Research On The Mechanism Of Cytc/Apaf-1/Caspase9/Caspase3 Pathway In Inhibiting Apoptosis Of Spermatogenic Cells By Heshouwuyin

Objection:To investigate the mechanism of Cytc/Apaf-1/Caspase9/Caspase3 pathway in inhibiting apoptosis of spermatogenic cells by Heshouwuyin.Method:1.The cultured cells were separated by combinatorial enzymatic methods and differential adherence methods.Sertoli cells were identified by Sudan IV staining.Spermatogonial stem cells were identified by detection ofα₆-integrin andβ₁-integrin.Spermatogenic cells were identified by alkaline phosphatase staining and HE staining.2.The aging model of spermatogenic cells was established by the method of free radical oxidative damage and was identified byβ-galactosidase staining.Flow cytometry was used to detect apoptosis of spermatogenic cells.3.To screen an optimal siRNA transfection time,we use immunofluorescence technique to detect Cy3 expression.In order to screen the dominant interference sequence,Western blot and RT-qPCR were used to detect the expression level of Apaf-1 in spermatogenic cells among three different siRNA interferences.According to the purpose of the experiment,Spermatogenic cells are mainly divided into NCG,AG,NC-siRNA（Normal control siRNA group）,SWYG（Heshouwuyin group）,Apaf-1-siRNA group,and Apaf-1-siRNA+SWYG（Apaf-1-siRNA+Heshouwuyin group）.4.The expression of mitochondrial apoptosis pathway genes Cytc,Caspase9 and Caspase3 were detected by immunofluorescence technique,Western blot and RT-qPCR.5.The changes of spermatogenic cells mitochondrial membrane potential were detected in each group.Results:1.The result of Sudan IV staining showed that the purity of the Sertoli cells was 90%.2.The activity of SA-β-gal in aging group was higher than that in the normal control group,and the phenomenon was reversed after the intervention of Heshouwuyin.It indicates that the spermatogenic cells aging model was successfully established.The trend of spermatogenic cell apoptosis detection is consistent with it.3.Apaf-1-siRNA screeningAfter detecting by Western blot and RT-qPCR,it proves that the expression levels of Apaf-1 mRNA and protein in the siRNA3 interference group were lower than those in other interference groups（P<0.05）.So it was confirmed that siRNA3 is the optimal sequence.4.The regulation effect of Heshouwuyin on Cytc、Caspase9、Caspase3The expression level of Cytc in each groupDetecting protein expression levels by Western blot,and the conclusions were as follows:The protein expression level of Cytc in AG was higher than that in NCG（P<0.05）.The Cytc protein expression levels in SWYG and Apaf-1-siRNA+SWYG were lower than that in AG（P<0.05）.The Cytc protein expression levels was no difference between NC-siRNA,Apaf-1-siRNA group and AG（P>0.05）.Compared with the Apaf-1-siRNA group,the Cytc protein expression levels of the SWYG and Apaf-1-siRNA+SWYG were decreased（P<0.05）.There was no significant difference between SWYG and Apaf-1-siRNA+SWYG（P>0.05）in Cytc protein expression level,and the expression trend was consistent with the immunofluorescence.Detecting the mRNA expression levels by RT-qPCR,and the conclusions were as follows:The mRNA expression level of Cytc in AG was higher than that in NCG（P<0.05）.The mRNA expression levels of Cytc in SWYG and Apaf-1-siRNA+SWYG were lower than that in AG（P<0.05）.The mRNA expression of Cytc was no significant difference between NC-siRNA,Apaf-1-siRNA group and AG（P>0.05）.Compared with the Apaf-1-siRNA group,the mRNA expression levels of Cytc of the SWYG and Apaf-1-siRNA+SWYG were decreased（P<0.05）.There was no difference between SWYG and Apaf-1-siRNA+SWYG（P>0.05）in Cytc mRNA expression（P>0.05）.The expression level of Caspase9 and Caspase3 in each groupThe protein expression levels of Caspase9 and Caspase3 in AG were higher than that in NCG（P<0.05）.The Caspase9 and Caspase3 protein expression levels were no difference between NC-siRNA and AG（P>0.05）.The Caspase9 and Caspase3 protein expression levels in SWYG,Apaf-1-siRNA group and Apaf-1-siRNA+SWYG were lower than that in AG（P<0.05）.Compared with the Apaf-1-siRNA group,the Caspase9 and Caspase3 protein expression levels of Apaf-1-siRNA+SWYG were decreased（P<0.05）.There was no difference between SWYG and Apaf-1-siRNA group in Caspase9 protein expression,so did Caspase3（P>0.05）.The expression trend was consistent with the immunofluorescence.Detecting the mRNA expression by RT-qPCR,and the conclusions were as follows:The mRNA expression levels of Caspase9,Caspase3 in AG were higher than that in NCG（P<0.05）.There was no significant difference between NC-siRNA and AG（P>0.05）in Caspase9,Caspase3 mRNA expression levels（P>0.05）.The Caspase9 and Caspase3 mRNA expression levels in SWYG,Apaf-1-siRNA group and Apaf-1-siRNA+SWYG were lower than that in AG（P<0.05）.Compared with the Apaf-1-siRNA group,the Caspase9 and Caspase3 mRNA expression levels of Apaf-1-siRNA+SWYG were decreased（P<0.05）.There was no significant difference between SWYG and Apaf-1-siRNA group in Caspase9 and Caspase3mRNA expression levels（P>0.05）.5.Mitochondrial membrane potential detection resultsThe mitochondrial membrane potential in AG was lower than that in NCG（P<0.05）.The mitochondrial membrane potential in SWYG and Apaf-1-siRNA+SWYG were higher than that in AG（P<0.05）.The mitochondrial membrane potential was no difference between NC-siRNA,Apaf-1-siRNA group and AG（P>0.05）.Compared with the Apaf-1-siRNA group,the mitochondrial membrane potential of the SWYG and Apaf-1-siRNA+SWYG were decreased（P<0.05）.There was no significant difference between SWYG and Apaf-1-siRNA+SWYG in mitochondrial membrane potential（P>0.05）.Conclusion:Heshouwuyin can inhibit the apoptosis of spermatogenic cells through the Cytc/Apaf-1/Caspase9/Caspase3 pathway.