从成纤维样滑膜细胞表观遗传学分子修饰探讨乌头汤对寒湿痹阻证型类风湿关节炎的治疗机理

The aberrant proliferation and invasiveness of fibroblast-like synoviocytes (FLS) play a key role in the pathogenesis of rheumatoid arthritis (RA). Abnormal epigenetic regulation is an important reason. Therapy of the integrated traditional Chinese and western medicine for RA, applying the clinical model of the combination disease and syndrome, lacks the study on the mechanisms and advantages of this mode. According to our previous study that Wutou decoction influenced the epigenetic modifications of peripheral blood mononuclear cells in collagen-induced arthritis rats, the hypothesis “Wutou decoction may alleviate RA through the multi-link and multi-target regulation of the epigenetic modifications of RA-FLS of cold-dampness-stasis” is proposed. The emphasis will be put on the common pattern of cold-dampness-stasis and the sera containing Wutou decoction will be applied for treatment. The aim of the project was to investigate the effects of Wutou decoction on DNA methylation, histone modification, regulatory proteases and aberrant modifications of RA-related gene promoters in RA-FLS of cold-dampness-stasis pattern and non-cold-dampness-stasis pattern. Meanwhile, the project will compare the epigenetic features of RA with different patterns and the advantages and disadvantages of Wutou decoction from different perspectives. Furthermore, we will explore the multi-link and multi-target complicated mechanisms of Wutou decoction in the treatment of RA of cold-dampness-stasis pattern, and explain the advantages and mechanisms of therapeutic model of combination of disease and syndrome.

在类风湿关节炎（RA）发病过程中，成纤维样滑膜细胞（FLS）异常的增殖力和侵袭性起着非常关键的作用，表观遗传学多环节调控紊乱是其重要原因。中西医结合治疗RA，常采用病证结合的临床模式，但缺乏该模式的优势和机理的研究。本研究在前期工作中发现“乌头汤能影响CIA大鼠PBMCs表观遗传修饰”的基础上，提出“乌头汤可能通过多环节、多靶点调控寒湿痹阻型RA的FLS表观遗传分子修饰而发挥其治疗优势”的假说。拟以RA常见证型(寒湿痹阻型)为研究重点，采用针对该证型的乌头汤含药血清为干预手段，以寒湿痹阻型和非寒湿痹阻型RA病人的FLS为研究对象，体外观察乌头汤对DNA甲基化、组蛋白修饰、调控蛋白酶及与RA相关基因的启动子异常修饰的影响，从表观遗传学的不同层面，比较两种证型的表观遗传特征及乌头汤对两种证型作用效果的优劣，探索乌头汤治疗寒湿痹阻型RA多环节、多靶点的复杂机制，阐释病证结合治疗模式的优势和机理

乌头汤能影响成纤维样滑膜细胞的表型及表观遗传学修饰，从而抑制滑膜血管生成，达到治疗类风湿关节炎的作用。本课题组通过MTT实验，划痕实验和Transwell实验，流式检测，管形成实验分别探究了乌头汤对成纤维样滑膜细的增殖活性，迁移与侵袭能力，细胞凋亡，诱导HUVEC血管形成能力等表型的影响。通过RT-qPCR、westernblot、免疫荧光，揭示了乌头汤可以下调HDAC7，影响组蛋白乙酰化，从而抑制FLS的增殖及减少促血管生成因子的分泌，进而抑制滑膜血管新生达到治疗RA的目的，并且验证这不是通过影响经典通路JAK2-STAT3的磷酸化发挥作用。我们在动物实验中，通过检测滑膜血管生成和HDAC7表达情况，进一步验证乌头汤可以减轻CIA大鼠关节肿胀，抑制滑膜血管的形成，改善关节骨质的破坏，并且这可能与影响HDAC7的表达有关。综上所述，本研究为乌头汤治疗类风湿关节炎机制探究的奠定了坚实的工作基础，为乌头汤临床应用提供了理论基础。同时，对乌头汤可能通过影响成纤维样滑膜细胞的表观遗传学修饰发挥作用提供了重要的启示性线索。项目资助发表核心论文四篇，SCI6篇。培养硕士4名，博士3名。项目投入经费57万元，支出48.9619万元，各项支出基本与预算相符。剩余经费8.0381万元，计划用于本项目研究的后续支出。