Microcystin-LR Induced Sertoli Cells Apoptosis Via The Mitochondrial Caspase-dependent Pathway

ObjectiveTo explore whether microcystin-LR induced Sertoli cells apoptosis via the mitochondrial caspase-dependent pathway by detecting changes of apoptosis rate, the contents of reactive oxygen species, the mitochondrial membrane potential and the relative expression of apoptosis related proteins in Sertoli cells. The results will provide scientific basis for the protection, prevention and treatment of reproductive system damage caused by MCs.Methods1. Sertoli cells, isolated from preadolescence (18-20d) SD rat testis, were purified to establish testicular Sertoli cells in primary culture cell model.2. CCK-8 assay:CCK-8 assay was used to measure the proliferation inhibition rate of Sertoli cells cultured with different concentrations(0,1,5,10,20,40,60μg/ml) of MC-LR for 24h. ICso was calculated. Then ICso/4, IC50/2 and ICso of MC-LR were as the exposure concentration in follow-up experiments.3. Sertoli cells were divided into control group (0μg/ml MC-LR), ICso/4, ICso/2, IC50 of MC-LR, caspase inhibitor-zVADfmk (50μmol/L)+ICso MC-LR, antioxidant NAC (10mmol/L)+IC50 MC-LR and cultured for 24h, respectively.4. Apoptosis assay:The apoptosis rates of each treatment group were measured by Annexin V-FITC and PI combined with flow cytometry.5. ROS detection:The changes in the fluorescence intensity of ROS were measured by ROS kit combined with fluorescence microscope and flow cytometry.6. Mitochondrial membrane potential:The changes of mitochondrial membrane potential were measured by JC-1 kit combined with flow cytometry.7. Protein detection:The protein expression of Cyt-c, Caspase-9, Cleaved-Caspase-9 and Caspase-3 were analyzed by Western Blot.Results1. (1,5,10,20,40,60μg/ml) MC-LR could inhibit the proliferation of Sertoli cells in a concentration-dependent manner, with the increasing of MC-LR concentration, cell proliferation was inhibited obviously (P<0.05), when compared with the control group (Oμg/ml MC-LR). The IC50 of MC-LR was 32μg/ml. Then 8μg/ml,16μg/ml,32μg/ml were selected as the exposure concentration in follow-up experiments.2. MC-LR could induce Sertoli cells apoptosis. The apoptosis rates was 3.900% ± 0.200%,6.000% ±0.100% and 27.266%±0.115% respectively. With the increasing of MC-LR concentration, the rates of cell apoptosis were significantly increased (P<0.05).3. MC-LR could increase the fluorescence intensity of ROS. Fluorescence microscope observation and flow cytometry results showed that:with the increasing of MC-LR concentration, the intracellular fluorescence intensity gradually increased (P<0.05).4. MC-LR could decrease the MMP, with the increasing of MC-LR concentration, the MMP was significantly decreased (P<0.05).5. MC-LR could increase the expression of Cyt-c, Caspase-9, Cleaved-Caspase-9 and Caspase-3, with the increasing of MC-LR concentration, the expression were significantly increased (P<0.05).6. Caspase inhibitor-zVADfmk could decrease the apoptosis rate of 32μg/ml MC-LR group from 27.266%±0.115% to 9.633%±0.057%(P<0.05), and reduce the expression of four apoptosis related proteins(P<0.05).7. Antioxidant NAC could decrease the apoptosis rate of 32μg/ml MC-LR group from 27.266%±0.115% to 13.200%±0.100%(P<0.05), obviously reduce the content of ROS and inhibit the decline of MMP, and significantly decrease the expression of four apoptosis related protein(P<0.05).Conclusion1. Microcystin-LR can inhibit Sertoli cell growth within a certain range of concentrations and induce apoptosis.2. Mitochondrial caspase-dependent pathway may be one of the key apoptosis pathway in Sertoli cells apoptosis induced by MC-LR.3. Reactive oxygen species was involved in the Sertoli cell apoptosis induced by...