从TLR4-MyD88-NF-κB信号通路研究丹蛭降糖胶囊调控免疫炎症反应防治糖尿病心肌病的分子机制

Diabetic cardiomyopathy (DCM) is a major cause of death for patients with diabetes mellitus (DM). Studies showed that pure glycemic control could not completely reduce the risk of the occurrence of cardiovascular complications and death of patients with DM. Toll like receptors, as an important factor of immuno-inflammatory activation, is the important pathophysiological foundation in the occurrence and development of DM. Research showed that TLR4-MyD88-NF- κB signaling pathway could activate the network of immuno-inflammatory cytokines of myocardial tissue, induced diabetic myocardial injury, and was perceived as the potential therapeutic target of DCM. Traditional Chinese Medicine holds that the deficiency of qi and yin, blood stasis and collateral obstruction is the main pathogenesis of diabetic cardiovascular diseases. Replenishing qi, nourishing Yin and promoting blood circulation is viewed as the basic therapeutic method. Based on the effectiveness of DZJTC on cardiovascular complications of diabetes and the inhibition of TLR4、NF-κB、TNF-α expressions of diabetic model rats by previous research, this project intends to use TLR4-MyD88-NF-κB signaling pathway as the starting point. Both in vivo and in vitro methods are used. Modern molecular biology techniques are applied to observe the molecule mechanism of immuno-inflammatory cytokines on the regulation of DCM and the effect of DZJTC, study the molecular target of DZJTC on the treatment and prevention of DCM, and provide experimental evidence for its clinical application.

糖尿病心肌病（DCM）是糖尿病患者死亡的主要原因之一，研究表明单纯强化血糖控制，并不能完全降低糖尿病心血管并发症发生和死亡的风险。Toll样受体作为免疫炎症激活的关键调控环节，是糖尿病发生发展的重要病理生理学基础。实验表明，TLR4-MyD88-NF-κB信号通路能够激活心肌组织免疫炎症因子网络，诱发糖尿病心肌损伤，是DCM的潜在治疗靶点。中医认为气阴两虚、瘀血阻络是糖尿病心血管病变的主要病机，益气养阴活血法是其基本治法。课题组前期研究表明丹蛭降糖胶囊能够有效防治糖尿病心血管并发症，显著抑制糖尿病模型大鼠TLR4、NF-κB、TNF-α等蛋白表达。本项目拟在此基础上，以TLR4-MyD88-NF-κB信号通路为切入点，采用体内外结合的实验方法，运用现代分子生物学技术，观察免疫炎症因子调节DCM的分子机制及丹蛭降糖胶囊干预效应，研究丹蛭降糖胶囊防治DCM的分子靶点，为其临床应用提供实验依据。

糖尿病心肌病（DCM）是由糖尿病代谢功能紊乱所引起心肌细胞坏死、间质纤维化等一系列病理改变。免疫和炎症之间的交互作用，参与糖尿病心肌损伤病理调节，是DCM潜在的调控靶点。丹蛭降糖胶囊（DJC）作为安徽中医药大学第一临床医学院院内制剂，临床被广泛应用于2型糖尿病（T2DM）心血管并发症的防治并取得满意疗效。课题组在前期研究基础上，分别以高脂饲料联合链脲佐菌素制备的T2DM大鼠模型，高糖诱导的大鼠H9C2心肌细胞和原代心肌成纤维细胞模型为研究对象，采用体内外结合的实验方法，研究TLR4-MyD88-NF-κB信号通路参与调节DCM的病理机制及丹蛭降糖胶囊干预效应，揭示DJC防治DCM的分子机制。.研究结果显示：①DJC可显著改善糖尿病模型大鼠心脏结构和功能损伤，有显著的防治DIC作用（心脏重量指数、HE及Masson、血流动力学）；②DJC防治DIC作用与抑制TLR4-MyD88-NF-κB信号通路过度表达有关（Western blot和RT-qPCR检测心肌组织TLR4、MyD88、NF-κB p65的蛋白和基因，ELISA测血清TNF-α、IL-1β和IL-6）；③DJC可通过调节TLR4-MyD88-NF-κB信号通路蛋白和基因表达，显著抑制高糖诱导心肌细胞凋亡（TUNEL、流式细胞术），调节凋亡蛋白caspase3、Bax、Bcl-2）表达；④DJC可通过调节TLR4-MyD88-NF-κB信号通路蛋白和基因表达，显著抑制高糖诱导心肌纤维化（MTT、划痕实验、Vimentin、Collagen I、CollagenIII 和TGF-β1蛋白表达）.研究结果表明：DJC可通过TLR4-MyD88-NF-κB信号通路，干预糖尿病诱发的免疫炎症反应，抑制高糖诱导心肌细胞凋亡、心肌成纤维细胞和胶原增殖，改善心脏结构和功能损伤，发挥防治DIC的作用，是临床糖尿病心肌病的有效防治手段。