基于miR146a-TLR4/NF-κB信号通路研究新风胶囊治疗佐剂性关节炎大鼠心脏病变的免疫机制

Morbidity and mortality rates are higher in rheumatoid arthritis (RA) patients than in the general population. Many studies have shown that heart disease is one of the most common causes of death in RA. It is necessary to study deeply on RA heart disease. Inflammatory is the key pathogenesis of RA heart disease , TLR4/NF- κB signal pathway is the common pathway for the various inflammatory reaction, the pathway can promote the expression of cytokines,that has closely associated with heart disease of RA.MiR146a,as a negative feedback regulation play an important role in the inflammatory immune reaction mediated by TLR.Our research group according to the " treat arthralgia from the spleen " theory, consider that the spleen dampness, deficiency of Qi and blood, phlegm and blood stasis is the basic pathogenesis of RA heart disease, and propose the hypothesis that the Xinfeng capsule can improve cardiac lesions in AA rats by regulate the expression of miR146a and one or more links in the TLR4/NF- B pathway. This topic design in vitro and in vivo experiments to verify the hypothesis.In vivo experiments,adjuvant arthritis rats as the research object, is divided into Xinfeng Capsule of high, middle, low dose groups, Tripterygium glucosides tablet group, MTX group, model group and normal group,observe the change of rats cardiac tissue morphology, ultrastructure of myocardium and cardiac function, use ELISA to detecting the expression level of TNF-α and IL-1of rats serum;Stem-loop real-time fluorescence quantitative PCR to detect the miR-146a expression levels in serum and heart tissue;TLR4, MyD88, IRAK1, TRAF6, NF- κB, TNF- α, IL-1 mRNA levels of serum and myocardium of rats were measured by real-time quantitative PCR ; TLR4, MyD88, IRAK1, TRAF6, NF-κ B protein expression in myocardial tissue detected by Western Blotting. Analyse the relationship between indicators and miR-146a, cardiac function, study the effect of Xinfeng Capsule to AA rat cardiac lesions and miR146a, TLR4/NF- κB signal pathway.In vitro experiment ,cultured rat myocardial cells as the research object, exogenous TNF- stimulation method, serum intervention measures, divided into blank control group, TNF- αstimulation group, Xinfeng Capsule serum group, miRNA-146a inhibitor group, miRNA-146a missense chain group, TLR4 specific inhibitor group, NF- κB inhibitor group.The expression level of miR-146a in the myocardial cells was detected by Stem-loop RT-qPCR;The expression level of TLR4, MyD88, IRAK1, TRAF6, NF- κB, TNF-α of myocardial cell were detected by immunohistochemical; The protein expression of TLR4, MyD88, IRAK1, TRAF6, NF- κB, TNF- α of myocardial cell were detected by Western Blotting. The expression of IRAK1, TRAF6 were detected by real-time quantitative PCR.This experimental study the immune regulation mechanism of Xinfeng Capsule on miR146a and TLR4/NF-κ B signal pathway, which provides the experimental basis for the Chinese medicine treatment of RA heart disease.

RA心脏病变发生率高，死亡率高，发生机制复杂，缺乏有效的治疗手段，有必要对RA心脏病变进行系统深入研究。目前认为炎症为RA心脏病变的重要发病机制，TLR4/NF-κB信号通路是机体各种炎症反应的共同通路，该通路能促进各种炎性细胞因子基因表达，与RA心脏病变紧密相关，miR146a是TLR信号转导通路的抑制因子，在TLR介导的炎症免疫反应中起负反馈调节作用。本课题组根据"从脾治痹"理论，认为脾虚湿盛，气血亏虚，痰瘀互结是RA心脏病变的基本病机，提出益气健脾通络中药复方新风胶囊通过干预miR146a的表达，调控TLR4/NF-κB信号通路的某一个或多个环节来改善AA大鼠心脏病变的假说，并通过体内、体外实验，运用RT-qPCR、细胞转染、Western blot、免疫组化等技术，研究新风胶囊对miR146a及TLR4/NF-κB信号通路关键分子的影响，为中医药治疗RA心脏病变提供实验依据。

研究背景：类风湿关节炎（RA）心脏病变发生率高，死亡率高，发生机制复杂，目前认为炎症为RA心脏病变的重要发病机制，TLR4/NF-κB信号通路是机体各种炎症反应的共同通路，该通路能促进各种炎性细胞因子基因表达，与RA心脏病变紧密相关，miR146a是TLR信号转导通路的抑制因子，在TLR介导的炎症免疫反应中起负反馈调节作用。本课题组根据“从脾治痹”理论，认为脾虚湿盛，气血亏虚，痰瘀互结是RA心脏病变的基本病机，且临床采用健脾化湿通络中药复方新风胶囊对于RA心脏病变的改善效果显著。.主要研究内容：本研究观察AA大鼠滑膜、心肌组织形态学变化，检测AA大鼠心功能变化，采用RT-qPCR、细胞转染、Western blot、免疫组化等技术检测各组大鼠血液、滑膜及心肌组织miR146a及TLR4/NF-κB信号通路关键分子的变化。 .研究结果：（1）XFC能明显抑制AA大鼠关节炎症反应：XFC能降低AA大鼠足趾肿胀度和关节炎指数，减少炎性细胞对关节滑膜的浸润，减轻局部充血和肿胀，改善关节功能；（2）XFC能明显改善AA大鼠心功能水平：XFC通过降低心功能参数LVSP、LVEDP、HR，提高±dp/dtmax、CI，改善大鼠左室心肌收缩功能，提高心功能水平。（3）XFC能显著改善AA大鼠心肌组织形态学变化：XFC能够减轻心肌纤维间隙水肿，通过抑制炎性介质对心肌组织的刺激减轻心肌纤维增粗程度，维持心肌细胞形态和功能；（4）XFC可抑制心肌、滑膜及血清miR146a表达，抑制其所介导的TLR4/NF-κB信号通路活性；（5）XFC能抑制心肌、滑膜及血清TLR4、MyD88、IRAK1、TRAF6、NF-κB、TNF-a表达，阻断炎性信号通路的激活。.研究意义：本项目意义不仅丰富和完善中医药治疗RA心功能降低的理论，还能在中医药治疗RA心功能降低领域产生重大的理论突破，开辟一条中医药治疗RA关节病变及心功能异常的新途径。