Study On The Mechanism Of Insulin Pathway In Delaying Senescence Of Rat Leydig Cells By Heshouwuyin

Objection:To investigate the regulation mechanism of Insulin pathway of Heshouwuyin in delaying the senescence of rat testis Leydig cells.Method:1.The testicular Leydig cell senescence model was established by using the method of free radical oxidative damage.The activity of ?-galactosidase in Leydig cells in each group was detected by the method of ?-galactosidase staining,and the cell cycle distribution of Leydig cells in each group was determined by the method of propidium iodide staining.So as to determine whether the senescence model of Leydig cells in testis was successfully established.2.Immunofluorescence was used to observe the expression sites of key genes of Insulin/IGF-1 signaling pathway IGF-1,INSR,IRS1,IRS2 and IGFBP3 in Leydig cells of testis.3.Protein expression levels of IGF-1,INSR,IRS1,IRS2 and IGFBP3,the key genes of Insulin/IGF-1 signaling pathway in Leydig cells of testis,were detected by Western blot.4.mRNA expression levels of IGF-1,INSR,IRS1,IRS2,and IGFBP3 key genes of insulin/igf-1 signaling pathway in testicular Leydig cells were detected by RT-qPCR.5.The senescence testicular Leydig cells were treated with the Insulin receptor/IGF-1 receptor specific inhibitor bms-754807,and the expression of the downstream gene bcl-2 in the Insulin/IGF-1 signaling pathway was detected by RT-qPCR,Flow cytometry was used to detect the level of apoptosis in each group.Results:1.The cytoplasm of ?-galactosidase positive cells was blue,the activity of ?-galactosidase in the senescent group was higher than that in the normal group(P < 0.05),and the rate of-galactosidase positive cells in Leydig cells of the senescent group was more than 60%.The percentage of G1 cells in the aging group was higher than that in the normal2.group(P < 0.05),and that in the S and G2 stages was lower than that in the normal group(P < 0.05).3.Results of Immunofluorescence and Western blot showed that the positive products fluoresce in red.The average optical density of IGF-1,INSR,IRS1 and IRS2 in the aging group was significantly lower than that in the normal group(P<0.05),and the expression of IGFBP3 was not detected.The average optical density of IGF-1,INSR,IRS1 and IRS2 in Leydig cells after treatment with Heshouwuyin was significantly higher than that in the senescence group(P<0.05).Semi-quantitative analysis by Western Blot showed that the expression levels of IGF-1,INSR,IRS1 and IRS2 in each group were consistent with the results of fluorescence immunostaining(P<0.05),and the protein expression of IGFBP3 was not detected.4.RT-qPCR results showed that the mRNA expression levels of IGF-1,INSR,IRS1 and IRS2 in the aging group were significantly lower than those in the normal group(P < 0.05),and the mRNA expression levels of IGFBP3 were significantly higher than those in the normal group(P < 0.05).The mRNA expression levels of IGF-1,INSR,IRS1 and IRS2 in Heshouwuyin group were significantly higher than that in the cell senescence model group(P < 0.05),and the mRNA expression levels of IGFBP3 were significantly lower than that in the cell senescence model group(P < 0.05).5.After treatment of testicular Leydig cells with Insulin receptor /IGF-1 receptor specific inhibitor BMS-754807,the mRNA expression of downstream gene bcl-2 of Insulin/IGF-1 signaling pathway was significantly lower than that of the aging group(P < 0.05).The results of flow cytometry showed that the apoptosis rate of senescent Leydig cells was significantly down-regulated by the intervention of Heshouwuyin(P < 0.05),and the apoptosis rate increased after the treatment of senescent Leydig cells with the inhibitor BMS-7548079 and Heshouwuyin(P < 0.05).Conclusion:1.Heshouwuyin could improve the expression of IGF-1,INSR,IRS1,IRS2 mRNA and prote2.Heshouwuyin could reduce the mRNA of IGFBP3 and delay the senescence of rat testicular Leydig cells.3.Heshouwuyin regulates the downstream gene bcl-2 mRNA of rat testicular Leydig cells through the Insulin/IGF-1 signaling pathway.