Objectives: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects.
Methods: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations.
Results: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group.
Conclusions: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.
目的: 本研究旨在探讨电针联合骨髓间充质干细胞（BMSCs）移植对宫腔粘连（IUA）大鼠子宫内膜的影响，以及两者联合治疗修复子宫内膜的可能机制。方法: 成年未交配雌性SD大鼠随机分为空白组、模型组、细胞组和联合组，每组10只。采用机械搔刮联合脂多糖感染的双重损伤法建立大鼠IUA模型。造模成功后第1、3、7天，模型组大鼠尾静脉注射磷酸盐缓冲液，细胞组仅尾静脉注射BMSCs悬液进行BMSCs移植，联合组大鼠移植与细胞组等量的BMSCs并进行电针治疗，每日针刺“关元”、电针双侧“足三里”和“三阴交”20 min，持续3个动情周期。每组5只大鼠在干预结束后取子宫组织，采用HE染色检测大鼠子宫内膜厚度以及腺体数目；采用Masson染色检测子宫内膜纤维化面积；采用免疫组织化学法检测血管生成标志物血管内皮生长因子（VEGF），增殖细胞核抗原（PCNA），雌激素受体（ER）的表达；Western blot法检测容受性相关分子同源框蛋白A10（HoxA10）、白血病抑制因子（LIF）的蛋白表达量。每组剩余的5只大鼠在干预后合笼并通过胚胎着床数来评估子宫功能的恢复情况。结果: 与空白组相比，模型组大鼠的子宫内膜变薄（P<0.001）、腺体数量减少（P<0.001），子宫内膜纤维化面积增加（P<0.001），子宫内膜组织中的VEGF、PCNA、ER阳性表达，子宫内膜容受性相关分子HoxA10和LIF的表达，子宫损伤侧的胚胎着床数均降低（P<0.001）。与模型组相比，联合组以上指标均逆转（P<0.001，P<0.01）；细胞组的子宫内膜增厚（P<0.001）、子宫内膜纤维化面积减少（P<0.001）。与细胞组相比，联合组子宫内膜厚度增加（P<0.01）、腺体数量增多（P<0.05），子宫内膜纤维化面积减少（P<0.05），子宫内膜中的VEGF、PCNA和ER阳性表达，子宫内膜HoxA10和LIF的表达，子宫损伤侧的胚胎着床数量均显著升高（P<0.001，P<0.05，P<0.01），治疗效果较细胞组更好。结论: 电针联合BMSCs协同修复受损子宫内膜，促进子宫内膜形态的改善，减轻纤维化，促进血管生成和基质细胞增殖，提高子宫内膜的容受性，并最终有利于胚胎的着床。.
Keywords: Angiogenesis; Bone marrow mesenchymal stem cells; Combined treatment; Electroacupuncture; Endometrial receptivity; Intrauterine adhesion; Matrix cell proliferation.