 were upregulated by MC-LR. However, RES pretreatment ameliorated MC-LR-induced apoptosis and SIRT1 inhibition, and downregulated the MC-LR-induced increase in p53 and Ku70 acetylation, Bax expression, and caspase-3 activation. In addition, RES reversed the MC-LR-mediated reduction in Ku70 binding to Bax. The present study indicated that the administration of RES could ameliorate MC-LR-induced Sertoli–germ cell apoptosis and protect against reproductive toxicity in rats by stimulating the SIRT1/p53 pathway, suppressing p53 and Ku70 acetylation and enhancing the binding of Ku70 to Bax.

p53‐Dependent pathway and the opening of mPTP mediate the apoptosis of co‐cultured Sertoli‐germ cells induced by microcystin‐LR

“…The identification of co‐cultured Sertoli‐germ cells was performed in our previous studies and is not shown here . The inhibition rate of MC‐LR on co‐cultured Sertoli‐germ cells for 24 hours was detected by CCK‐8.…”

“…The inhibition rate of cell proliferation was detected by CCK‐8 kit as described previously . In detail, 100 μL cell suspension was added to 96‐well plate with a minimum inoculum density of 1000 cells/well.…”

“…FITC of 5 μL and PI staining solution of 10 μL were added, which were detected by flow cytometry within 1 hour after the mixture was incubated for 15 minutes in the dark. The experimental protocol is reference to our previous research …”

“…The determination of MMP was performed as described previously . Briefly, the medium containing MC‐LR was removed from cells and digested with 0.25% trypsin.…”

“…As performed in the previous studies, after treatment with MC‐LR alone or MC‐LR + the corresponding inhibitor (PFT‐α or CsA) for 24 hours, total RNA was extracted from co‐cultured Sertoli‐germ cells by TRIzol lysis buffer (Ambion, Beijing, China). Then, 1 μg RNA was used for the synthesis of cDNA according to the instruction of RevertAid first Strand cDNA Synthesis kit (Thermo Scientific, China).…”