The Study On The Mechanism Of Asperosaponin ? Regulate PR,PSG1 And Apoptosis In Trophoblast Cell

BackgroundSpontaneous abortion(SA)refers to the miscarriage that occurs within20 weeks during clinical pregnancy(18 weeks after fertilization)which is a common reproductive disorder in obstetrics and gynecology.For a long time,spontaneous abortion has been studied by many academic professors,especially as the country fully opens two-child policy implementation,the incidence of spontaneous abortion is increasing,but the pathogenesis is not completely clear.Trophoblast cells play an important role in early pregnancy,there are a lot of research shows that the dysfunction of trophoblast cell can lead to spontaneous abortion,such as insufficient invasiveness,the increased apoptosis and proliferation deficiency of trophoblast cells can cause spontaneous abortion.Therefore,trophoblast cells are usually used to study study mechanism of spontaneous abortion.Progesterone is a routine examination target in pregnancy.Progesterone is the first choice for clinical treatment of incipient abortion,while without PR,progesterone function could not work.Therefore,PR plays an important role in pregnancy.Pregnancy specific protein(PSG)is produced by syncytial trophoblasts during pregnancy.It is a protein family.By Gene-Chip screening of early stage of our team,PSG1 is one of the most obvious difference genes between the SA patients and the early pregnant women.Shou-Tai pill is a common prescription for tonifying kidney and preventing miscarriage in clinic,and the teasel is a representative medicine of them.Our previous study has showed that Asperosaponin ? can improve PR expression through the Notch pathway in decidual cell.Therefore,based on the previous research of our team,this paper adopts the ATCC cell library species for human early pregnancy trophoblastic cell line HTR8/Svneo cell function experiments,mainly studies whether Asperosaponin ? can affect the trophoblast cells which is a model of spontaneous abortion and apoptosis after intervention of RU486 or not,as well as studies whether PR or PSG1 has a relationship with apoptosis in trophoblast cells,and then explained the mechanism of spontaneous abortion.ObjectiveStudy the expression differences of PR,PSG1,p-Akt,Bax,Bcl-2 m RNA and protein between villus tissues of patients with spontaneous abortion and villus tissues of normal pregnancy patients.To establish a spontaneous abortion and apoptosis model on HTR8/Snveo trophoblast cells through inducing RU486.To discuss the effect of Asperosaponin ? on PR,PSG1 expression and apoptosis on the spontaneous and apoptosis model trophoblast cells.To discuss the relationship among PR,PSG1,Akt pathway and apoptosis and clarify the role of the Asperosaponin ? on PR and PSG1 expression.Methods1.In vivo study: To use clinical screening and validate the differences in gene and protein expression of PR,PSG1 and apoptosis in villus were tested by qRT-PCR and Western Blot.2.In vitro study: Using RU486 to induce to establish the abortion and apoptosis cell model.Different concentrations of RU486 were used to induce trophoblastic cells at different time.Apoptosis was detected by CCK8proliferation-apoptosis test,flow cytometry and Hoechst33258 fluorescence staining.The expression of PR,PSG1,p-Akt and apoptotic protein was detected by Western Blot to validate establishment of the cell model.The effect of asperosaponin ? on expression of PSG1,PR and apoptosis in trophoblast cells of SA model cell.Based on trophoblasts which induced by RU486 model of spontaneous abortion and apoptosis,the CCK8,Annexin V/PI staining,Hoechst33258 staining were used to detect the effect of asperosaponin ? on apoptosis of trophoblast cells,and the expression of PR,PSG1,p-Akt and apoptotic protein was detected by Western Blot.PR and PSG1 regulate the mechanism of apoptosis through the PI3K/Akt singal pathway.By transfection of PR si RNA and PSG1 si RNA,the transfection efficiency was detected by qRT-PCR,and the expression of PR,PSG1 and apoptosis genes were observed.To observe the changes of PR,PSG1 and apoptosis proteins in trophoblast cells by Wwstern Blot.PI3K/Akt inhibitor was used to intervene trophoblast cells,and observe the expression of PR,PSG1 and apoptosis protein in trophoblast by Wwstern Blot.The targeting effect of asperosaponin ? on PR and PSG1 protein.Using asperosaponin ? to work on transfected PR or PSG1 si RNA trophoblast cells respectively.To observe the changes of PR,PSG1 and apoptosis proteins in trophoblast cells by Wwstern Blot.Results1.In vivo study.The expression of PR and PSG1 in the villi tissue of SA patients was decreased.In the Akt pathway,the expression of Akt and Bcl-2were downregulated while the expression of Bax was up-regulated.qRT-PCR detected the expression of PR,PSG1 and Bcl-2 m RNA in villi of SA patients were significantly lower than those in normal early pregnancy,while Bax m RNA increased,the difference was statistically significant(P < 0.001).The expression of PR,PSG1,p-Akt and Bcl-2 in villi tissue of SA patients was significantly lower than those of normal early pregnant women,while Bax protein was increased significantly by Western Blot,the difference was statistically significant(P<0.05).2.In vitro study: Using RU486 to induce to establish the abortion and apoptosis cell model.By CCK8,flow cytometry,Hoechst33258 fluorescence staining and Western Blot methods were used to treat HTR8/Svneo trophoblast cells with different concentrations of RU486 at different time,to establish spontaneous abortion and apoptosis models.The results showed that the higher concentration of RU486 or the longer time of RU486 acted on trophoblast cell was,the lower of cell growth activity was,and early and total apoptosis rate was higher,the PR,PSG1 as well as Bcl-2 had decreased expression while Bax increased expression,showed that the PI3K/Akt pathway was inhibited,the differences were statistically significan(P < 0.05).Therefore,RU486 can reduce the expression of PR and PSG1 in the trophoblastic cells,inhibit the Akt pathway and promote apoptosis,and the model establishment is successful.Finally,the best concentration of RU486 to induce the model cell was 50?mol/L,CCK8 and Annexin V/PI double staining,flow cytometry and Hoechst33258 staining showed the best intervention time to induce the model was 24 h,and the best time for WB detection was 8h.The effect of asperosaponin ? on expression of PSG1,PR and apoptosis in trophoblast cells of SA model cell.By CCK8,Annexin V/PI double staining flow cytometry,Hoechst33258 fluorescence staining and Western Blot,observeing asperosaponin ? effects on RU486 induced abortion and apoptosis model of trophoblast cell,all the results showed that the ASA VI can increase the growth of trophoblast cells,reduce early apoptosis rate,the differences were statistically significant(P<0.05).Asperosaponin ? can increase pregnancy protein PR,PSG1 expression and activate PI3K/Akt pathway to up-regulate expression of the anti apoptotic protein Bcl-2,down-regulate the expression of Bax.PR and PSG1 regulate the mechanism of apoptosis through the PI3K/Akt singal pathway.After transfection of PR si RNA in trophoblast cells,the expression of Bcl-2 and p-Akt protein was decreased,while expression of Bax was increased.The difference was statistically significant(P<0.05),however,PSG1 protein was not statistically significant(P > 0.05),indicating that PR did not directly regulate PSG1.After transfection of PSG1 si RNA,the expression of PR,Bcl-2 and p-Akt was decreased,the expression of Bax was increased.The difference was statistically significant(P<0.05),indicating that PSG1 could regulate the expression of PR,and then regulate the Bcl-2 and Bax by Akt pathway.After inhibiting Akt,the expression of Bcl-2 was decreased,and the expression of Bax was increased.The difference was statistically significant(P < 0.05),indicating that Akt could regulate the apoptosis function of trophoblast cells.The targeting effect of asperosaponin ? on PR and PSG1 protein: After transfection PR,PSG1 si RNA to trophoblast cells,using asperosaponin ? to treat can enhance the expression of PR and PSG1 in target of trophoblast cells.Conclusion1.In SA patients villi tissues,the expression of PR,PSG1 were decreased,while the expression of apoptosis was increased.2.RU486 can induce HTR8/Svneo cells to establish spontaneous abortion and apoptosis cell model in vitro.The more conconcentration of RU486 and the longer time of RU486 on HTR8/Svneo cells,the lower cell viability and more apoptosis was.The cell is concentration-dependent and time-dependent to the RU486.The best concentration of RU486 to induce the model cell was 50?mol/L.3.PSG1 can regulate PR expression,and PR can activate Akt pathway to regulate Bcl-2 and Bax expression,affecting apoptosis.It is suggested that the expression of PR and PSG1 have an important effect on apoptosis in SA.4.Asperosaponin ? has some progesterone-like effect,which can obviously promote cell proliferation and reduce apoptosis of the spontaneous abortion model trophoblast cells.Asperosaponin ? can increase PR and PSG1 expression and activate Akt signaling pathway to up-regulate the Bcl-2 expression and reduce Bax expression,preventing and treating miscarriage.