基于Wnt/β-catenin通路介导的上皮间质转化探讨电针治疗宫腔粘连大鼠子宫内膜纤维化的作用机制

Mechanism of electroacupuncture in treating uterine endometrial fibrosis in intrauterine adhesions rats based on Wnt/β-catenin pathway-mediated epithelial-mesenchymal transition

目的:观察电针对宫腔粘连(IUA)大鼠子宫内膜Wnt/β-连环蛋白(β-catenin)信号通路与上皮间质转化(EMT)相关蛋白的影响,探讨其修复IUA子宫内膜的可能机制。方法:雌性SD大鼠随机分为空白组、模型组、电针组、ICG-001组,每组10只。采用机械搔刮联合脂多糖感染双重损伤法制备IUA模型。电针组针刺“关元”,电针双侧“足三里”“三阴交”,20 min/次,1次/d;ICG-001组腹腔注射β-catenin抑制剂ICG-001(5 mg/kg),1次/2 d;以上干预均连续进行3个动情周期。每组5只大鼠干预后取材,HE染色法观察IUA大鼠子宫内膜形态、子宫内膜厚度及腺体数目变化,Masson染色法观察子宫内膜组织纤维化程度,免疫组织化学法检测子宫内膜组织转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、结缔组织生长因子(CTGF)、Ⅰ型胶原蛋白(Col-Ⅰ)、糖原合酶激酶3β(GSK-3β)、β-catenin、E-钙粘蛋白(E-cadherin)、N-钙粘蛋白(N-cadherin)及波形蛋白(Vimentin)的阳性表达,Western blot法检测子宫内膜组织中GSK-3β、β-catenin、E-cadherin、N-cadherin及Vimentin蛋白相对表达量;每组剩余5只大鼠,干预后与雄鼠合笼,记录各组大鼠子宫胚胎着床数目。结果:HE染色示模型组子宫内膜组织部分坏死或缺失,电针组有所恢复,且较ICG-001组更好。与空白组相比,模型组大鼠子宫内膜组织腺体数目与子宫内膜厚度,子宫内膜组织中E-cadherin和GSK-3β蛋白表达与阳性表达降低(P<0.0001,P<0.001,P<0.01),子宫内膜纤维化面积比值,子宫内膜组织中TGF-β1、α-SMA、FN、CTGF、Col-Ⅰ阳性表达,N-cadherin、Vimentin及β-catenin蛋白表达与阳性表达升高(P<0.0001,P<0.001,P<0.01),胚胎着床数目减少(P<0.0001);与模型组相比,电针组与ICG-001组大鼠子宫内膜组织腺体数目与子宫内膜厚度,子宫内膜组织E-cadherin与GSK-3β蛋白表达及阳性表达升高(P<0.001,P<0.05,P<0.01),子宫内膜纤维化面积比值,子宫内膜组织TGF-β1、α-SMA、FN、CTGF、Col-Ⅰ阳性表达,N-cadherin、Vimentin、β-catenin蛋白表达与阳性表达降低(P<0.001,P<0.01,P<0.05),胚胎着床数目增多(P<0.001);与电针组相比,ICG-001组以上指标差异均无统计学意义。结论:电针可能通过抑制Wnt/β-catenin信号通路逆转EMT进程,降低内膜组织纤维化程度,从而促进IUA子宫内膜修复。

Objective To observe the effect of electroacupuncture(EA)on the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition(EMT)-related proteins in rats with intrauterine adhesions(IUA),so as to explore the possible mechanisms of EA in repairing endometrial damage in IUA.Methods Female SD rats were randomly divided into blank,model,EA,and ICG-001 groups,with 10 rats in each group.The IUA model was established by using mechanical scraping combined with lipopolysaccharide infection for double injury.In the EA group,“Guanyuan”(CV4)was needled and EA(2 Hz/15 Hz,1—2 mA)was applied to“Zusanli”(ST36)and“Sanyinjiao”(SP6)on both sides.In the ICG-001 group,ICG-001(5 mg/kg),the inhibitor ofβ-catenin was intraperitoneally injected.After intervention,samples were taken from 5 rats in each group,and uterine endometrium morphology,endometrial thickness,and gland counts were observed using HE staining.Masson staining was used to assess the degree of fibrosis in the endometrial tissue.Immunohistochemistry was used to detect the positive expression of transforming growth factorβ1(TGF-β1),α-smooth muscle actin(α-SMA),fibronectin(FN),connective tissue growth factor(CTGF),type I collagen(Col-Ⅰ),glycogen synthase kinase-3β(GSK-3β),β-catenin,E-cadherin,N-cadherin,and Vimentin in the endometrial tissue.Western blot was used to detect the relative expression of GSK-3β,β-catenin,E-cadherin,N-cadherin,and Vimentin proteins in the endometrial tissue.Another 5 rats from each group were placed in cages with male rats after intervention to record the number of embryo implantations.Results Necrosis and loss of endometrial tissue in the model group observed after HE staining were alleviated in the EA group,better than those in the ICG-001 group.Compared with the blank group,the numbers of glands and endometrial thickness in the uterine endometrial tissue,relative expression and positive expression of E-cadherin and GSK-3βproteins in the uterine endometrial tissue,and embryo implantation numbers were reduced(P<0.0001,P<0.001,P<0.01)in the model group,while fibrosis area ratio in the uterine endometrial tissue,TGF-β1,α-SMA,FN,CTGF,Col-Ⅰpositive expressions,N-cadherin,Vimentin,andβ-catenin proteins expression and positive expression were increased(P<0.0001,P<0.001,P<0.01).Compared with the model group,the number of glands and endometrial thickness,E-cadherin and GSK-3βproteins expression and positive expression,and embryo implantation numbers were increased(P<0.001,P<0.05,P<0.01)in the EA and ICG-001 groups,while the fibrosis area ratio in the uterine endometrial tissue,TGF-β1,α-SMA,FN,CTGF,Col-Ⅰpositive expression,and N-cadherin,Vimentin,andβ-catenin proteins expression and positive expression were decreased(P<0.001,P<0.01,P<0.05).Compared with the EA group,the differences of the above-mentioned indicators in the ICG-001 group were not statistically significant.Conclusion EA may reverse the EMT process and reduce the degree of fibrosis in endometrial tissue by inhibiting the Wnt/β-catenin signaling pathway,thereby promoting the repair of endometrial damage in IUA.