| ObjectiveTo investigate whether the p53-dependent pathway is involved in the regulation of apoptosis induced by microcystin-LR and the role of mitochondria in the sertoli-germ cells of SD rats,cell apoptosis rate,mitochondrial membrane potential,apoptosis-associated protein in p53-dependent pathway and mitochondrial outer membrane protein VDAC1 were detected.It provides a novel molecular mechanism and theoretical basis for the reproductive toxicity of sertoli-germ cells in SD rats induced by microcystin-LR.Methods1.The extraction and culture of primary sertoli-germ cells:to establish the testicular support-spermatozoa co-cultured cell systems,22 days male SD rats were selected for isolating testes and extracting sertoli-germ cells.2.Inhibition of Cell Proliferation:the inhibition of cell proliferation was measured with CCK-8 kit to calculate the median inhibitory concentration(IC50)of sertoli-germ cells after exposed to various concentrations of MC-LR（0,1,5,10,20,40,60μg/mL）for 24 hours,and IC50,1/2IC500 and 1/4IC50were selected in the following experiments.3.The determination of apoptosis rate:The apoptosis rate in MC-LR groups and MC-LR+corresponding inhibitor（PFT-αand CsA）groups were detected by AnnexinV-FITC/PI staining and flow cytometry.4.Mitochondrial Membrane Potential Assay:The mitochondrial membrane potential in MC-LR groups and MC-LR+corresponding inhibitor（PFT-αand CsA）groups was measured by JC-1 staining and flow cytometry.5.The isolation and extraction of mitochondria.:Mitochondria were isolated from sertoli-germ cells after treated with MC-LR alone or MC-LR+corresponding inhibitor（PFT-αand CsA）for 24 hours by the mitochondrial isolation kit,which was used to extract mitochondria and cytoplasmic proteins in the next steps.6.Apoptosis-related Protein Assays:Western blotting was used to detect the relative expressions of p53,PUMA,p21,MDM2 and Cytc proteins in MC-LR groups and MC-LR+corresponding inhibitor（PFT-αand CsA）groups.7.VDAC1 mRNA level:Real time-PCR was used to detect the expression of VDAC1 mRNA in MC-LR groups and MC-LR+CsA groups.Results1.MC-LR can inhibit the proliferation of sertoli-germ cells in dose-depentent after teated with various concentrations MC-LR except for 1μg/mL MC-LR for 24hours and the differences were significant compared to control groups（P<0.05）.The IC500 of MC-LR on sertoli-germ cells was 36μg/ml and 36μg/mL,18μg/mL and 9μg/mL were selected for the following experiments.2.MC-LR have a dose dependent induction of apoptosis to sertoli-germ cells after exposed to different concentrations of MC-LR（0,9,18,36μg/mL）for 24 h and the difference was significant compared to control groups（P<0.05）.The apoptosis rate was significantly lower after pretreatment with PFT-αor CsA for 2 h in 36μg/mL groups than that in 36μg/mL MC-LR alone,and the differences were significant（P<0.05）.3.MC-LR induce a dose-dependent decline of mitochondrial membrane potential in sertoli-germ cells after exposed to different concentrations of MC-LR（0,9,18,36μg/mL）for 24 h.The cell mitochondrial membrane potential was significantly higher after pretreatment with PFT-αor CsA 2 h earlier in the 36μg/mL MC-LR group,than that in the 36μg/mL MC-LR alone,and the difference was significant（P<0.05）.4.Sertoli-germ cells have a higher level of proapoptotic proteins such as p53,PUMA,P21,clevead-PARP and clevead-caspase-3 and a low level of MDM2 with respect to control groups after exposed to different concentrations of MC-LR（0,9,18,36μg/mL）for 24 h.However,the expression of proapoptotic proteins p53,PUMA,P21,clevead-PARP and clevead-caspase-3 were declined after pretreatment with PFT-αfor 2 hours in 36μg/mL MC-LR group compared with 36μg/mL MC-LR alone.Furthermore,the release of Cytc from the mitochondria to the cytoplasm was inhibited.5.VDAC1 located on mitochondrial outer membrane was upregulated in the level of protein and mRNA in sertoli-germ cells after exposed to different concentrations of MC-LR（0,9,18,36μg/mL）for 24 hours（P<0.05）.However,there was no obvious effect on the expression of VDAC1 protein though the realese of Cytc from mitochondria to the cytoplasm was inhibited after pretreatment with CsA 2 hours in the 36μg/mL MC-LR group than that in 36μg/mL MC-LR alone（P<0.05）.Conclusion1.p53-dependentpathwaymediatedMC-LR-inducedapoptosisinrat sertoli-germ cells2.MC-LR induced the opening of mPTP and promoted Cyt c release in rat sertoli-germ cells3.Cell cycle arrest may be involved in the occurrence of rat sertoli-germ cells apoptosis... |
