The Effect Of PGE2 On Apoptosis Of Rat Ovarian Granulosa Cells Induced By MEHP And Its Mechanism

Objective:To investigate the effect of DEHP's metabolite product MEHP on the apoptosis of rat ovarian granulosa cells.To explore the role of PGE2 in mehp-induced apoptosis of rat ovarian granulosa cells and its mechanism,so as to provide scientific basis for the prevention of dehp-induced female reproductive toxicity and the protection of female reproductive health.Methods:Use DMEM-F12 medium + 10% fetal bovine serum cultivate primary culture of rat ovarian granulosa cells,by immunofluorescence method to tested ovarian granulosa cells of rats for purity,CCK 8 method to detect cell survival,according to the result of preliminary experiment set the experimental group and MEHP infected doses : Blank control group,solvent control group(0.25 ‰ DMSO),200 ?M MEHP group,250 ?M MEHP group,300 ?M MEHP group and 350 ?M MEHP group.The apoptosis rate was detected by flow cytometry.The protein expression levels of apoptosis-related proteins(BAX,BCL-2,Caspase 3,Caspase 7),PGE2 receptors proteins(PTGER 2 and PTGER 4)and downstream genes of PGE2(G?s?cAMP?HAS2?TNFAIP6)were detected by Western Blot.MRNA expression levels of apoptotic genes(BAX,BCL-2),PGE2 receptor genes(PTGER 2 and PTGER 4)and downstream genes of PGE2(G?s,cAMP,HAS2,TNFAIP6)were detected by Real-time PCR.PGE2 hormone levels were detected by ELISA.To investigate the effects of MEHP exposure on PGE2 hormone and apoptosis in rat ovarian granulosa cells.Exogenous PGE2 was used to supplement PGE2 hormone in primary cultured rat ovarian granulosa cells,the role and mechanism of PGE2 in the apoptosis of rat ovarian granulosa cells were analyzed.The experiment was divided into three groups: Blank control group,350?M MEHPpoisoning group,10?M PGE2 group,and 350?M MEHP+10?M PGE2 group.The mRNA expression levels of apoptotic genes(BAX,BCL-2),PGE2 receptor genes(PTGER 2 and PTGER 4)and downstream genes of PGE2(G?s,cAMP,HAS2,TNFAIP6)were detected by Real-time PCR.IBM SPSS24.0 was used for statistical analysis.The measurement data of normal distribution were presented ±s,one-way anova was used for comparison between groups,and s-n-k test was used for pair-wise comparison between groups.The test level was ? = 0.05.Results:1.After being exposed to 0,16,32,64,125,250,500 and 1000 ?M MEHP for 48 h,the cell survival rate of the rat ovarian granulosa cells gradually decreased.The cell survival rate of the 500 and 1000 ?M MEHP groups was significantly lower than that of the control group,and the difference was statistically significant(P< 0.05).The IC50 of MEHP on rat ovarian granulosa cells was 373.3?M.2.The apoptosis rate of ovarian granulosa cells in 350?M MEHP group was significantly higher than that in control group(P< 0.05).3.With the increase of MEHP infected dose,ovarian granulosa cells of rats PGE2 hormone expression level gradually decline,statistically significant difference(P< 0.05),and 300?M MEHP rat ovarian granulosa cells PGE2 hormones were significantly lower than the control group and 200,250 ?M MEHP,350 ?M MEHP rat ovarian granulosa cells PGE2 hormones were significantly lower than the rest of the group(P< 0.05).4.The BAX/ BCL-2 ratio of ovarian granulosa cells in the 300?M MEHP group was significantly higher than that in the control group(P< 0.05).The mRNA expression of BAX in the 350?M MEHP group was significantly higher than that in the control group,the DMSO group and the 200?M MEHP group(P< 0.05).The mRNA expression levels of BCL-2 in ovarian granulosa cells in 250,300 and 350?M MEHP were significantly lower than those in control group and DMSO group(P<0.05).The expression level of Cle-Caspase 3 protein in rat ovarian granulosa cells of MEHP infection group was significantly higher than that of control group(P<0.05).The expression levels of Cle-Caspase 7 in 250,300 and 350?M MEHP groups were significantly higher than those in the control group,the DMSO group and the 200 ?M MEHP group(P<0.05).5.The expression levels of PTGER 4 protein in ovarian granulosa cells of rats in the 300 and 350 ?M MEHP poisoning groups were significantly lower than those in the control group and the DMSO group(P<0.05).The mRNA expression level of PTGER 2 was significantly lower in the mehp-infected group than in the control group.The mRNA expression level of PTGER 2 in the 350 ?M MEHP was significantly lower than that in the other groups(P<0.05).The mRNA expression level of PTGER4 in rat ovarian granulosa cells in MEHP infection group was lower than that in control group and DMSO group,and the differences were statistically significant(P<0.05).6.The HAS2 protein expression levels in 250,300 and 350 ?M MEHP groups were significantly lower than those in the control group,the DMSO group and the 200?M MEHP group(P<0.05).The TNFAIP6 protein expression level of rats' ovarian granulosa cells in 350 ?M MEHP group was significantly lower than that in the control group,the DMSO group,the 200 ?M MEHP group and the 250 ?M MEHP group(P< 0.05).The expression level of G?s mRNA at 350?M MEHP group was significantly lower than that of the control group,the DMSO group and the 200?M MEHP group(P<0.05).The cAMP mRNA expression levels of rat ovarian granulosa cells were significantly lower in 250,300 and 350?M MEHP than in the control group(P< 0.05).The mRNA expression levels of HAS2 in ovarian granulosa cells in each dose group were significantly lower than those in the control group and the DMSO group(P<0.05).The mRNA expression levels of TNFAIP6 mRNA in ovarian granulosa cells of rats in the 300 and 350 ?M MEHP group were significantly lower than those in the control group,the DMSO group and the 200?M MEHPgroup(P<0.05).7.The mrna expression of BAX mRNA in ovarian granulosa cells of rats in 350 ?M MEHPgroup was significantly higher than that in control group and PGE2+MEHP group(P<0.05).The mRNA expression level of BCL-2 in ovarian granulosa cells of 350 ?M MEHP was significantly lower than that of control group and PGE2+MEHP group(P<0.05).The expression of BCL-2 mRNA in PGE2 group was significantly higher than that in control group(P<0.05).8.The mRNA expression levels of PTGER 2 and PTGER 4 genes in ovarian granulosa cells of the MEHP group were significantly lower than those in the control group(P<0.05).The mRNA expression levels of PTGER 2 and PTGER 4 genes in rat ovarian granulosa cells in the PGE2 group were significantly higher than those in the control group(P<0.05).The mRNA expression levels of PTGER 2 and PTGER 4 genes in ovarian granulosa cells of rats in the PGE2+MEHP group were significantly higher than those in the MEHP group,and mRNA expression levels of PTGER 2 were significantly lower than those in the PGE2 group(P<0.05).9.Compared with the control group,the mRNA expression levels of G?s,cAMP,HAS2 and TNFAIP6 genes in ovarian granulosa cells of the MEHP group were significantly reduced(P< 0.05).The mRNA expression levels of G?s,cAMP,HAS2 and TNFAIP6 genes in the PGE2 group were significantly higher than those in the control group(P< 0.05).The mRNA expression levels of G?s,cAMP,HAS2 and TNFAIP6 genes in the PGE2+MEHP group were significantly higher than those in the MEHP group(P< 0.05),and the mRNA expression levels of HAS2 and G?s genes were significantly lower than those in the PGE2 group(P<0.05).Conclusion:1.MEHP exposure can reduce the survival rate of rat ovarian granulosa cells and promote the apoptosis of ovarian granulosa cells.2.MEHP exposure can affect the expression levels of apoptosis-related genes in rat ovarian granulosa cells,up-regulate the expression levels of cle-caspase 3 and cle-caspase 7 proteins,up-regulate the expression levels of BAX mRNA,and down-regulate the expression levels of BCL-2 protein and BCL-2 mRNA.3.MEHP exposure inhibited the secretion of PGE2 hormone in rat ovarian granulosa cells,down-regulated the mRNA and protein expression levels of PGE2 receptor gene(PTGER 2,PTGER 4),and decreased the mRNA and protein expression levels of downstream genes G?s,cAMP,HAS2,and TNFAIP6.4.When exogenous PGE2 was used to increase the PGE2 hormone level in the MEHP infected group,the expression levels of PGE2 receptor and PGE2 downstream genes increased.The expression of apoptosis-related gene BCL-2 mRNA increased,while the expression of BAX mRNA decreased.It suggested that PGE2 could inhibit mehp-induced apoptosis of ovarian granulosa cells.5.MEHP may promote the apoptosis of ovarian granulosa cells by down-regulating the secretion of PGE2 hormone.Exogenous PGE2 inhibited mehp-induced ovarian granulosa cell apoptosis by upregulating BCL-2 and down-regulating BAX.