基于Rho／ROCK信号通路的桂楼药对宫腔缓释给药降低子宫腺肌病异位内膜细胞侵袭力的研究

Adenomyosis (AM) is a gynecological stubborn disease, with characteristics that tumor remotely attack and has high recurrence rate, Intrauterine administration is an effective treatment. Rho / ROCK signaling pathway is closely related to cell invasiveness. The research group has confirmed the clinical efficacy of "Tongmai Xiaozheng" method in AM and carried out the study on the intrauterine drug of its prescription--Gui Zhi and Chong Lou and Rho / ROCK pathway. Based on this, it is hypothesized that "the Rho / ROCK pathway has a key role in the pathogenesis of AM and Gui Zhi , Chong Lou has the potential to reduce endometrial invasiveness through this pathway." In this study, we will establish AM rat model and clinical AM in vitro cells. Gui Zhi, Chong Lou drug have intervention on the components, observe the ultrastructure of the cells, detect VEGF, COX-2, NF-κB, MMP-2/9, TIMP, RhoA , B, C and ROCKI, II, MLCP, G-actin, Bcl-2, Bax, Caspase-3/9, F-actin, ROCKI-MBSThr-697 phosphorylation and mRNA expression and proliferation / apoptosis rate, Observe the blocking effect of the pathway, analyze the characteristics of the Rho / ROCK signaling pathway in the ectopic endometrium of AM, and clarify the key target of reducing the invasiveness of the AM intima by intrauterine administration of "Tongmai Xiaozheng" method.

子宫腺肌病(AM)是妇科疑难病，具有肿瘤远隔侵袭且高复发特征，宫腔给药是有效治法。Rho/ROCK信号通路与细胞侵袭性密切相关，课题组已证实AM“通脉消癥”法临床疗效，开展其组方桂楼药对宫腔给药及Rho/ROCK通路研究。基于此，提出假说“Rho/ROCK通路在AM发病机制中有关键作用，桂楼药对可能通过该通路降低内膜细胞侵袭性而获得疗效”。本研究将建立AM大鼠模型和临床AM体外培养细胞，桂楼药对组分干预后，观察细胞超微结构，检测VEGF、COX-2、NF-κB、MMP-2/9、TIMP，RhoA、B、C及ROCKI、II、MLCP、G-actin、Bcl-2、Bax、Caspase-3/9、F-actin、ROCKI-MBSThr-697磷酸化和mRNA表达及增殖/凋亡率，观察通路阻断效果，分析AM异位内膜Rho/ROCK通路特征，明确“通脉消癥”法宫腔给药降低AM内膜细胞侵袭力关键靶点。

研究背景：子宫腺肌病（AM）是妇科常见疑难病，持续进展易复发，具有似肿瘤异常增殖的特点，是妇科常见的疑难病种，严重影响女性的身心健康和生活质量，Rho/ROCK信号通路与AM侵袭转移密切相关，且前期已证实AM“通脉消癥”法临床疗效确切。因此，探索AM的发生发展机制，进一步明确其治疗关键干预环节，是抑制AM侵袭转移及复发的重要研究内容。研究内容：依靠课题组前期建立的无创垂体移植法AM大鼠模型和提取的人子宫腺肌病异位内膜（AMDC）原代细胞，以课题组临床证实有效的中成药联用方案桂枝茯苓胶囊+宫血宁胶囊+散结镇痛胶囊中的代表药物（桂枝、重楼、三七）为核心。根据中药有效组分理论，提取桂枝总挥发油、重楼总皂苷、三七总皂甙，制成中药有效组分宫内缓释系统，并通过实验证实其基本治疗效果及潜在作用机制。重要结果、关键数据及其科学意义：体内实验结果，HE染色显示桂楼药对灌胃组与桂楼药对有效组分宫内缓释系统组的大鼠子宫肌层均可见少量腺体侵入深内膜层及少量纤维化病灶，但较模型组及空白缓释棒组完整连续，表明造模稳定及药物干预的有效性；桂楼药对灌胃及桂楼药对有效组分宫内缓释系统可显著降低AM大鼠血清中VEGF、NF-κB炎性细胞因子的表达，下调促进细胞侵袭运动能力的MMP-2、MMP-9、RhoA、RhoB、ROCK1及ROCK2表达，同时上调抑制运动能力的TIMP-1，从而抑制病灶细胞的异位侵袭，并通过下调BCL-2和上调BAX及Caspase-3促进病灶组织的凋亡，抑制疾病的进展。以上结果证实了桂楼药对有效组分宫内缓释系统通过下调Rho/ROCK信号通路，抑制异位内膜侵袭和迁移，调控异位病灶增殖凋亡的失衡，从而减轻AM的发生发展。体外实验显示，桂楼药对有效组分可抑制AMDC细胞的侵袭和迁移，并促进其凋亡，可下调RhoA、RhoB、RhoC、ROCK1、ROCK2的蛋白及mRNA表达水平；激动剂U46619可显著上调Rho/ROCK信号通路，增加细胞侵袭与迁移，桂楼药对有效组分则对U46619激活的Rho/ROCK信号通路发挥挽救作用，这证实了桂楼药对有效组分在抑制AMDC细胞侵袭、迁移及调控Rho/ROCK信号通路方面的有效性，为中医药精准治疗AM提供科学依据。