基于α7nAchR介导的胆碱能抗炎通路探讨电针干预早发性卵巢功能不全的机制

Premature ovarian insufficiency (POI) is threatens to the reproductive health of women with childbearing age seriously, and the pathogenesis is still unclear. Cellular immune dysfunction is one of the main pathological mechanisms of POI. Acetylcholine plays an important role in immune regulation, and its mediated cholinergic anti-inflammatory pathway (CAP) is involved in neuro-endocrine-immune network, regulating various diseases in the body. Acupuncture can improve ovarian endocrine function, promote ovulation, and regulate CAP pathway to inhibit pro-inflammatory factors. At present, there are few reports on whether acupuncture regulates immune POI and ovarian function through CAP signaling pathway. The previous study of applicant have found that the molecules related to the CAP pathway, such as α7nAChR and AChE, were abnormally expressed in granulosa cells of subclinical POI patients. It is suggested that CAP pathway may be involved in the pathogenesis of POI. In this project, we aim to explore the immune phenotype and CAP pathway in POI patients from systemic and ovarian local microenvironment perspective, to demonstrate the mechanism of CAP pathway involved in the regulation of estrogen synthesis in granulosa cells through vitro cell experiments; to establish POI mouse model and clarify the target of electro-acupuncture on Guanyuan(CV4) and Sanyinjiao(SP6) to regulate the neuro-endocrine-immune network, further to improve ovarian function, and provide a theoretical basis for clinical prevention and treatment of POI.

早发性卵巢功能不全（POI）严重威胁育龄期女性的生殖健康，其致病机制尚不明确，其中细胞免疫异常是POI的重要病因。乙酰胆碱在免疫调节中发挥重要作用，其介导的胆碱能抗炎通路（CAP）参与多种疾病的神经-内分泌-免疫网络调节，但在POI中相关研究尚少。针灸可改善卵巢内分泌功能，促进排卵，同时能够调节CAP通路抑制促炎性因子分泌，目前有关针灸通过CAP信号通路调控免疫性POI研究鲜有报道。申请人前期研究发现CAP通路相关分子α7nAChR、AChE在亚临床期POI患者颗粒细胞中表达异常，提示CAP通路可能参与POI发病。本项目拟从全身性及卵巢局部微环境明确POI患者免疫异常表型、CAP通路异常，通过体外细胞实验分析颗粒细胞中CAP通路调控雌激素合成的机制，进一步通过免疫性POI小鼠模型阐明电针关元、三阴交穴调控CAP通路改善卵巢功能的靶点和作用途径，为临床POI预防治疗提供理论依据。

早发性卵巢功能不全（premature ovarian insufficiency，POI）是指发生在女性40岁之前发生卵巢功能衰竭，发病率1%-3%，是不孕的重要原因，其发病机制较为复杂，目前半数以上患者病因尚未明确，其中4%-30%患者可由自身免疫性因素引起，自身免疫失调诱导POI发生的机制尚不明确，对其确诊指标及治疗方法也缺乏依据。研究表明针灸能够影响排卵，与女性生殖的关系密不可分，但目前有关针灸调控免疫性POI的机制研究尚少。胆碱能抗炎通路（cholinergic anti-inflammatory pathway，CAP）是近年发现的在神经免疫网络中发挥炎症调节作用的重要抗炎通路，介导多种免疫性疾病的发生，其中α7nAChR是CAP通路上重要的烟碱型乙酰胆碱受体，本课题探讨电针是否能够通过CAP信号通路抑制POI炎症、改善卵巢功能。本课题通过筛选并验证生化异常期POI患者与卵巢功能正常者卵巢颗粒细胞及卵泡液中CAP通路重要分子的表达差异，进一步体外实验探索阐明α7nAChR介导的胆碱能抗炎通路调控颗粒细胞雌激素合成的机制，通过诱导自身免疫性POI小鼠模型，采用电针干预模型小鼠，探讨电针治疗POI的可能机制。结果发现bPOI患者卵巢颗粒细胞及卵泡液中胆碱能抗炎通路重要因子α7nAChR、AChE表达异常，POI发病可能与胆碱能抗炎通路抑制有关；体外实验及动物实验结果提示电针能够改善小鼠卵巢功能，能够激活α7nAChR介导的CAP信号通路，促进雌二醇合成关键酶CYP19A1蛋白表达，提高颗粒细胞雌二醇合成能力。本课题从临床意义着手，以临床指南为研究依据，以临床研究为基础，借助自身免疫性POI动物模型，揭示电针对防治POI的作用机理，为POI免疫学病因及机制研究提供线索，为电针在临床防治POI的应用提供思路及依据。