The Effect Of CaMKⅡ On Apoptosis Of PCOS Ovarian Granulosa Cells

Background:Polycystic ovarian syndrome(PCOS)is a common female endocrine and metabolic disorder that seriously affects the reproductive health of women in their reproductive years.The typical pathological features of patients with PCOS are high androgen levels,polycystic ovarian changes and anovulation or sporadic ovulation.Studies have shown that abnormal ovulation in PCOS patients is associated with high levels of androgens,elevated levels of reactive oxygen species(ROS)in granulosa cells of the luminal follicle resulting in oxidative stress(OS)and apoptosis.Calmodulin-dependent protein kinases Ⅱ(CaMK Ⅱ)is a protein phosphokinase regulated by Ca2+ and ROS levels and regulates the phosphorylation of many proteins.In cells whose phosphorylation level activation is interdependent with ROS levels,elevated ROS levels have a promotive effect on intracellular CaMKⅡ phosphorylation,while continued activation of CaMKⅡ phosphorylation causes upregulation of cellular ROS levels,oxidative stress and apoptosis.Objective:The aim of this study was to investigate the mechanism of CaMKⅡ in the regulation of androgen-induced oxidative stress and apoptosis in PCOS granulosa cells and to verify the alleviating effect of CaMKⅡ phosphorylation inhibitor KN93 on polycystic phenotype and granulosa cell apoptosis in PCOS mice,so as to provide valuable therapeutic targets and theoretical basis for the alleviation and treatment of PCOS.Method:The study was conducted to investigate the interaction between androgens,CaMKⅡ,ROS and apoptosis in granulosa cells of PCOS patients,human ovarian cell line-KGN cells and DHEA-induced PCOS mice on the basis of clinical samples,cells and animal models by immunofluorescence,protein immunoblotting,ELISA,TUNEL and immunohistochemistry.Results:(1)Relative to normal controls,PCOS patients had significantly higher androgen levels in follicular fluid,upregulated granulosa cell ROS levels,increased apoptosis,and significantly higher CaMK Ⅱ phosphorylation levels.It is suggested that granulosa cell apoptosis in PCOS patients may be related to CaMK Ⅱ phosphorylation levels.(2)Androgen-induced rise in calcium levels,upregulation of CaMKⅡ phosphorylation levels,rise in ROS levels,oxidative stress and mitochondrial pathway apoptosis in KGN cells;inhibition of CaMKⅡ phosphorylation by KN93 could alleviate DHT-induced rise in ROS levels,oxidative stress levels and apoptosis.It suggests that androgens may have induced CaMKⅡautophosphorylation activation further promoting upregulation of ROS levels,oxidative stress and apoptosis by promoting intracellular calcium ion levels.(3)Treatment of PCOS mice with KN93 resulted in the alleviation of granulosa cell apoptosis and the restoration of ovarian function in PCOS mice.It indicates that CaMKII is an important target downstream of hyperandrogenism,and targeted inhibition of CaMKII can play a role in alleviating PCOS.Conclusion:CaMKII is an indirect target of hyperandrogenism in the granulosa cells of PCOS ovaries.Hyperandrogenism induces CaMKII phosphorylation through upregulation of calcium ion levels in granulosa cells,which in turn triggers a series of cascade reactions such as upregulation of ROS levels and ultimately promotes apoptosis.Specific inhibition of CaMKII phosphorylation alleviated the apoptosis of ovarian granulosa cells in PCOS mice and restored the ovarian function of PCOS mice to some extent.This study provides a valuable therapeutic target and theoretical basis for the remission and treatment of PCOS.