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[摘要]
【目的】观察调任通督针法对多囊卵巢综合征 (PCOS) 大鼠的治疗作用及机制。 【方法】将雌性SD大鼠随机分为正常组、模型组、二甲双胍组、针刺组和针刺+Nigericin[NOD样受体含pyrin结构域蛋白3 (NLRP3) 激活剂]组,每组10只。除正常组,其他各组大鼠采用皮下注射脱氢表雄酮 (DHEA) 法构建PCOS模型。干预后,采用苏木素-伊红 (HE) 染色法观察卵巢和子宫结构形态,酶联免疫吸附法 (ELISA) 检测血清总睾酮 (T) 、促黄体生成素 (LH) 、性激素结合蛋白 (SHBG) 、促卵泡激素(FSH) 、空腹胰岛素 (FINS) 水平,比色法检测空腹血糖 (FPG) 并计算胰岛素抵抗指数 (HOMA-IR) 和游离睾酮 (FAI) ,采用实时定量聚合酶链反应 (RT-qPCR) 法检测卵巢组织胰岛素受体底物1(IRS-1) 、NLRP3、凋亡相关斑点样蛋白 (ASC) 、半胱氨酸天冬氨酸蛋白酶 (caspase) -1、白细胞介素 (IL) -1β和IL-18 mRNA表达,采用蛋白免疫印迹 (Western Blot) 法检测卵巢组织IRS-1、NLRP3、ASC、caspase -1前体 (pro-caspase-1) 、裂解型caspase-1 (cleaved caspase-1) 、IL-1β和IL-18蛋白表达。【结果】(1) 与正常组比较,模型组黄体数量和子宫内膜厚度显著降低,囊性卵泡数量显著增加 (P<0.05) ;与模型组比较,二甲双胍组和针刺组黄体数量和子宫内膜厚度显著增加,囊性卵泡数量显著降低 (P<0.05) 。 (2) 与正常组比较,模型组大鼠FSH、LH、T、FAI、FINS、FPG、HOMA-IR水平均显著升高,而SHGB水平显著降低 (P<0.05) ;与模型组比较,二甲双胍组和针刺组大鼠血清FSH、LH、T、FAI、FINS、FPG、HOMA-IR水平均显著降低,而SHGB水平显著升高 (P<0.05) 。 (3) 与正常组比较,模型组卵巢组织中IRS-1、NLRP3、ASC、caspase-1、IL-1β和IL-18 mRNA和蛋白表达水平均显著降低 (P<0.05) ;与模型组比较,二甲双胍组和针刺组卵巢组织中IRS-1、NLRP3、ASC、caspase-1、IL-1β和IL-18 mRNA和蛋白表达水平均显著升高 (P<0.05) 。 (4) 调任通督针对PCOS大鼠卵巢功能、子宫内膜容受性、性激素分泌、IR和NLRP3信号通路相关上述指标的影响均能够被Nigericin消除。 【结论】调任通督针法能够改善PCOS大鼠卵巢形态和功能,增加子宫内膜厚度,调节性激素代谢紊乱和胰岛素抵抗,其机制可能与抑制NLRP3炎症小体信号传导通路有关。
[Key word]
[Abstract]
Objective To observe the therapeutic effect and mechanism of regulating conception vessel and unblocking governor vessel acupuncture in rats with polycystic ovary syndrome(PCOS) . Methods Female SD rats were randomly divided into normal group,model group,metformin group,acupuncture group and acupuncture+Nigericin [NOD-like receptor pyrin domain-containing protein 3 (NLRP3) activator] group,with 10 rats in each group. Except for the normal group, the PCOS model was constructed by subcutaneous injection of dehydroepiandrosterone (DHEA) in rats of all groups. After the intervention,the structural morphology of ovary and uterus was observed by hematoxylin-eosin (HE) staining, the levels of serum total testosterone (T),luteinizing hormone (LH),sex hormone-binding protein (SHBG),follicle-stimulating hormone (FSH) and fasting insulin (FINS) were detected by enzyme-linked immunosorbent assay (ELISA),the level of free plasma glucose(FPG) was detected by colorimetry,and the insulin resistance index (HOMA-IR) and free testosterone (FAI) were calculated,the mRNA expressions of insulin receptor substrate 1 (IRS-1),NLRP3,apoptosis-associated speck-like protein (ASC),caspase-1,interleukin (IL) -1β and IL-18 in ovarian tissues were detected by using real-time fluorescence quantitative PCR (RT-qPCR) . And protein immunoblotting (Western Blot) was used to detect protein expressions of IRS-1,NLRP3,ASC,caspase-1 precursor (pro-caspase-1),cleaved caspase-1,IL-1β and IL-18 in ovarian tissues.Results (1) Compared with the normal group,the number of corpus luteum and endometrial thickness were significantly decreased,and the number of cystic follicles was significantly increased in the model group (P<0.05);compared with the model group,the number of corpus luteum and endometrial thickness were significantly increased,and the number of cystic follicles was significantly decreased in the metformin group and the acupuncture group(P<0.05). (2) Compared with the normal group,the levels of FSH,LH,T,FAI,FINS,FPG,and HOMA-IR in rats in the model group were significantly increased,while the level of SHGB was significantly decreased (P<0.05) ;compared with the model group,the levels of serum FSH, LH, T, FAI, FINS, FPG, and HOMA-IR were all significantly decreased in the metformin group and the acupuncture group,while SHGB level was significantly increased (P<0.05) . (3) Compared with the normal group,the mRNA and protein expression levels of IRS-1,NLRP3,ASC,caspase-1,IL-1β and IL-18 in the ovarian tissues were significantly reduced in the model group (P<0.05);compared with the model group,the mRNA and protein expression levels of IRS-1,NLRP3,ASC,caspase-1,IL-1β,and IL-18 in the ovarian tissues were significantly reduced in the metformin group and the acupuncture group(P<0.05) . (4) The effects of regulating conception vessel and unblocking governor vessel acupuncture on ovarian function, endometrial receptivity, sex hormone secretion, IR and NLRP3 signaling pathway-related above indexes in PCOS rats were eliminated by Nigericin. Conclusion Regulating conception vessel and unblocking governor vessel acupuncture can improve ovarian morphology and function, increase endometrial thickness,regulate sex hormone metabolism disorders and insulin resistance in PCOS rats,and its mechanism may be related to the inhibition of NLRP3 inflammasome signaling pathway.
[中图分类号]
R285.5
[基金项目]
广东省中医药局科研项目 (编号:20211323);国家中医药管理局全国名老中医药专家传承工作室建设项目 (国中医药人教函〔2022〕75号)