The Effects Of Heshouwuyin On Genes Expression Of IRS1 IGF-1 IGFBP3 In Insulin Pathway About Aging Rat Germ Cells

Objective:Explore the effects of Heshouwuyin on the key genes of IRS1,IGF-1,IGFBP3 in insulin signaling pathway of aging rat germ cells.Methods: In vivo experiment1.In this study,30 male Wistar rats,12 months old,were randomly divided into three groups: young control group(YCG,10),natural aging group(NAG,10),Heshouwuyin group(SWYG,10).18-month-old Wistar rats served as NAG and 12-month-old rats served as YCG.SWYG were comprised with 18-month-old rats treatment Heshouwuyin(4.8 g/100 g body weight)intragastrically for 60 days.Then testis of YCG were isolated in the age of 12 months,the other two groups were in the age of 18 months.2.Observing the expression of IRS1,IGF-1,IGFBP3 in testis To observe the location of the key genes IRS1,IGF-1,IGFBP3 by immunofluorescence;To detect IRS1,IGF-1,IGFBP3 protein expression changes by Western blot and protein expression changes in mRNA in testicular tissue.In vitro studies1.The germ cell separation,purification,culture and identification Combination of enzymatic separation of cells,Percoll density gradient centrifugation and adherent method difference purified cells.With Sudan IV dye solution identification of Sertoli cells and to calculate the purity of Sertoli cells;Fulgent,Alkaline phosphatase and HE to identified germ cells.2.Creative aging model Culture germ cells for 7 d,add final concentration of 50 ?mol/L H2O2 and 100 ?mol/L FeSO4 respectively,culture for 8 h,change liquid,continuing to culture germ cells for 72 h.The ?-galactose glucoside enzyme staining detects the ?-galactosidase expression in germ cells.3.The experimental group and processing According to the experimental purpose divided into 3 groups: normal control group(NCG),cultivate born after 7 d of germ cells,continue to culture for 80 h;aging model group(AMG),up to 7 d in germ cells to add in the final concentration of 50 ?mol/L H2O2 and 100 ?mol/L FeSO4 respectively,intervention 8 h,in liquid,continue to culture 72 h;Heshouwuyin group(SWYG),up to 7 d in germ cells to add the final concentration of 50 ?mol/L H2O2 and 100 ?mol/L FeSO4,respectively,intervention 8 h,in liquid,continue to add 10% medicated serum and culture for 72 h.4.Using Western blot and qRT-PCR to observe the changes of protein expression and mRNA expression level relatively in germ cells.Results: In vivo experiment results:1.Immunofluorescence results IRS1 positive product present red particles,located in cells cytomembrane,cell nucleus with DAPI staining show blue,IRS1 mainly expressed in the spermatocyte.Compared with the YCG,IRS1 positive rate of NAG was obviously lower(P < 0.01),IRS1 positive rate of SWYG was obviously higher than NAG(P < 0.01);IGF-1 positive product present green fluorescent,located in cell cytoplasm,cell nucleus with DAPI staining show blue,IGF-1 mainly expressed in the sperm cells.Compared with the YCG,NAG of IGF-1 positive rate significantly decreased(P<0.01),IGF-1 of SWYG were significantly higher than NAG(P < 0.01);IGFBP3 positive product present green fluorescent,located in cell cytoplasm,cell nucleus with DAPI staining show blue,IGFBP3 mainly expressed in spermatogonia cells.Compared with YCG,IGFBP3 expression was obviously increased in NAG(P < 0.01),compared with the NAG,SWYG was obviously lower(P < 0.01).2.Western blot results The results show that compared with the YCG,the expression of IGFBP3 in NAG increased significantly(P < 0.01),IRS1,IGF-1 expression decreased significantly(P < 0.01);SWYG compared with NAG,IGFBP3 expression quantity decreased significantly(P < 0.01);IRS1,IGF-1 expression quantity increased significantly(P < 0.01).3.qRT-PCR results The results show that compared with the YCG,the expression of IGFBP3 in NAG increased significantly(P < 0.01),IRS1,IGF-1 expression decreased significantly(P < 0.01);SWYG compared with NAG,IGFBP3 expression quantity decreased significantly(P<0.01);IRS1,IGF-1 expression quantity increased significantly(P < 0.01).By in vivo experiment results analysis,HSWY can regulate the insulin signaling pathways through IRS1,IGF-1,IGFBP3 expressions,slow down the aging process of testicular tissue of rats.In order to further analysis of HSWY to regulate the mechanism of aging rat spermatogenic function,we choose the method of sertoli cells and germ cells co-culture to detecte the changes of IRS1,IGFBP3,IGF-1 expression in germ cells.In vitro experiment results:1.Sudan IV dyeing results show that the sertoli cells purity rate reached more than90%.2.?-galactosidase dyeing results The positive product of ?-galactosidase is blue and located in the cytoplasm.Compared with NCG,AMG positive rate significantly increased(P < 0.01);Compared with AMG,the positive rate of SWYG decreased significantly(P < 0.01).3.Western blot and qRT-PCR results Compared with the NCG,IGFBP3 expression of the AMG was obviously raised(P < 0.01),the expression of IRS1,IGF-1 significantly decreased(P < 0.01);HSWY compared with AMG,the expression of IGFBP3 significantly decreased(P < 0.01),the expression of IGF-1,IRS1 significantly increased(P < 0.01).Conclusion:1.HSWY can delay aging of rat testicular tissue by improving IRS1,IGF-1 expression and reducing IGFBP3 expression in insulin pathway.2.HSWY can reduce aging markers expression of ?-galactose glucoside enzyme in germ cells.3.HSWY can decrease aging in germ cells by improving IRS1,IGF-1 expression and reducing IGFBP3 expression in insulin pathway.Above all,HSWY by regulating the expression of IRS1,IGF-1,IGFBP3 in insulin signaling pathway,slow down the aging process of testicular tissue and inhibit the germ cell aging.