The Methods Of Bushen And Shugan Improve The Endometrial Receptivity Through VEGFR-2 Regulating The MAPK Signaling Pathway

Objective:Preliminary studies have confirmed that Bushen and Shugan treatments can improve pregnancy outcomes and the endometrial receptivity in patients with IVF-ET(in vitro fertilization-embryo transfer,IVF-ET).This study aimed to explore the mechanisms of Bushen and Shugan methods improving the endometrial receptivity,to compare Bushen with Shugan in mechanismthe of the similarities and differences.Methods:1 The culture of Human endometrial micro vascular endothelial cellsHuman endometrial micro vascular endothelial cells(HEMEC)were incubated at 37? and 5%CO2 incubator with ECM containing 10%fetal bovine serum,then refreshed culture medium every other day until the culture is approximately 90%confluent to Subculture.2 Preparation of serum containing drugsThe serum containing Bushenzhuyun recipe or Xiaoyaowan pill:At the age of 20-30 years old 8 female volunteers,healthy body,regular menstruation,signing the informed consent form,took Bushen Zhuyun recipe or Xiaoyao pill orally for 4 days according to the instruction respectively.In the fourth days early morning volunteers on fasting took all days doses,sampled venous blood 5ml 1h later.venous blood at room temperature were static for 2h,after centrifugation at 3000 rpm for 10 min,the supernatants were collected,filter-sterilized with 0.22?m sterile filter and stord at-20?until ready for used.Normal serum:4 female volunteers were selected,at the age of 20-30 years old,in good health,with menstrual regularity,after fasting sampled venous blood 5ml,the method was as above.3 Groups3.1 HEMECs were treated with serum-low culture medium for 24 hours,then incubated with VEGF(40ng/ml)for different time(Oh,6h,12h,24h),Western blot and qRT-PCR analysis were performed to detect the expressions of VEGFR-2,PCNA,CyclinDl and MMP9.Endothelial cells were treated with serum-low culture medium for 24 hours,then incubated with VEGF(40ng/ml)for short different time(0,15min,30min,60min),Western blot was performed to detect the changes of MAPK signaling pathway(p-ERK,ERK,p-JNK,JNK,p-P38,P38).3.2 HEMECs were incubated with serum containing drugs(Bushen Zhuyun recipe or Xiaoyao pill)or VEGF for indicated times,Western blot and qRT-PCR analysis were performed to detect the expressions of VEGF and VEGFR-2.3.3 Aftre incubated with serum containing drugs(Bushen Zhuyun recipe or Xiaoyao pill),HEMECs were treated with VEGF or not.Grouped were as follows:control group,VEGF group,Bushen group,Bushen+VEGF group,Shugan group,Shugan+VEGF group.Western blot and qRT-PCR analysis were performed to detect the expressions of PCNA,CyclinD1,MMP9,and MAPK sinaling pathway.3.4 Aftre incubated with serum containing drugs(Bushen Zhuyun recipe or Xiaoyao pill)or not,HEMECs were treated with inhibitor(PD98059,SP600125,or SB203580),and then treated with VEGF.Grouped were as follows:control group,VEGF group,Inhibitor+VEGF group,Bushen group or Shugan group,Bushen+VEGF group or Shugan+VEGF group,Bushen+inhibitor+VEGF group or Shugan+inhibitor+VEGF group.Westem blot analysis was performed to detect the expressions of PCNA,CyclinDl,MMP9,and MAPK sinaling pathway.The VEGF,VEGFR-2,PCNA,CyclinDl and MMP9 mRNA expressions in all groups were analyzed by qRT-PCR.The expressions of VEGF,VEGFR-2,PCNA,CyclinD1,MMP9,ERK,p-ERK,JNK,p-JNK,P38 and p-P38 protein were detected by western blot.Cells proliferation was determined using CCK8 assay.Cells migration was observed by Scratch-wound assay.Endothelial cells tubule formation experiment assay observed angiogenesis.Results:1 The effects of VEGF on HEMECs proliferation,migration and angiogenesis.HEMECs were treated with VEGF for different time(Oh,6h,12h,24h),Western blot and qRT-PCR analysis were performed to detect the expressions of VEGFR-2,PCNA,CyclinDl and MMP9.The results showed that VEGFR-2,PCNA,CyclinD1 and MMP9 were increased in a time-dependent manner,reaching a maximum at 12h(P<0.05),and maintain in a high level at 24h.Cells proliferation was determined using CCK8 assay.We observed that the OD level of the group treated with VEGF for 12h was increased higher than that in the Oh,6h and 24h groups(P<0.05).Scratch-wound assay showed that VEGF promote HEMECs migration.Endothelial cell tube formation assay was performed,and we observed that VEGF induced HEMECs angiogenesis.HEMECs were treated with VEGF for short different time(Omin,15min,30min,60min),Western blot analysis was performed to detect the changes of MAPK signaling pathway.the results showed that VEGF stimulated markedly p-ERK phosphorylation on Ser residues within 15min higher than other groups(P<0.05),the expressions of p-JNK and p-P38 protein were increased within 15 min,reaching a maximum at 30min(P<0.05,compared with Omin).2 The effects of Bushen method and Shugan method on the expressions of VEGF and VEGFR-2.The results showed that compared with control group,Bushen method and Shugan method increased the the expressions of VEGF and VEGFR-2(P<0.05),also the expressions of VEGF,VEGFR-2 protein and mRNA in the Bushen and Shugan groups were higher than that in the VEGF group(P<0.05),but there were no differences between the Bushen and Shugan groups(P>0.05).3 Bushen method and Shugan method increased the expressions of PCNA,CyclinD1 and MMP9.After incubated with serum containing drugs(Bushen Zhuyun recipe or Xiaoyao pill),HEMECs were treated with VEGF or not.Western blot and qRT-PCR analysis were performed to detect the expressions of PCNA,CyclinD1 and MMP9.We observed that compared with control group,the expressions of PCNA,CyclinD1 and MMP9 protein and mRNA in VEGF,Bushen,Bushen+VEGF,Shugan,Shugan+VEGF groups were increased significantly(P<0.05).The expressions of PCNA,CyclinD1,MMP9 mRNA and protein in Bushen+VEGF group were higher than Bushen group(P<0.05),these expressions in Shugan+VEGF group were higher than that in Shugan group(P<0.05).Meanwhile,the expressions of PCNA protein and mRNA in Bushen group were higher than Shugan group(P<0.05),and the expressions of MMP9 potein and mRNA in Shugan group were higher than Bushen group(P<0.05).there were no differences in the expressions of CyclinDl protein and mRNA between the Bushen and Shugan groups(P>0.05),CCK8 assay was performed.Compared with VEGF group,the OD level in Bushen,Bushen plus VEGF,Shugan,Shugan plus VEGF groups were increased significantly(P<0.05).Scratch-wound assay and Endothelial cell tube formation assay were performed.We observed that Bushen and Shugan methods can increase the migration of endothelial cells,promote endothelial cell tube formation.Further,we examined which signaling pathway in response to Bushen and Shugan groups.Western blot analysis showed that compared with control group,the expressions of p-ERK,p-JNK and p-P38 protein in VEGF,Bushen,Bushen+VEGF,Shugan,Shugan+VEGF groups were increased significantly(P<0.05).The results indicated that Bushen and Shugan methods can activate ERK,JNK and P38 signaling pathways.4 Bushen method and Shugan method increased the expressions of PCNA,CyclinD1 and MMP9 through MAPK signaling pathway.4.1 Bushen method increased the expressions of PCNA,CyclinD1 and MMP9 through MAPK signaling pathway.Aftre incubated with serum containing drugs(Bushen Zhuyun recipe)or not,HEMECs were treated with inhibitor(PD98059,SP600125,or SB203580),and then treated with VEGF.Western blot was performed to dectect the MAPK signaling pathway.Compared with control group,the protein expressions of p-ERK,p-JNK and p-P38 in VEGF,Bushen,Bushen+VEGF groups were increased significantly(P<0.05).The results indicated that Bushen method can activate MAPK signaling pathway.Compared with the Bushen group,the protein expressions of PCNA and CyclinDl in Bushen+PD98059+VEGF group were reduced significantly(P<0.05).the protein expressions of CyclinDl and MMP9 in Bushen+SP600125+VEGF group were lower than that in Bushen group(P<0.05).the protein expressions of CyclinDl and MMP9 in Bushen+SB203580+VEGF group were lower than that in Bushen group(P<0.05).The results indicated that Bushen method promote CyclinDl expression through ERK,JNK and P38 pathways,induce PCNA expression through ERK pathway,and increase MMP9 expression through JNK and P38 pathways.4.2 Shugan method increased the expressions of PCNA,CyclinD1 and MMP9 through P38 and JNK signaling pathways.Aftre incubated with serum containing drugs(Xiaoyao pill)or not,HEMECs were treated with inhibitor(PD98059,SP600125,or SB203580),and then treated with VEGF.Western blot was performed to dectect the MAPK signaling pathway.Compared with control group,the protein expressions of p-ERK,p-JNK and p-P38 in VEGF,Shugan,Shugan+VEGF groups were increased significantly(P<0.05).The results indicated that Shugan method can activate MAPK signaling pathway.We obesreved that the protein expressions of PCNA,CyclinDl and MMP9 in Shugan+PD98059+VEGF and Shugan groups had no significant difference(P>0.05).The protein expression of CyclinDl in Shugan+SP600125+VEGF group was lower than that in Shugan group(P<0.05).compared with the Shugan group,the protein expressions of PCNA,CyclinDl and MMP9 in Shugan+SB203580+VEGF group were reduced signifcantly(P<0.05).The results indicated that Shugan method promote PCNA and MMP9 expressions through P38 pathway,induce CyclinDl expression through JNK and P38 pathways.Conclusions:1 VEGF and VEGFR-2 are the initial factors located upstream of the MAPK signaling pathway,which can activate MAPK(ERK,JNK,P38)signaling pathway,can promote expressions of PCNA,CyclinD1 and MMP9,thus to promote angiogenesis and improve endometrial receptivity.2 The methods of Bushen and Shugan can promote expressions of VEGF and VEGFR-2.3 The methods of Bushen and Shugan can increase the expressions of PCNA,CyclinDl and MMP9,which are conductive to the improvement of endothelial cell proliferation and migration.But Bushen method improving expression of PCNA in HEMECs is better than Shugan method;Shugan method improving expression of MMP9 in HEMECs is better than Bushen method.So Bushen method in promoting cell proliferation is better than Shugan method,while Shugan method in promoting cell migration is superior to Bushen method.4 Bushen and Shugan methods can activate MAPK signaling pathway to promoting endometrial receptivity.The mechanism may induce VEGF with its receptor VEGFR-2 to form complex,then promote ERK,JNK and P38,phosphorylation,thus to regulate signal transduction.5 Bushen and Shugan methods can target multiple gene to promote HEMECs proliferation and migration,ultimately promote angiogenesis.Bushen method can promote PCNA expression by activating ERK signaling pathway,activate ERK,JNK,P38 signaling pathways to promote the expression of CyclinDl,activate JNK and P38 signaling pathways to promote the expression of MMP9.Shugan method can promote PCNA,MMP9 expressions by activating P38 signaling pathway and activate JNK,P38 signaling pathways to promote the expression of CyclinD1.