基于lncRNA/MMP3研究清热祛湿活血法抑制类风湿关节炎骨质侵蚀的分子机制

Rheumatoid arthritis (RA) is a common chronic autoimmune disease characterized by bone destruction. Damp-heat stasis obstruction is the core pathogenesis of bone erosion in active stage of RA, and Qingre Qushi Huoxue therapy is an important treatment method.The previous project of National Natural Science Foundation found that the empirical formula of Qingre Qushi Huoxue, Bizongxiao decoction, played an important role in inhibiting RA bone erosion by regulating the key node protein MMP3 of signal network. LncRNA is closely related to bone erosion of RA. Tumor studies have found that lncRNA can regulate the expression of MMP3, but it has not been reported in RA studies. Our previous study found that lncRNA was closely related to MMP3 in synovial tissue of RA model rats. The research object of this project is RA rat model and the synovial fibroblasts. By using advanced techniques such as transfection in vivo, plasmid construction, cell transfection and double luciferase reporter gene and so on, the project indends to screen lncRNA which targeting MMP3 regulated by Qingre Qushi Huoxue formula, Bizongxiao decoction and the main active ingredients. This project aims at illustration the pharmacodynamic substance basis and deep mechanism of anti-RA bone erosion of Qingre Qushi Huoxue formula, Bizongxiao decoction by interfering with lncRNA/MMP3. It will provide new therapeutic targets for the clinical treatment of RA bone erosion, and provide a new scientific basis for the treatment of RA active stage bone erosion with Qingre Qushi Huoxue therapy.

类风湿关节炎（RA）是常见的以骨质破坏为特征的慢性自身免疫病，湿热瘀阻是RA活动期骨质侵蚀的核心病机，清热祛湿活血法是其重要治法，前一个国自基金研究发现清热祛湿活血经验方-痹肿消汤通过调控信号网络的关键节点蛋白MMP3发挥抑制RA骨质侵蚀的作用。lncRNA与RA骨质侵蚀关系密切，肿瘤研究发现lncRNA可调控MMP3的表达，但在RA研究中尚未见报道，课题组前期研究发现RA模型鼠滑膜组织中lncRNA与MMP3密切相关。本项目拟以RA大鼠模型和滑膜成纤维细胞为研究对象，采用动物体内转染、质粒构建、细胞转染、双荧光素酶报告基因等先进技术筛选清热祛湿活血方药痹肿消汤及主要有效成分调控靶向MMP3的lncRNA，阐明其通过干预lncRNA/MMP3抑制RA骨质侵蚀的药效物质基础和深层作用机制，为临床治疗RA骨质侵蚀提供新的治疗靶点，为清热祛湿活血法治疗RA活动期骨质侵蚀提供新的科学依据。

为阐明清热祛湿活血方药痹肿消汤通过调控lncRNA/MMP3抑制RA骨质侵蚀的作用机制，本研究采用动物实验学，细胞生物学，分子生物学，生物信息学等领域的先进技术筛选调控靶向RA骨质侵蚀的关键蛋白MMP3的lncRNA，并从细胞和动物水平通过功能缺失或过表达研究阐明lncRNA通过调控MMP3在RA骨质侵蚀中的作用机制，明确清热祛湿活血方药痹肿消汤对lncRNA/MMP3的调控作用，研究结果表明：（1）运用 UPLC-MS/MS 法对痹肿消汤中的化学成分进行定性与定量分析，确定对香豆酸、阿魏酸、迷迭香酸、熊果酸等8种成分可能是痹肿消汤主要有效成分；（2）成功复制了CIA动物模型，并从TNF-α和IL-β水平、病理学、影像学检测发现痹肿消汤及有效成分对模型大鼠有干预作用；（3）基于lncRNA和mRNA转录组学筛选出lncRNA ENSRNOT00000092834等4条lncRNA可能是痹肿消汤抑制RA骨质侵蚀的潜在靶点；（4）构建目标lncRNA 敲除及过表达质粒，RT-qPCR验证质粒转染后ENSRNOT00000084631、ENSRNOT00000092834能稳定表达，通过流式细胞术、ELISA等实验方法证实了其可抑制CIA-FLS细胞凋亡，促进RA相关的炎症因子TNF-α、IL-1β、IL-6表达增加；（5）动物和细胞实验发现痹肿消汤及其有效成分可通过靶向抑制ENSRNOT00000092834影响MMP3的表达；（6）构建目标基因MMP3的敲除及过表达质粒，发现MMP3过表达后TNF-α、Ang-1、MMP13、MMP9、VEGF等蛋白相对表达量均上调，敲降MMP3后上述蛋白表达下调，动物和细胞实验发现痹肿消汤及其有效成分可通过干预ENSRNOT00000092834/MMP3通路治疗RA。结论：（1）ENSRNOT00000092834和ENSRNOT00000084631可能是痹肿消汤抑制RA骨质侵蚀的潜在靶点;(2) 清热祛湿活血方药痹肿消汤可以通过干预ENSRNOT00000092834调控MMP3的表达；(3)清热祛湿活血方药痹肿消汤通过调控ENSRNOT00000092834/MMP3通路下调TNF-α、Ang-1、MMP13、MMP9、VEGF的表达抑制局部关节炎症、减少关节骨质破坏、抑制滑膜血管增生。