The mechanism of Se in regulating the proliferation and apoptosis of sheep Leydig cells through the miR-200a/NRF2 pathway

This study aimed to investigate the mechanism by which Se in regulates the proliferation and apoptosis of sheep Leydig cells via the miR-200a/NRF pathway. The cells were isolated and purified from the testes of 8-month-old sheep via a Percoll density gradient. After the cells were treated with different concentrations of Se (0, 2.0, 4.0, 6.0, and 8.0 μmol/L of Se) for 18 h, the miR-200a levels was detected. MiR-200a mimics and inhibitors were transfected into the cells, resulting in five groups (control, NC mimics, miR-200a mimics, NC inhibitor and miR-200a inhibitor). Cell viability and antioxidant status were measured via CCK8 and antioxidant assays, respectively. The abundances of pro-apoptotic (BAX, CASPASE 3 and CASPASE 8), cell cycle (P21, P27 and CDK1), and NRF2-related (NRF2, HO-1, NQO1 and KEAP1) genes were detected by real-time PCR and Western blot analysis. The results revealed that miR-200a mimics group presented greater (P < 0.05) abundances of NRF2, HO-1 and NQO1 mRNA transcripts and proteins. Compared with those both in the NC mimics and the miR-200a inhibitor groups, the activities of GSH-Px and SOD, as well as cell viability in the miR-200a mimics group were significantly greater (P < 0.05). In contrast, the ROS levels, MDA content and abundances of KEAP1, P21, P27 and apoptosis-related genes mRNA transcripts and proteins were decreased (P < 0.05). The highest (P < 0.05) miR-200a expression level was detected in the Se_6.0 group. Compared with that in the Se (6.0 μmol/L) group, cell viability in the Se + miR-200a inhibitor group was lower (P < 0.05). The abundances of NRF2, HO-1 and NQO1 in the Se + miR-200a inhibitor group were lower (P < 0.05) than those in the Se (6.0 μmol/L) group but greater (P < 0.05) than those in the inhibitor group, while KEAP1 displayed the opposite trend (P < 0.05). These results indicate that Se can activate the NRF2 antioxidant signaling pathway to regulate the proliferation and apoptosis of sheep Leydig cells and that miR-200a plays a vital role in this process. The regulatory effect of Se on male reproduction and spermatogenesis may be related to the number of Leydig cells. This study aimed to provide experimental data for Se regulation of spermatogenesis.