Androgen Up-regulates Klotho To Promote Apoptosis Of Granulosa Cells And Influences The Development Of PCOS

Background: Polycystic ovary syndrome(PCOS),an endocrine metabolic syndrome affecting 9%-15% of childbearing age women,mainly characterized by hyperandrogenism,anovulation or ovulation,insulin resistance,obesity and metabolic syndrome.Hyperandrogenism is one of the most important potential mechanisms of PCOS.Androgen acts via the androgen receptor(AR),however,the exact mechanism of androgen/AR signaling regulates granulosa cells(GCs)apoptosis is unknown.Klotho as a longevity gene which can bind to AR in kidney.Klotho also is found that associated with the apoptosis of GCs in PCOS.But whether androgen directly regulates klotho via AR in GCs to affect cell apoptosis is still unknown.This study aims to study the effect of androgen/AR signaling regulates klotho expression in GCs,which may provide a basis for clinical detection indicators and treatment methods.Method: 1)Construction of PCOS rat model: using vaginal smears monitor changes in the estrous cycle of PCOS rat model,automatic biochemistry analyzer to detect the level of testosterone(T)and blood lipids,hematoxylin-eosin(HE)staining to observe the changesof ovarian tissue morphology,immunohistochemistry(IHC)and western-blot(WB)were used to detect the expression of AR and klotho in the ovary of each group;2)KGN cells were incubated with TP(5~100 nmol/L)for 12-24 hours,and the cell viability was detected by the CCK-8 kit;3)Hoechst and cell flow were used to detect apoptosis after intervening the expression of AR in KGN cells.IHC and WB were used to detect the expressions of AR and klotho;4)After using siKlotho to interfere with klotho expression in KGN cells,Hoechst and cell flow cytometry were used to detect KGN apoptosis;5)Using ChIP to detect the combination of AR and klotho promoter.Results:1)Compared with the control group,the estrous cycle dysfunction,serum T was significantly increased(P<0.01),dyslipidemia such as HDL decreased(P<0.05)and CHO increased(P<0.05),ovarian polycystic changes and luteal formation decreased or even disappeared in TP-treated rats.After treatment with flutamide,the estrous cycle was restored,serum T was significantly reduced(P<0.01),dyslipidemia was improved,follicles at various levels were visible in the ovary and a large amount of corpus luteum was produced.2)Compared with the control group,the expressions of AR and klotho were increased in PCOS rats(P<0.05),but flutamide can reverse this situation.3)Detecting the viability and apoptosis of KGN cells treated with TP by CCK-8,Hoechst and cell flow cytometry.We found that TP inhibited the proliferationactivity of KGN cells in a concentration-dependent manner and induced cell apoptosis.After treatment with flutamide or silencing klotho,KGN cell apoptosis was significantly inhibited,suggesting that blocking AR or klotho expression can improve KGN cell apoptosis.4)ChIP data showed that AR didn't specifically bonds to the klotho promoter.Conclusion: Androgen/AR signaling indirectly increases klotho expression to promote the apoptosis of GCs.