SF-1 Mediates The Pharmacological Serum Of Siwu Mixture To Interfere With The Activity And Expression Of Cyp19a1 In Rat Ovarian Granulosa Cells After Cisplatin Injury

Objective:Ovary is one of the major reproductive organs in women.During the development of follicles,granulocytes and follicular cells are synthesized and regulated.Granulocyte and follicular cell proliferation or dysfunction can lead to increased follicular atresia,thus accelerating the arrival of menopause,that is,shorten the reproductive period of women,leading to premature ovarian failure.In this study,cisplatin(CDDP)was used as a model of ovarian granulosa cells to establish a chemotherapeutic model of ovarian premature senility by cell experiments.To observe the effect of four substances on the secretion of E2 from granulosa cells and the expression of Cyp19al gene(P<0.05).The effect of four substances on the expression of TGF-?3-Smad3-SF-1 in the pathogenesis of ovarian premature senescence induced by cisplatin was demonstrated,and the mechanism of action was observed.On the treatment of premature ovarian failure to provide experimental basis.Method:1.The expression of Cyp 19al in cisplatin was regulated by pharmacokinetic serumOvarian granulosa cells were the main cells secreted by ovarian estrogen synthesis.The level of E2 in the supernatant was determined by radioimmunoassay.The changes of E2 levels were observed after the intervention of cisplatin and pharmacological serum.Cypl9al is a rate-limiting enzyme for estrogen production.In this study,the expression levels of Cyp 19al in granulosa cells were observed by Western Blot and RT-PCR from protein and mRNA levels to understand the mechanism of Shenwu Mixture in the secretion of E2 in granulosa cells.2.The effect of the Shenwu Mixture pharmacological serum on the activation of Cyp 19al gene promoter by SF-1Smad3 interacts with SF-1 to facilitate the regulation of Cyp 19al expression by combining SF-1 with the P? promoter of Cypl9al.Using chromatin immunoprecipitation(ChIP)experiments,The Cyp19al P? fragment was prepared by binding the SF-1 antibody to the endogenous SF-1 protein of the granulosa cells,and the content of Cypl9al P? was detected by PCR gel electrophoresis after ultrasonography.In the immunoprecipitation test(CoIP),SF-1 antibody was used as the immunoprecipitated antibody and the Smad3 antibody was used as the immunoblot antibody to observe the changes of Smad3 level by SF-1 specific precipitation.3.The effect of Siwu Mixture on the transcriptional initiation of TGF-?3-regulated target gene Cypl9alSince Smad3 is TGF-?3 downstream protein,TGF-?3 regulates the expression of Cyp19al by activating the Smad protein in combination with SF-1.Therefore,we added exogenous TGF-?3 to stimulate the action of cisplatin granulosa cells in the experiment.First,we observed the level of E2 secretion in each group,and clarified the effect of the four drugs on the secretion of E2 in granulosa cells after TGF-?3 regulation.Second,the expression of Cypl9al in cisplatin was observed.The expression of Cyp19al in TGF-?3 was detected by the pharmacological serum of Siwu mixture.Result:1.The level of E2 in supernatant of granulosa cells was decreased by radioimmunoassay,and the level of E2 in cisplatin group was decreased.The level of E2 in Siwu mixture was significantly higher than that of cisplatin group.Westem blot and RT-PCR showed that the expression of Cyp19al protein in the Siwu groups was higher than that in cisplatin group.2.he expression level of Cyp19al PII in cisplatin group was the lowest,and the expression level of Cypl9al P? was increased after the intervention of four substances.The results of CoIP showed that the expression level of Smad3 of Siwu groups was significantly increased compared with that of cisplatin group.3.After adding exogenous TGF-?3 to granule cells,compared with control group,The level of E2 and the expression of Cyp19al was decreased,the level of E2 secretion and the expression of Cypl9al were increased after the Siwu pharmacological serum.Conclution:The pharmacological serum of the Siwu mixture can enhance the secretion of E2 in granulosa cells by affecting the expression of Cyp19al gene,it also can promote the transcription of Cypl9al gene promoter by SF-1 to regulate Cyp19al expression.The activity and expression of Cypl9al may be related to TGF-?3-Smad3-SF-1 signal transduction pathway.