基于PI3K/Akt/mTOR通路探讨缺氧诱导细胞自噬稳态失衡在子宫腺肌病中的作用机制及加味芍药甘草汤的干预研究

Adenomyosis(AM) is a kind of gynecopathy that estrogen endometrial glands and stroma invasion into the uterine muscle colonization with the adjacent muscle cell hyperplasia and hypertrophy. It’s also a common clinical disease that affects women's physical and mental health and women's fertility. Traditional Chinese medicine has a unique advantage in treating of the disease. Studies have shown that the abnormal proliferation and metastasis of the lesion cells in patients with adenomyosis can lead to hypoxia,induce the lesion’s cell autophagy homeostasis disequilibrium, decrease the expression of autophagy associated factors:Beclin1;Atg3;Atg7;LC3Ⅱ.and activate correlation factors in PI3K/Akt/mTOR signal pathway,as a result it can promote the plant and growth of endometrium.With the support of two national natural science funds, the research group found that the Modified Shaoyaogancao Decoction could relieve the contraction of smooth muscle and promote the apoptosis of adenomyosis. So whether Modified Shaoyaogancao Decoction can relieve the contraction of smooth muscle, improve local oxygen lesions, adjust the autophagy steady state of imbalance, promote apoptosis lesions, achieve the effect of treatment of uterine adenomyosis need to be validated.The study aims at exploring the pathogenesis of adenomyosis and the intervention mechanism of the Modified Shaoyaogancao Decoction from the perspective of hypoxia induced autophagy homeostasis imbalance,and then providing reference for further clinical medication.

子宫腺肌病以子宫内膜的腺体和间质异位至深部子宫肌层，并伴随相邻肌细胞增生和肥大为特征，是临床上常见的严重影响女性身心健康及女性生育力的妇科病。中医药治疗该病有独特优势。研究表明，子宫腺肌病患者病灶细胞异常增殖和转移等生物学行为可导致病灶组织缺氧，诱发病灶细胞自噬稳态失衡，使自噬相关Beclin1、Atg3、Atg7、LC3Ⅱ表达下降，PI3K/Akt/mTOR信号通路相关因子激活，从而促进了异位内膜的种植、生长，促进了子宫腺肌病的发生发展。课题组前期在两项国家自然科学基金的支助下发现加味芍药甘草汤可减缓平滑肌收缩，促进子宫腺肌病病灶细胞凋亡。加味芍药甘草汤是否可以通过舒缓局部平滑肌，改善病灶局部缺氧状态，调节失衡的自噬稳态，促进病灶细胞凋亡，达到治疗子宫腺肌病的作用？本课题拟从缺氧诱导细胞自噬稳态失衡的角度，探讨子宫腺肌病的发病机制，以及加味芍药甘草汤的干预机制，为临床用药提供参考。

子宫腺肌病在严重影响患者身心健康的同时，也向存在治疗局限与风险的现代医学提出了挑战。中医药治疗子宫腺肌病时具有缓解症状、促进生育的临床疗效，成为关注和研究的热点。本课题通过免疫组化和ELISA检测病灶组织、在位内膜中缺氧、细胞自噬等相关指标的表达。培养原代细胞并建立缺氧模型，CCK-8及Maestro Z检测细胞增殖；转录组学富集分析细胞中自噬与通路的相关性和差异性；Western Blot检测自噬相关蛋白LC3、p62、Beclin-1和迁移、侵袭相关蛋白MMP-2、MMP-9、VEGF的表达。腹腔移植建立人来源型子宫腺肌病裸鼠模型并模型鉴定；透射电镜检测自噬体超微结构；RT-qPCR检测了移植瘤内Beclin-1mRNA、LC3 mRNA、PI3K mRNA、AKT mRNA、mTORC1mRNA、HIF-1αmRNA表达； WB检测移植瘤内Beclin-1、LC3-I/II、Atg 7、Atg 3、p-PI3K、p-AKT、mTORC1、HIF-1α蛋白的表达。结果发现：1.与对照组相比，子宫腺肌病组在位内膜LC3、Beclin-1、Atg7蛋白表达降低，p62、mTOR、p-mTOR (Ser2448)、p-mTOR (Ser2481)、HIF1α蛋白表达增加；SOD含量下降，MDA、LDH的含量升高。2. 转录组学显示差异转录的基因主要参与细胞周期的调控，以及脂质代谢、遗传信息的复制与修复、信号转导、细胞生长与死亡过程；与HIF-1信号通路、FoxO信号通路、mTOR信号通路、Rap1信号通路密切相关。加味芍药甘草汤介导mTOR通路的活化而促进原代细胞以及人子宫内膜基质原代细胞的细胞自噬水平，抑制细胞迁移、侵袭相关蛋白的表达，进一步增强缺氧状态下原代细胞的自噬水平，抑制细胞增殖。3. 加味芍药甘草汤有效缩小腹腔内移植病灶的体积；促进病灶细胞自噬体和溶酶体产生；上调病灶组织内Beclin-1mRNA、LC3 mRNA表达水平，下调PI3K mRNA、AKT mRNA、mTORC1mRNA、HIF-1αmRNA表达水平，细胞自噬相关蛋白的表达升高，HIF-1α和PI3K/AKT/mTOR信号通路等蛋白表达下降。综上，子宫腺肌病异位病灶组织为一个缺氧且自噬性死亡稳态失衡的环境。同时，加味芍药甘草汤下调HIF-1α以抑制PI3K/AKT/mTOR通路上相关蛋白的表