从Keap1/Nrf2/ARE抗氧化通路探讨补肾活血汤抑制免疫性POF卵巢颗粒细胞凋亡的分子机制

The incidence of premature ovarian failure (POF) is increased year by year and POF could seriously damage women's health. Traditional Chinese Medicine has shown a certain advantage in the treatment of POF. Our research group have found that Bushenhuoxue Decoction has reliable treatment efficacy on POF. However, the pharmacological mechanisms of Bushenhuoxue Decoction have been not studied systematically. According to the latest domestic and international reports, immune system disorder is an important cause of POF. Apoptosis of ovarian granulosa cells is closely related to the occurrence of immune POF. Keap1/Nrf2/ARE anti-oxidation signaling pathway plays a key role in regulating apoptosis of ovarian granulosa cells. According to the results of previous experiments, we have found that Bushenhuoxue Decoction can not only improve immune POF, but also inhibit apoptosis of ovarian granulosa cell and activate Keap1/Nrf2/ARE signaling pathway. Therefore, our research group puts forward the scientific hypothesis that Bushenhuoxue Decoction may inhibit the apoptosis of ovarian granulosa cells by activating the Keap1/Nrf2/ARE antioxidant signaling pathway, thereby exerting therapeutic effects on immune POF. Based on the establishment of immune POF mice model and ovarian granulocyte cell apoptosis model, the molecular pharmacological mechanisms of Bushenhuoxue Decoction in treating immune POF by using molecular biotechnologies (such as gene konck out) via the Keap1/Nrf2/ARE signaling pathway in vivo and in vitro.

卵巢早衰（POF）发病率逐年上升，严重影响女性健康，中医药在治疗POF方面显示出一定优势。本课题组前期证实补肾活血汤对POF有确切临床疗效，但其药理机制尚未系统阐明。国内外最新研究发现免疫系统紊乱是POF的重要诱因，卵巢颗粒细胞凋亡与免疫性POF发生密切相关，Keap1/Nrf2/ARE抗氧化信号通路在调控卵巢颗粒细胞凋亡方面发挥关键作用。本项目通过预实验发现补肾活血汤不仅能改善免疫性POF，而且能抑制卵巢颗粒细胞凋亡以及激活Keap1/Nrf2/ARE信号通路。在此基础上，我们提出了“补肾活血汤可能通过激活Keap1/Nrf2/ARE抗氧化信号通路，抑制卵巢颗粒细胞凋亡，从而对免疫性POF发挥疗效”的科学假说。通过建立免疫性POF小鼠模型和卵巢颗粒细胞凋亡模型，运用基因敲除等分子生物学技术，以Keap1/Nrf2/ARE信号通路为视角，体内外探讨补肾活血汤治疗免疫性POF的分子药理机制。

卵巢早衰（premature ovarian failure，POF）是一种常见的难治性妇科疾病，多种病因均可诱导卵泡破坏或耗竭，从而导致卵巢功能不全。POF发病率逐年上升，严重影响妇女身心健康，目前临床上缺乏安全有效的治疗药物。近年来，传统中医药在改善POF方面得到了广泛关注，显示出一定优势。本课题组研究的补肾活血汤具有补肾宁心、活血调经的功效，前期临床研究证实补肾活血汤对POF有确切疗效，但其药理机制尚未系统阐明。POF发病机制复杂，有新近报道表明患者免疫系统紊乱是POF的重要诱因，卵巢颗粒细胞凋亡与免疫性POF发生密切相关，Keap1-Nrf2-ARE抗氧化信号通路在调控卵巢颗粒细胞凋亡方面发挥关键作用。因此，本项目以Keap1-Nrf2-ARE抗氧化信号通路为角度来阐释补肾活血汤的分子药理机制。首先，利用超高效液相色谱和质谱联用技术（UPLC-ESI-Q-TOF-MS/MS），研究了补肾活血汤的物质基础和药物质量控制。其次，通过构建ZP3多肽诱导的免疫性POF小鼠模型，研究发现补肾活血汤传统水提液可以改善卵巢早衰病理状态，改善雌激素水平和机体氧化应激状态，激活卵巢组织中Keap1-Nrf2-ARE抗氧化信号通路。再者，构建了Nrf2信号缺失的人卵巢颗粒氧化损伤细胞模型，研究发现补肾活血汤含药血清一定程度上可以改善细胞损伤状态，激活Keap1-Nrf2-ARE抗氧化信号通路。总之，本项目通过多种实验手段证明补肾活血汤能够激活Keap1-Nrf2-ARE抗氧化信号通路，抑制卵巢颗粒细胞凋亡，改善卵巢早衰状态，从而对免疫性POF发挥疗效。本项目为研究免疫性POF的微观病理机制提出了新观点，为阐释补肾活血汤的分子药理提供了新视野，为补肾活血汤的临床应用提供了新证据。