| Controlled ovary hyperstimulation(COH)is an important part of In vitro fertilization and embryo transfer(IVF-ET).Studies have confirmed that COH may damage the structures and functions of oocyte chromosomes,and affect the expression of genes in embryos.Thus it reduces the quality and implantation potential of embryos and leads to significantly low rates of implantation and pregnancy.Chinese medicine has a remarkable effect in IVF-ET treatment.Our early studies confirmed that herbs replenishing qi and blood and reinforcing liver and kidney can improve the embryo implantation rate and pregnancy rate in IVF-ET.This study focuses on the effect of Jinghou Zengzhi Recipe(JHZZR)on apoptosis in ovarian cumulus cells(CCs)of COH rats.There is a close relationship between CCs and oocytes in human beings.CCs directly affect the development and maturation of oocytes and the quality of embryos.Whereas oocytes can secrete oocyte secreted factors(OSFs)to regulate the growth and differentiation of CCs around them.Oocytes and CCs both play very important roles in influencing each other in the process of oocyte maturation.Growth differentiation factor 9(GDF9)is an important member of transforming growth factor β(TGF β)family.As an OSF,it is one of the important factors taking part in the growth and development progress of follicles.Thus the expression of GDF9 can forecast the quality of oocytes and embryos.Bim(Bcl-2 interacting mediator of cell death)is a member of BH3-only family,which belongs to Bcl-2 family.It expresses in many kinds of organizations including reproductive cells in the body.Bim protein plays an important role in starting up and regulating the progress of apoptosis.This paper mainly discusses the effect of JHZZR,which has the function of replenishing qi and blood,on apoptosis in ovarian CCs of COH rats and the mechanism of Yiqixue Recipe on regulating GDF9 and Bim secretion.Meanwhile,this paper will try to verify the possible signaling pathways JHZZR affects the secretion of GDF9.So as to provide molecular theoretical basis for its wide clinical use,hoping to find out a way to improve the quality and development potential of oocytes,and to further improve the embryo implantation rate and pregnancy rate in IVF-ET.ObjectiveTo observe the effect of JHZZR,which has the function of replenishing qi and blood,on the secretion of GDF9 and Bim in ovaries of COH rats.To study the mechanism of Yiqixue Recipe on apoptosis in Ovarian CCs,and to verify its possible signaling pathways.Methods1 Animal studyDevelop COH rat model.Use JHZZR to intervene.Natural ovulation rats were used as blank control,and COH rats as positive control.TUNEL and qPCR were used to study the effect of different doses of JHZZR on the apoptosis in ovarian granulosa cells and the expression of Bim gene in the ovary of COH rats.2 Cell study2.1 Prepare different concentration of serum containing JHZZR.Use JHZZR serum to intervene ovarian granulosa cells of COH rats cultured in vitro.qPCR was used to detect the expression of Bim gene in granulosa cells.To select an optimal acting concentration and an optimal acting time of JHZZR serum for the follow-up study.2.2 Develop COH rat model.Obtain ovarian COCs and CCs for in vitro culture.Use JHZZR serum and GDF9 receptor blockers to intervene.qPCR and Westernblot were used to detect the expression of GDF9 gene and protein and Bim gene in COCs and CCs.2.3 Develop COH rat model.Obtain ovarian COCs and CCs for in vitro culture.Use JHZZR serum and inhibitors of PI3K,PKA,p38MAPK,SMAD and NF-κB signaling pathway to intervene.qPCR and Westernblot were used to detect the expression of GDF9 gene and protein and Bim gene in COCs.Results1 The effect of JHZZR on the apoptosis in ovarian granulosa cells and the expression of Bim gene in the ovary of COH rats1.1 JHZZR can reduce the apoptosis rate of ovarian granulosa cells of COH ratsThe apoptosis rates of ovarian granulosa cells in JHZZR low-dose group(LD group),JHZZR medium-dose group(MD group)and JHZZR high-dose group(HD group)were significantly lower than that in positive control group(PC group)(P<0.05),and HD group have no significant difference comparing with the blank control group(BC group)(P>0.05).This shows that high,medium and low doses of JHZZR can reduce the apoptosis rate of ovarian granulosa cells of COH rats,and high dose of JHZZR can reduce the apoptosis rate nearly to the natural ovulation rats’.The apoptosis rates of ovarian granulosa cells of HD group,MD group and LD group decreased gradually,which suggests that the apoptosis rate of ovarian granulosa cells of COH rats is negatively correlated with the dose of JHZZR.1.2 JHZZR can reduce the expression of Bim gene in the ovary of COH ratsThe expression of Bim gene in LD group,MD group and HD group were significantly higher than that in BC group(P<0.05),but were lower than that in PC group(P<0.05).These show that low,medium and high doses of JHZZR can reduce the expression of Bim gene in the ovary of COH rats.The expression of Bim gene in LD group,MD group and HD group decreased gradually,but there were no statistical differences(P>0.05).These show that all doses of JHZZR have nearly the same effect.2 JHZZR serum can suppress the apoptosis of ovarian CCs of COH rats by regulating the expression of GDF9 and Bim through p38MAPK signaling pathway and NF-κB signaling pathway2.1 The optimal acting concentration and acting time of JHZZR serum to inhibit the expression of Bim gene in ovarian CCs of COH rats24 hours group:The expression of Bim gene in LD group,MD group and HD group were significantly lower than BC group(P<0.05).These show that low,medium and high doses of JHZZR serum can significantly reduce the expression of Bim gene.The expression of Bim gene in LD group,MD group and HD group decreased gradually,and there were statistical differences among them(P<0.05).These indicate that the effect of JHZZR serum on reducing the expression of Bim gene is positively correlated with its concentration.The high dose of JHZZR serum has the optimal effect-48 hours group:The expression of Bim gene in LD group,MD group and HD group decreased gradually.HD group was significantly lower than MD group,LD group and BC group(P<0.05).But there were no statistical differences among MD group,LD group and BC group(P>0.05).These indicate that only high dose of JHZZR serum can maintain the expression of Bim gene at the low level after 48 hours’ intervention,but medium and low dose of JHZZR serum had no satisfactory effect.Thus we can infer that the optimal acting concentration is high dose and the optimal acting time is 24 hours for JHZZR serum to suppress the expression of Bim gene in ovarian CCs of COH rats.2.2 The effect of JHZZR serum on the expression of GDF9 and’ Bim in the ovarian COCs and CCs of COH rats2.2.1 JHZZR serum can increase the expression of GDF9 gene and protein in the ovarian COCs and CCs of COH ratsThe expression of GDF9 gene in COCs-JHZZR serum group(COC-JS group)was higher than that in COCs-blank serum group(COC-BS group)and COCs-JHZZR serum+GDF9 receptor blockers group(COC-JS+GDF9rb group)(P<0.05).The expression of GDF9 gene in CCs-JHZZR serum group(CCs-JS group)was higher than that in CCs-blank serum group(CCs-BS group)and CCs-JHZZR serum+GDF9 receptor blockers group(CCs-JS+GDF9rb group)(P<0.05).These indicate that JHZZR serum can increase the expression of GDF9 gene in the ovarian COCs and CCs of COH rats.The expression of GDF9 protein in COC-JS group was higher than that in COC-BS group(P<0.05),and was also higher than that in COC-JS+GDF9rb group,but there was no statistical difference(P>0.05).The expression of GDF9 protein in CCs-JS group was higher than that in CCs-BS group and CCs-JS+GDF9rb group(P<0.05).These indicate that JHZZR serum can increase the expression of GDF9 protein in the ovarian COCs and CCs of COH rats.The expression of GDF9 gene in COC-JS group was significantly higher than that in CCs-JS group(P<0.05).This indicates that COCs are better for the expression of GDF9 gene than CCs which with no oocytes.2.2.2 JHZZR serum can reduce the expression of Bim gene in the ovarian COCs and CCs of COH ratsThe expression of Bim gene in COC-JS group and COC-JS+GDF9rb group were lower than that in COC-BS group(P<0.05).The expression of Bim gene in CC-JS group and CC-JS+GDF9rb group were lower than that in CC-BS group(P<0.05).These indicate that JHZZR serum can reduce the expression of Bim gene in the ovarian COCs and CCs of COH rats.The expression of Bim gene in COC-JS group was lower than that in COC-JS+GDF9rb group,but there was no statistical difference(P>0.05).The expression of Bim gene in CC-JS group was significantly lower than that in CC-JS+GDF9rb group(P<0.05).These indicate that affected by GDF9 receptor blockers,the expression of GDF9 gene was reduced.So it was failed to maintain the expression of Bim gene at a low level.It is verified that GDF9 have the ability to maintain the expression of Bim gene at a low level in CCs.2.3 JHZZR serum regulates the expression of GDF9 and Bim in ovarian COCs of COH rats through p38MAPK signaling pathway and NF-κB signaling pathwayThe expression of GDF9 gene and protein in JS+PI3K inhibitors group(JS+iPI3K group),JS+PKA inhibitors group(JS+iPKA group),JS+SMAD inhibitors group(JS+iSMAD group)were higher than that in blank serum group(BS group)(P<0.05),but had no statistical difference compared with JHZZR serum group(JS group)(P>0.05).The expression of GDF9 gene and protein in JS+p38MAPK inhibitors group(JS+ip38MAPK group)and JS+NF-κB inhibitors group(JS+iNF-κB group)were lower than that in JS group(P<0.05),but had no statistical difference comparing with BS group(P>0.05).These indicate that JHZZR serum can increase the expression of GDF9 gene and protein in ovarian COCs of COH rats through p38MAPK signaling pathway and NF-κB signaling pathway.The expression of Bim gene JS+iPI3K group,JS+iPKA group,JS+iSMAD group were lower than that in BS group(P<0.05),but had no statistical difference comparing with JS group(P>0.05).The expression of Bim gene in JS+ip38MAPK group and JS+iNF-κB group were higher than that in JS group(P<0.05),but had no statistical difference comparing with BS group(P>0.05).These indicate that JHZZR serum can suppress the expression of Bim gene in ovarian COCs of COH rats through p38MAPK signaling pathway and NF-κB signaling pathway.Conclusion①Jinghou Zengzhi Recipe,which has the function of replenishing qi and blood,can facilitate the secretion of GDF9 in ovarian COCs of COH rats.Then GDF9 affects the CCs around through paracrine function,maintains the apoptosis of CCs at a low level to improve the function of COCs and the quality of ovum.②Jinghou Zengzhi Recipe can suppress the expression of Bim in ovarian CCs and COCs of COH rats through many ways.One of them is to improve the secretion of GDF9 to maintain the expression of Bim at a low level and suppress the apoptosis of ovarian CCs.③ Jinghou Zengzhi Recipe regulates the expression of GDF9 and Bim in ovarian COCs and CCs of COH rats through p38MAPK signaling pathway and NF-κB signaling pathway. |
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