Research Of SRAGE On The Regulation Of AKT Phosphorylation And SP1 Expression In PCOS Granulosa Cells

Polycystic ovary syndrome(PCOS)is a systemic endocrine metabolic disorder,which affects almost 5-10% of women in reproductive age.The main pathophysiological changes of PCOS are insulin resistance,hyperandrogenemia and follicular dysplasia.PCOS can lead to infertility,and endocrine disorders.And the incidence of its long-term complications such as cardiovascular disease,diabetes and hypertension are increasing.Therefore,PCOS seriously affects the quality of life and physical health of women.The pathogenesis of PCOS is unclear,previous researches suggested that inflammation,genetic factors,oxidative stress,environmental factors and social psychological factors are involved in the occurrence and development of PCOS.Soluble receptor for advanced glycation end-products(sRAGE)is the endogenous secretory form of RAGE.sRAGE can inhibit multiple signal transduction pathways induced by AGE-RAGE axis by competing with RAGE.Vascular endothelial growth factor(VEGF)can act directly on oocyte maturation and early embryo development.VEGF can also regulate follicular development by regulating the formation of microvascular in theca cell layer and increasing the vascular permeability.Therefore VEGF is closely related to the occurrence and development of PCOS.The level of serum VEGF in patients with PCOS was significantly higher than that in non PCOS patients.The previous studies of our group have found that sRAGE,as a protective factor,can prevent the occurrence and development of PCOS by down regulating the expression of VEGF in granular cells.The expression of VEGF protein and mRNA decreased with the increasing of sRAGE concentration.However,the specific mechanism of how sRAGE down regulate VEGF expression remains unclear.Further research is needed to discover the mechanism.PI3K/AKT pathway is a classical signaling pathway involved in the regulation of cell metabolism.AKT phosphorylation is the center of this pathway.The PI3K/AKT pathway activates a variety of cytokines,including SP transcription factors.SP1 is the main transcription factor that regulates the expression of VEGF,and its binding with VEGF promoter plays a dominant role in the regulation of VEGF transcription.In U937 derived foam cells,CD147 can up regulate the expression of VEGF through PI3K/AKT pathway.In human ovarian epithelial cancer cells,resistin can enhance DNA binding activity of SP1 and VEGF promoter through the PI3K/AKT pathway,and then increase the expression of VEGF.Therefore,whether sRAGE can regulate the expression of VEGF by regulating the expression of transcription factor SP1 through the AKT pathway in granulosa cells of PCOS? There were no available research currently.The aim of this study was to investigate the effect of sRAGE on AKT phosphorylation and SP1 expression in PCOS ovarian granulosa cells,and to lay the foundation for further study of sRAGE mechanism.Objective1.To investigate the effect of sRAGE on AKT phosphorylation in PCOS ovarian granulosa cells.2.To investigate the effect of sRAGE on transcription factor SP1 expression in PCOS ovarian granulosa cells.Material and Methods1.Follicular fluid from 10 PCOS patients whom were treated with in vitro fertilization(IVF)in the reproductive medicine center of the First Affiliated Hospital of Zhengzhou University were collected.The granulosa cells were collected and cultured in vitro for 48 hours,and then treated with sRAGE at concentrations of 0 ?g/ml,0.6 ?g/ml and 1.2 ?g/ml respectively.The expression of AKT,pAKT and SP1 were detected after adding sRAGE 12 h and 24 h respectively.The protein expression of AKT,pAKT and SP1 was detected by Western blotting.The mRNA expression of SP1 was detected by real-time quantitative polymerase chain reaction(RT-qPCR).To investigate the effect of sRAGE on AKT phosphorylation and transcription factor SP1 expression in PCOS ovarian granulosa cells.Results1.After treated with sRAGE at concentrations of 0 ?g/ml,0.6 ?g/ml and 1.2 ?g/ml for 12 and 24 hours,there were no significant difference in the expression of AKT protein among three groups(P>0.05).While the expression of pAKT protein were significantly decreased with the increasing of sRAGE concentration,the differences were statistically significant(P<0.05).2.After treated with sRAGE at concentrations of 0 ?g/ml,0.6 ?g/ml and 1.2 ?g/ml for 12 hours,the expression of SP1 protein in 1.2 ?g/ml group was significantly lower than that of 0 ?g/ml group(P<0.05).After treatment for 24 hours,the expression of SP1 protein significantly decreased with the increasing of sRAGE concentration,the differences were statistically significant(P<0.05).3.After treated with sRAGE at concentrations of 0 ?g/ml,0.6 ?g/ml and 1.2 ?g/ml for 12 hours,the expression of SP1 mRNA in the three groups decreased with the increase of sRAGE concentration,but the difference was not statistically significant(P>0.05).After treatment for 24 hours,the expression level of SP1 mRNA significantly decreased with the increase of sRAGE concentration,the differences were statistically significant(P<0.05).Conclusions1.sRAGE can decrease the level of AKT phosphorylation in PCOS granulosa cells with dose and time dependence.2.sRAGE can down regulate the expression of transcription factors SP1 in PCOS granulosa cells with dose and time dependence.