The Role Of AGEs In Promoting Polycystic Ovary Syndrome By Regulating The Expression Of AMH In Granulosa Cells

Objective: Advanced glycation end products(AGEs)are significantly elevated in the blood and follicular fluid of women with polycystic ovary syndrome(PCOS),which are involved in the pathogenesis of PCOS.However,the mechanism of how AGEs affect the occurrence and development of PCOS is unclear.The serum levels of anti-Müllerian hormone(AMH)in women with PCOS are 2 to 3 times higher than women without PCOS.High concentrations of AMH are one of the important causes of anovulation in women with PCOS.A clinical investigation suggested that AGEs had a positive relation with AMH levels.Therefore,the regulation mechanism of AGEs to AMH play an important part in exploring the initiation and progression of PCOS.This paper investigates the effects of AGEs on the expression of AMH in ovary granulosa cells and increasing the risk of PCOS in model rats,and explore its possible mechanism.Method: 1)AGEs were used at different concentrations(0,25,50,100,200 μ g/m L)and different times(0,3,6,12,24,48h)to treat cultured ovarian granulosa cells(KGN cell line)in vitro.The expression of AMH and steroidogenic factor(SF1)were detected by Western blot and q RT-PCR.The proliferation and apoptosis of KGN cell lines were detected by MTT and Hoechst.2)The expression of key molecules such as AMH,SF1 and PI3 K,was intervened with si RNA or inhibitor in KGN cells,cell proliferation and apoptosis were detected by Western blot and TUNEL kit.3)40 female Sprague-Dawley(SD)rats were feed with normal diets and high-AGEs diets for 12 weeks.In the last 3 weeks,letrozole(1mg/Kg·d)were given to rats orally.The change of the estrous and ovarian weight ratio,and the effects of ovarian tissue structure of rats were observed by HE staining.QRT-PCR,Western blot,immunohistochemistry and immunofluorescence were used to detect the expression of AMH,SF1 and phosphorylated PI3 K in ovaries.Results: 1)In vitro cell experiments,AGEs increased the expression of AMH and SF1 in a dose-and time-dependent manner(P<0.05).Compared with the control group,the expression of PCNA,a proliferation-related protein,was down-regulated,while the ratio of apoptosis-related protein Bax/Bcl-2 was increased in AGEs group(P<0.05).Hoechst staining showed a significant increase of KGN cells apoptotic.2)After transient transfection of AMH significantly reversed AGE-induced granulosa cell apoptosis.The expression of AMH decreased after expression of SF1 was repressed.After PI3 K inhibitor treatment,the expression of SF1 and AMH were up-regulated,the expression of PCNA was up-regulated,and the ratio of Bax/Bcl-2was decreased.5)Compared with the control group,the estrous cycle dysfunction,ovarian polycystic changes,increased ovarian cortical fibrosis,increased expression of AMH,SF1 and phosphorylation of PI3 K and Akt in AGEs group and letrozole group,while the ratio of Bax/Bcl-2 increased and the expression of PCNA decreased in the AGEs group and letrozole group.Conclusions: 1)AGEs contribute to PCOS of rat by up-regulating AMH in ovarian granulosa cells,promoting the apoptosis and inhibit proliferation.2)SF1may be an important target of AMH expression mediated by AGEs,and its mechanism is related to the phosphorylation of PI3K/Akt signaling pathway.