Based On The FOXO3a Transcription Factor, The Protective Mechanism Of Erxian Decoction On Ovarian Granulosa Cells In Rats With Cisplatin Injury Was Investigated

ObjectivePremature ovarian failure(POF)is a common clinical gynecological disease which is characterized by non-physiological menstruation pause before 40 years.POF could lead to a series of reproductive and endocrine problems,even the loss of reproductive capacity.Especially with the fast pace and increasing pressure of modern life,the incidence of POF has been increasing year by year and shows younger trend.Chemotherapy is widely used in clinic as a common method for the treatment of autoimmune diseases and tumors on account of definite therapeutic effect.But the chemotherapeutic drugs kill not only target tumor cells but also the normal cells to some extent because of their poor selection.Many reports found that Cisplatin(CDDP),a commonly used clinical chemotherapy drug,has a certain degree of damage to the development of follicles and shows ovarian toxicity.Our laboratory has established an successful animal model of cisplatin induced ovarian failure.FOX03a transcription factor is one of the members of FOXO protein family which is involved in the regulation of cell proliferation,growth,differentiation,apoptosis and senescence.In addition,FOXO3a transcription factor is one of the important downstream proteins in phosphatidylinositol-3-kinase-protein-kinase B(PI3K-AKT)signaling pathways.It could inhibit cell cycle and cell apoptosis by regulating expressing level of pro-apoptotic protein Bim and nonspecific cyclin dependent kinase(CKI)P27kipl.The prescription of Erxian Decoction is commonly used for tonifying kidney.Modern pharmacological research shows that it can obviously improve the function of gonad,endocrine system,immune system and treat osteoporosis.It is widely used in perimenopausal syndrome,climacteric syndrome,hypertension,menopausal osteoporosis,prostatitis and other clinical diseases including POF.The experimental study of Erxian Decoction are limited in morphological observation and physiological and biochemical indexes of the animal model.Nevertheless,its molecular mechanism research has been rarely reported.It has not elucidated yet that the molecular mechanism of Erxian Decoction in the treatment of chemotherapy induced POF.Especially the molecular protection mechanism on cisplatin injured ovarian granulosa cells based on FOXO3a transcription factor has not been studied.The rat ovarian granulosa cells are separated and cultured in vitro in this research.The aim is to explore the mechanism of Erxian Decoction on proliferation and apoptosis of cisplatin injured ovarian granulosa cells based on FOXO3a-P27kip1/Bim pathway and provide a detailed theoretical basis for Erxian Decoction treating POF in clinical.MethodsExtract the original generation of rat ovarian granulosa cells and utilize cisplatin to create granular cells damage model.Cells are divided into normal,progynova,high dose,middle dose and low dose of Erxian Decoction pharmacological serum groups.Erxian Decoction’s intervention effect on CDDP injured ovarian granulosa cells were observed by detecting proliferation effect by MTT assay,measuring cell cycle and apoptosis effect by flow cytometry and testing the expression of FOXO3a,P27kiPl and Bim by immunohistochemical and Western blot analysis.LY294002 were used to explore the molecular pathway of the above effect of Erxian Decoction.Results1.Proliferation effect on cisplatin injured ovarian granulosa cells of Erxian Decoction Erxian Decoction pharmacological serum has obvious promoting effect on cisplatin injured rat ovarian granulosa cells.The effect of middle dose Erxian Decoction was most significant.The proliferation proliferative activity continued to 72h,and 48h showed the most obvious effect.So we chose 48h as the pharmacological serum treating time.2.Effect of Erxian Decoction pharmacological serum on Cell cycle and apoptosis of cisplatin injured rat ovarian granulosa cells(1)Each dose of Erxian Decoction pharmacological serum could influence cisplatin injured ovarian granulosa cell cycle:Cells in S phase increased while G0/G1 phase cells decreased and the proliferation index increased.There is no difference between groups treating by different dose of Erxian Decoction.But the proliferation effect of low dose group is relatively significant.(2)Each dose of Erxian Decoction pharmacological serum could reduce the apoptosis rate of cisplatin injured ovarian granulosa cells and the result of middle dose group is more apparent.3.Effect of Erxian Decoction pharmacological serum on expression level of FOXO3a,P27kip1,Bim(1)FOXO3a expressed in both cytoplasm and endonuclear.P27kipl mainly expressed in nucleus and Bim mainly expressed in the cytoplasm.Cisplatin induced high expression of FOXO3a and positive particles in nucleus increased obviously.It led to high expression of P27kipland Bim at the same time.The expression level of FOXO3a,P27kipl and Bim in different Erxian Decoction treating groups was significantly decreased.The average optical density analysis of FOXO3a,P27kipl and Bim shows that the model group increased significantly versus control group.While a significant reduce in Progynova group and all Erxian Decoction groups versus model group.The positive particles and average optical density of FOXO3a,P27kipl and Bim increased obviously in LY294002 inhibitor added groups compared with the corresponding groups.(2)Western blot results showed that FOXO3a,P27kipl,Bim expression level were significantly increased in model group.While their relative expression level was obviously decreased in positive control group and Erxian Decoction treating groups compared with the model group.Totally there is no difference among each Erxian Decoction treating groups.ConclusionErxian Decoction plays a significant role in protecting cisplatin injured ovarian granulosa cells.The mechanism shows in the following aspects:Erxian Decoction could promote the transformation from G0 phase to S phase and increase the rate of S phase cells the proliferation index.Erxian Decoction could also reduce the apoptosis rate of cisplatin injured ovarian granulosa cells.Erxian Decoction could down-regulate the expression level of FOXO3a,cell cycle arrest protein P27kipl and pro-apoptotic protein Bim.Therefore it can promote cell proliferation and inhibit cell apoptosis.Erxian Decoction has a protective effect on cisplatin injured ovarian granulosa cells.