调经促孕方对胚胎着床障碍性不孕症小鼠子宫内膜容受性及AREG/EGFR/HIF-1α信号通路的影响

Effects of Tiaojing Cuyun Prescription on Endometrial Receptivity and AREG/EGFR/HIF-1αSignaling Pathway in Embryo Implantation Dysfunction Infertility Mice

目的观察调经促孕方对胚胎着床障碍(EID)性不孕症小鼠子宫内膜容受性及AREG/EGFR/HIF-1α信号通路的影响,探讨其改善子宫内膜容受性的可能机制。方法80只昆明小鼠于妊娠第1日随机分为正常组、模型组、黄体酮组和调经促孕方组,每组20只,黄体酮组和调经促孕方组分别予相应药物灌胃。妊娠第4日,除正常组外,其余各组小鼠皮下注射米非司酮溶液(2.3 mg/kg)建立EID性不孕症模型,正常组皮下注射等体积丙二醇。妊娠第5日,每组随机取10只小鼠,剖腹取子宫,HE染色、Masson染色观察子宫内膜组织形态,扫描电镜观察子宫内膜胞饮突形态及数量,ELISA检测子宫内膜组织双调蛋白(AREG)含量,Western blot和免疫荧光检测子宫内膜组织表皮生长因子受体(EGFR)、p-EGFR、缺氧诱导因子(HIF)-1α表达。其余小鼠灌胃至妊娠第8日取材,记录平均胚胎着床点数。结果与正常组比较,模型组小鼠平均胚胎着床点数显著减少(P<0.01),子宫内膜腺体发育不良,胶原纤维增加,血管及胞饮突数量减少,子宫内膜组织AREG含量显著降低(P<0.01),p-EGFR、HIF-1α蛋白表达显著降低(P<0.05);与模型组比较,调经促孕方组小鼠平均胚胎着床点数显著增加(P<0.05),子宫内膜胶原纤维增生减少,间质内腺体、血管及胞饮突数量均明显增加,胞饮突发育饱满、均匀,子宫内膜组织AREG含量显著升高(P<0.01),p-EGFR、HIF-1α蛋白表达显著升高(P<0.05,P<0.01),免疫荧光检测结果与Western blot一致。结论调经促孕方能促进子宫内膜腺体和血管发育,增加胞饮突数量,改善EID性不孕症小鼠子宫内膜容受性,利于胚胎黏附及植入,从而提高妊娠率,其机制可能与激活AREG/EGFR/HIF-1α信号通路,改善子宫内膜血流灌注,恢复子宫内膜相对缺氧环境有关。

Objective To observe the effects of Tiaojing Cuyun Prescription on endometrial receptivity and AREG/EGFR/HIF-1αsignaling pathway of embryo implantation dysfunction(EID)infertility mice;To explore its mechanism on improving endometrial receptivity.Methods On the first day of pregnancy,80 Kunming mice were randomly divided into control group,model group,progesterone group and Tiaojing Cuyun Prescription group,with 20 mice in each group.Progesterone group and Tiaojing Cuyun Prescription group were given corresponding drugs by gavage.On the fourth day of pregnancy,mifepristone solution was subcutaneously injected(2.3 mg/kg)to establish the EID infertility model except the normal group,while the normal group was subcutaneously injected with equal volume of propylene glycol.On the fifth day of pregnancy,10 mice were randomly selected from each group,and the endometrium morphology were observed by HE and Masson staining,the pinopodes were observed by scanning electron microscope,the content of AREG in endometrial tissue was detected by ELISA,and the expressions of EGFR,p-EGFR and HIF-1αwere detected by Western blot and immunofluorescence,the rest of the mice were gavaged until the eighth day of pregnancy,and the average number of embryo implantation points was recorded.Results Compared with the normal group,the average number of embryo implantation points of model group significantly decreased(P<0.01),and the glandular development in the endometrium was poor,collagen fibers increased,and blood vessels and pinopodes decreased,the AREG content in endometrial tissue was significantly decreased(P<0.01),and the expressions of p-EGFR and HIF-1αprotein were significantly decreased(P<0.05).Compared with the model group,the average number of embryo implantation points in Tiaojing Cuyun Prescription group significantly increased(P<0.05),the proliferation of collagen fibers in the endometrium was reduced,and the number of glands,blood vessels and pinopodes in stroma significantly increased,the development of pinopodes were full and uniform,the AREG content in endometrial tissue significantly increased(P<0.01),and the expressions of p-EGFR and HIF-1αprotein significantly increased(P<0.05,P<0.01).The immunofluorescence results were consistent with Western blot.Conclusion Tiaojing Cuyun Prescription can promote the development of endometrial glands and blood vessels,enrich the number of pinopodes,improve the endometrial receptivity of EID infertility mice,facilitates embryo adhesion and implantation,and thus increase the pregnancy rate.Its mechanism may be related to activating AREG/EGFR/HIF-1αsignaling pathway,improving endometrial perfusion,and restoring the relatively hypoxic environment of the endometrium.