滋肾通络方调控miRNAs介导的Th细胞可塑性分化重建RA免疫稳态的机制

Rheumatoid arthritis (RA) is one of the clinical refractory diseases. Zishentongluo recipe, which has an accurate efficacy in curing RA, is one of the empirical formulas designed by the famous doctor- Zhou Zhong-ying. Recent studies suggest that miRNAs- mediated Th cells plasticity have features of totality and multi- link, which fit for the features of traditional Chinese medicine curing diseases. The mediated effects may be the mechanism of the recipe reconstructing the immune homeostasis. Our previous results showed that the recipe could regulate the expressions of some miRNAs that were abnormal in RA, and eight miRNAs among them may have relations with Th cells differentiation. Based on the results, this project presents a hypothesis that the three efficacy units of Zishentongluo recipe act on the different miRNAs targets and mediate the Th cells differentiation and plasticity through regulating the target genes and the micro enviorenment. The combination of the different efficacy units strengthen the overall effect on both the symptoms and root cause. To verify the hypothesis, we will collect the peripheral blood of RA patients which have “Yinxuluore” syndromes and found the collagen-induced arthritis models in mice. Then we will set up the method for the miRNA study of Gain-of-function and Loss-of-function. The key miRNAs that regulating Th cells differentiation will be investigated, and the interactions of miRNAs expression, Th cells differentiation and phenotypes will also be analyzed. Based on the results, the effects of Zishentongluo recipe and its “Zishenyangyin”, “Qufengchushi” and “Huatanquyu” units on the miRNAs-regulated Th cells plasicity will be observed from the different parts of the immune system (immune organs, circulation, local tissues) and the different links of Th cells differentiation (initial differentiation, redifferentiation). Through the studies, we can understand the differences and relevances between the different efficacy units and the recipe, and then explain the molecular mechanisms of Zishentongluo recipe on reconstructing immune homeostasis in RA. That will help to clarify the scientific nature of the prescription so as to provide scientific basis for clinical treatment.

类风湿关节炎(RA)是临床难治病，国医大师周仲瑛治疗RA的验方（滋肾通络方）疗效确切，新近研究表明miRNAs（miR）介导Th细胞可塑性分化具整体性和多环节作用，与中医药疗效特点相契合，可能是该方重建RA免疫稳态的重要机制。预实验发现该方调控RA多种异常表达的miR，且部分与Th分化相关。由此提出假说：滋肾通络方功效单元靶向不同miR，通过调控基因和微环境，介导Th可塑性分化平衡，协同增效达到“标本兼治”效果。项目采用RA阴虚络热证患者外周血和CIA小鼠的Th细胞，运用miR功能获得性/缺失性实验技术，筛选调控分化的关键miR，揭示“miR表达-Th分化-临床表型”内在联系；从免疫系统不同层面（免疫器官、循环、关节局部）、Th分化不同环节（初始分化、再分化），研究复方和滋肾养阴、祛风除湿、化痰祛瘀功效单元的个体效应和整体调节机制，阐明其组方配伍机制和治法方药的科学内涵，指导临床组方用药。

滋肾通络方（ZTF）是由国医大师周仲瑛教授治疗类风湿性关节炎（RA）的临床验方精简而来，由滋肾养阴（生地）配合祛风除湿通络（雷公藤、青风藤）和化痰袪瘀通络（僵蚕、三七）组成，前期已证实其对关节炎动物的治疗效应，功效活性组分雷公藤甲素、三七总皂苷和梓醇与各功效药物具有相似的作用效果。新近研究表明miRNAs介导CD4+细胞（Th）的可塑性分化具整体性和多环节作用，可能是ZTF发挥防治RA效应的机制。以此为切入点，项目组首先研究ZTF各功效单元和活性组分对CIA小鼠炎症表现的调节作用。随后，结合临床、动物和细胞实验研究，分析与Th可塑性分化相关的miRNAs，探讨ZTF通过作用于miRNAs来调控Th可塑性分化的效应和机制。研究结果表明：各功效单元均能不同程度减轻CIA关节炎表现，以全方效果最佳。RA患者Th中miR-26b和Let-7g显著降低，miR-143与RA疾病活动度负相关。miR-26b和Let-7g与Th17分化相关，miR-143与Treg分化相关；Let-7g和Treg再分化相关。转染miR-143、let-7g、miR-26b mimics均可缓解CIA小鼠关节炎症症状，影响Th分化。各功效单元和活性组分可干预不同免疫部位的Th分化，协同调控Th17/Treg平衡，但不同功效单元作用有别：滋肾养阴药物以调节Treg分化可塑性为主；祛风除湿药物以调节Th17分化为主；化痰祛瘀药物二者兼而有之。不同功效单元可通过作用于不同miRNA影响Th分化：滋肾养阴药物调节Let-7g和miR-27b；祛风除湿药物调节miR-26b和miR-143；化痰祛瘀药物调节miR-26b和miR-27b。其调控机制与影响不同的细胞因子和STAT3等通路有关。研究结果表明：miR-143、miR-26b、Let-7g对RA具有保护性作用，可作为RA的临床诊断和判断疾病转归的指标。调控Th17/Treg平衡是各功效单元配伍的协同效应和功效机制，不同功效单元通过差异作用于Th的初始分化和再分化阶段，协同恢复Th17/Treg平衡，重建RA免疫稳态。这种调控作用的实现与影响细胞因子、信号通路、miRNAs表达密切相关。研究阐释了滋肾养阴、祛风除湿、化痰祛瘀不同功效药物效应机制的差异和协同增效机理，为中医调节机体平衡治疗RA组方用药提供了科学依据。