Regulations And Mechanisms Of Electroacupuncture On Endometrial Angiogenesis During Peri-implantation Period In Controlled Ovarian Hyperstimulation Rats

Part?Regulations of electroacupuncture on endometrial angiogenic factors during peri-implantation period in controlled ovarian hyperstimulation ratsObjective:To investigate the effects of electroacupuncture on embryo implantation and endometrial angiogenesis during the peri-implantation period in controlled ovarian hyperstimulation(COH)rats.Method:120 female rats were randomly balanced assigned to 4 groups:normal(group N),model(group M),electroacupuncture(group EA)and electroacupuncture pretreatment(group PEA).To establish the COH model,female rats in groups M,EA and PEA were intraperitoneally injected with pregnant mare serum gonadotropin(PMSG).46 hours later,human chorionic gonadotropin(HCG)was injected,and the females were then mated with male rats.Rats in group N received an equal volume of phosphate-buffered saline(PBS).successful mating was confirmed by the presence of sperm in vaginal smears obtained the next day,which was designated the first day of gestation(D1).Beginning at the day of PMSG injection(for group EA)or three days before injection(for group PEA),electroacupuncture treatment was provided until D3.Rats from each group were sacrificed on D4,D6 or D8.Immunohistochemistry was used to measure the endometrial microvascular density(MVD),while immunofluorescence,real-time PCR and western blotting were used to investigate the mRNA or protein expression of vascular endothelial growth factor-A(VEGF-A)?angiopoietin-1(Ang-1)?fibroblast growth factor-2(FGF-2)?placental growth factor(PlGF)and Delta like canonical Notch ligand-1(DLL-1).Flow cytometry was used to assess the percentage of DBA~+uNK cells in endometrial lymphocytes.Results:1)The embryo number in group M was significantly lower than that in groups N and PEA,but had no significant difference with that in group EA.2)Compared with that in group M,the MVDs in groups N and PEA were significantly increased.However,the MVD in group EA was higher than that in group M on D6,but no significant difference was found.3)Compared with that in group M,the relative protein expression levels of VEGF-A and Ang-1 in groups N and PEA were significantly higher on D4,D6.But the expression level of FGF-2 had no significantly difference among the four groups,except between groups PEA and M on D6.However,the significant difference was not found between groups M and EA.4)VEGF-A mRNA expression levels were increased significantly in groups N and PEA compared with that in group M during peri-implantation,but the FGF-2 expression level was no significantly different among the four groups,except between groups PEA and M on D6.The mRNA expression level of Ang-1 was significantly lower in group M compared with that in group N on D6,group EA on D4 and group PEA on D4 and D6.5)In endometrial lymphocytes,the percentage of DBA+uNK cells in groups N and PEA was higher than that in group M on D6 and D8.But the percentage in group EA was only higher than that in group M on D8.6)No significant differences in PlGF and DLL-1 protein expression levels were found among the four groups on D4.However,the PlGF and DLL-1 protein expression levels in groups N and PEA were higher than that in group M on D6 and D8.7)No significant differences in PlGF and DLL-1 mRNA expression levels were found among the four groups on D4 and D6.However,the PlGF and DLL-1 mRNA expression levels in groups N and PEA were higher than that in group M on D8.Conclusions:electroacupuncture can facilitate embryo implantation and endometrial angiogenesis in COH rats.Part ? Regulations of electroacupuncture on the VEGFR2/PI3K/AKT and VEGFR2/ERK signalling pathways during the peri-implantation period in controlled ovarian hyperstimulation ratsObjective: To investigate the regulations of electroacupuncture on signalling pathways of endometrial angiogenesis during the peri-implantation period in controlled ovarian hyperstimulation(COH)rats.Method: 40 female rats were randomly balanced assigned to 4 groups: normal(group N),model(group M),electroacupuncture(group EA)and electroacupuncture pretreatment(group PEA).To establish the COH model,female rats in groups M,EA and PEA were intraperitoneally injected with pregnant mare serum gonadotropin(PMSG).46 hours later,human chorionic gonadotropin(HC G)was injected,and the females were then mated with male rats.Rats in group N received an equal volume of phosphate-buffered saline(PBS).successful mating was confirmed by the presence of sperm in vaginal smears obtained the next day,which was designated the first d ay of gestation(D1).Beginning at the day of PMSG injection(for group EA)or three days before injection(for group PEA),electroacupuncture treatment was provided until D3.Furthermore,another 30 female rats who received the same treatment as the rats in group PEA were injected with si-VEGFR2 or si-NC into uterine lumen.Rats from each group were sacrificed on D4,D6 or D8.Immunohistochemistry was used to measure the endometrial microvascular density(MVD).Western blotting was used to investigate the protein expression levels of VEGFR2?PI3K?p-AKT and p-ERK.Results: The endometrial protein expression levels of VEGFR2?PI3K?p-AKT?p-ERK in group M were significantly lower than those in groups N and PEA.The endometrial protein expression levels of PI3K?p-AKT?p-ERK in the uterus injected with si-VEGFR2 were also remarkably lower than those in the uterus injected with si-NC.The embryo number and MVD in the uterus injected with si-VEGFR2 were remarkably fewer than those in the uterus injected with si-NC.Conclusions: Electroacupuncture pretreatment facilitates embryo implantation and endometrial angiogenesis through the VEGFR2 mediated PI3K/AKT and ERK signalling pathways.