基于TLR4信号通路白喉乌头诱导耐受性树突状细胞抗类风湿关节炎及miRNA调控机制的研究

Dendritic cell (DC) is a major participant in rheumatoid arthritis. It promotes or promotes the development of inflammation by presenting synovial autoantigen and activating T cells. Therefore, there is a research hotspot of RA that inducing DC to inhibit the activation of dendritic cells by T cells and establishing the peripheral immune tolerance. Kazakh medicine suggests that wind-cold damp pathogen, obstruction of meridian is the basic pathogenesis of rheumatoid arthritis. Aconitum leucostomum vorosch is one of the most important curative medicine in Kazakh medicine to expelling wind and cold, dehumidification pain in the treatment of rheumatoid arthritis. Our previous research has proved that aconitum leucostomum vorosch can inhibit the maturity of DC, but the mechanism is still not clear. Current studies have demonstrated that the maturation and activation of DC are regulated by TLR4 signaling pathway. Therefore, this paper is based on aconitum leucostomum vorosch have effects on anti-rheumatoid arthritis through TLR4 signaling pathway to inhibit the maturation and activation of DC. The study have took CIA model and DC as the research objects, and aimed at clearing the anti-rheumatoid arthritis mechanism of aconitum leucostomum vorosch by blocking TLR4 and its downstream key factors. The study also aimed at expounding the epigenetic mechanism of aconitum leucostomum vorosch inhibited the maturation and activation of DC through TLR4 which implemented by the use of miRNA chip technology, and providing scientific basis for the anti-rheumatic arthritis function of aconitum leucostomum vorosch in Kazakh medicine.

树突状细胞（DC）是类风湿关节炎炎症的主要参与者，通过呈递滑膜自身抗原和活化T细胞来发起或促进炎症持续进展。目前将DC诱导为可抑制T细胞活化的耐受性树突状细胞，建立外周免疫耐受，是RA研究的热点。哈萨克医学认为风寒湿邪，痹阻经络是类风湿关节炎的基本病机。白喉乌头以祛风散寒、除湿止痛为功效是哈萨克医学中治疗类风湿关节炎的主要药物。我们前期工作已经证实白喉乌头可以抑制DC的成熟，但作用机制尚不明确。目前研究已经证实DC成熟与活化受TLR4信号通路的调控。因此，本课题假说为：白喉乌头基于TLR4信号通路抑制DC成熟与活化发挥抗类风湿关节炎作用。课题以CIA模型与DC为研究对象，通过阻断TLR4及其下游关键因子，明确白喉乌头抗类风湿关节炎的机制，通过miRNA芯片技术，阐明白喉乌头通过TLR4通路抑制DC成熟与活化的表观遗传学机制，为哈萨克民族医药白喉乌头抗风湿作用提供科学依据。

白喉乌头临床上用于治疗类风湿关节炎，前期研究证明白喉乌头能改善胶原诱导关节炎大鼠炎症及影响树突状细胞成熟，但其具体作用机制尚不明确。本课题中我们首先发现白喉乌头能降低关节肿胀度、减轻滑膜细胞增生、血管增生及炎性细胞浸润，减少促炎因子、增加抑炎因子表达水平，证明了白喉乌头及其单体对胶原诱导性关节炎大鼠的治疗作用。其次提取小鼠骨髓单核细胞并诱导BMDCs生成，采用脂多糖和低中高浓度的DLTD进行干预，观察细胞增殖情况；流式细胞术观察共刺激因子CD80、CD86的表达；PCR、ELISA检测炎性因子表达。结果显示DLTD抑制BMDCs共刺激因子CD80、CD86的表达，降低BMDCs分泌的细胞因子的表达，且DLTD还显著抑制了BMDCs激活的T细胞分泌的细胞因子的表达，证实了DLTD可以抑制BMDCs成熟及活化，从而诱导T细胞耐受。我们进一步运用PCR和Western-Blot检测TLR4-Myd88依赖通路中相关基因mRNA和蛋白水平，结果显示DLTD降低TLR4、Myd88、IRAK4、TRAF6和Ikk2的mRNA和蛋白水平，验证了DLTD通过TLR4-Myd88依赖通路抑制BMDCs成熟的成熟及活化，并且影响T细胞的激活。由于BMDCs成熟及活化与miRNA调控有关，我们利用miRNA表达谱芯片检测技术考察DLTD对LPS诱导BMDCs成熟及活化的影响。结果提示基因芯片技术显示在“Fold Change >1.5，P<0.05”的条件下，经LPS诱导分化的成熟和正常组未成熟BMDCs有348个小RNA表达失调，与LPS组相比，经DLTD给药后有54个小RNA表达失调。生物信息学分析预测正常组与LPS组三个数据库共有靶基因1076个，LPS组与DLTD组共有靶基因63个，预测miR-511-3p共有靶基因17个，其中Calcr, Fzd10 and Eps8与BMDCs成熟密切相关，证实了基于TLR4信号通路DLTD抑制BMDCs成熟及活化受miRNA及下游靶基因调控。通过以上实验验证了白喉乌头总生物碱及其单体对胶原诱导型关节炎大鼠有治疗作用，其单体1通过TLR4-MyD88依赖通路抑制BMDCs成熟及活化，同时受miRNA及下游靶基因调控。为哈萨克民族医药的抗类风湿关节炎作用研究提供新的理论依据。