RIP1-RIP3坏死小体通过自噬流途径介导2型糖尿病心肌纤维化的机制研究

Diabetes mellitus is firmly established as a major threat to human health in the 21st century due to its alarming rise in incidence over the past two decades,which has attracted considerable attention. Type 2 diabetes mellitus (T2DM) accounts for the 90% of all cases of diabetes. Diabetic fibrosis is not only the main complication of diabetes, but the main cause of death. Diabetes mellitus is associated with a specific cardiomyopathy, include impaired myocardial relaxation and increased diastolic dysfunction, at last, it can cause cardiac infarction. Growing evidence indicates that fibroblasts can promote their proliferation and transformation through a variety of mechanisms, and provide the pathological basis for myocardial fibrosis, autophagy are also involved in the pathogenesis of diabetes. However, the underlying mechanisms are not completely understood. .The aim of the present study is to measure the improvement of cardiac function and attenuate myocardial fibrosis. The steps are divided three parts. Part 1: observe cardiac function changes of type 2 diabetic mellitus (T2DM) using echocardiography, myocardial fibrosis were analyzed histologically and ELISA, autophagy related proteins were assessed by western blot or Co immunoprecipitation. Part 2: analyse the relations of diabetic mellitus and myocardial autophagic flux through RIP1 knockdown and RIP3 knockout, including RIP1, RIP3, LC3, LAMP2, p62, Cathepsin B, ULK1, Beclin 1 and Cathepsin D. The effects of autophagy were determined by transmission electron microscopy and immunoblot analysis, we also use autophagic flux to summarize the impact of extracellular autophagosome accumulation on the development of type 2 diabetic mellitus and its associated pathologies. Part 3: observe the relationship of RIP1-RIP3 and autophagic flux, and using transfected high-glucose cells with RIP1 and RIP3 knockdown to evaluated their effects on cell activity using a clonogenic survival assay, investigate the feasibility and mechanism of RIP1 specific inhibitor Nec-1 for drug therapy, and to screen the reasonable concentration and time point..In conclusion, we have demonstrated that diabetes mellitus decreased autophagic activity, RIP1-RIP3-autophagic flux pathway plays an important role in diabetic cardiomyopathy. Our investigation of the study revealed a novel function regulating a critical cellular event that significantly impacts diabetic biology and therapy.

成纤维细胞可通过多种机制促进自身增殖和转化，为纤维化提供病理基础。已发现自噬水平下降是导致2型糖尿病（T2DM）心脏功能障碍的重要因素，但心肌成纤维细胞自噬水平在T2DM心肌纤维化中的作用及机制尚缺少研究。预实验发现T2DM心肌成纤维细胞自噬流进程受阻，坏死性凋亡关键酶RIP1、RIP3显著升高，药理和基因抑制RIP1减轻心肌纤维化和改善自噬流进程，提示坏死性激酶可能是改善T2DM心脏自噬流的重要调控因子。本项目拟多角度反映成纤维细胞自噬流改变在T2DM心肌纤维化中的作用，应用RNA干扰、基因敲除和药理学技术验证RIP1是否依赖RIP3改变自噬流以此影响T2DM心肌纤维化。同时建立以RIP1-RIP3坏死小体-自噬流为主的调控网络，观察RIP1特异性抑制剂Nec-1的药物治疗效果及作用机制，筛选Nec-1药物治疗的合理浓度和时间点，为开展T2DM心脏疾病的预防和治疗提供新思路和新靶点。

心肌纤维化是2型糖尿病（T2DM）心脏病变中的常见类型和必然过程，已知自噬水平下降是导致心肌纤维化的重要因素，但调节糖尿病心肌自噬水平的具体机制尚缺少研究。本项目通过验证自噬流在T2DM心肌纤维化发生发展中的作用地位，探讨RIP1-RIP3-坏死小体调控的自噬流在T2DM心肌纤维化中的作用机制，并使用RIP1特异性阻断剂Nec-1在在体糖尿病大鼠模型进行验证，证实RIP1-RIP3调控的自噬流在T2DM心肌纤维化的作用。本项目成功建立T2DM大鼠心脏模型和高糖高脂诱导的心脏成纤维细胞损伤模型，发现糖尿病组自噬标志蛋白表达升高，纤维化指标增加，坏死性凋亡激酶关键蛋白RIP1、RIP3、p-RIP1、p-RIP3表达均增加。基因敲低RIP1、RIP3减少高糖高脂培养的心脏成纤维细胞损伤和死亡，减少collagen I/III含量和细胞增殖，同时减少高糖高脂培养的心脏成纤维细胞自噬标志蛋白表达，改善细胞自噬流进程和增强溶酶体膜稳定性。在体实验中发现，Nec-1治疗组大鼠心脏功能改善，同时减少自噬溶酶体标志蛋白LC3-II、P62、active-Cathepsin D表达，减少collagen I/III含量。本项目通过探讨以RIP1-RIP3-坏死小体-自噬流为主的调控网络，验证RIP1-RIP3-坏死小体-自噬流的作用，在此基础上观察RIP1特异性阻滞剂Nec-1在糖尿病心肌纤维化治疗效果和作用机制，为糖尿病心血管并发症的预防和治疗提供新的视野，相关研究结果还将为自噬性死亡学说提供直接证据。