RIP1/RIP3/MLKL信号通路调控的程序性细胞坏死在糖尿病心肌病中的作用

Necroptosis is an alternative mode of programmed cell death. In the necroptosis pathways, receptor-interacting protein 1/ receptor-interacting protein 3/ mixed lineage kinase domain-like protein (RIP1/RIP3/MLKL) is play a vital role. Cell death is a fundamental process involved in the pathogenesis of diabetic cardiomyopathy (DCM). In our previous study, the cardiomyocyte showed the characteristic of necroptosis. Moreover, the protein levels of RIP1, RIP3 and MLKL were overexpression in the myocardium of DCM. Injection of RIR1-siRNA lentivirus (RIP1-siRNA) reduced the protein expression of RIP3 and MLKL. Consistent with this, the cardiac function was improved. Thus, we hypothesized that necroptosis, regulated by RIP1/RIP3/MLKL pathway, might participate in the pathogenesis of DCM. In this study, the rat model of DCM by lenti-RIP1 (up-regulation) and RIP1-siRNA (down-regulation) intravenous injection will be established to reveal the influence of RIP1/RIP3/MLKL pathway on cardiomyocyte necroptosis and cardiac function. In vitro, H9C2 cardiomyocyte cell line will be respectively infected with lenti-RIP1, RIP1-siRNA, RIP3-siRNA and MLKL-siRNA to further clarify the molecular mechanisms of necroptosis in DCM. Our research will provide a new theoretical support on understanding the pathological mechanism of DCM and may be helpful in designing a novel therapeutic target against DCM in patient with diabetes.

受体相互作用蛋白1/受体相互作用蛋白3/混合系列蛋白激酶样结构域（RIP1/RIP3/MLKL）是程序性细胞坏死（necroptosis）的核心调控通路。我们的前期研究示DCM大鼠左室心肌呈现necroptosis形态，且RIP1、RIP3、MLKL蛋白表达升高。RIP1-siRNA慢病毒干预DCM大鼠使心功能好转。我们推测，DCM大鼠心肌组织RIP1/RIP3/MLKL通路异常表达可诱发necroptosis，促进DCM。我们拟予lenti-RIP1及RIP1-siRNA慢病毒干预各组大鼠，比较心肌RIP3、MLKL表达及necroptosis比例等，揭示RIP1对DCM的影响；予lenti-RIP1、RIP1-siRNA、RIP3-siRNA及MLKL-siRNA慢病毒转染心肌细胞，探讨RIP1/RIP3/MLKL诱发necroptosis可能机制。本课题旨在为DCM防治提供实验依据。

背景：心肌细胞死亡是糖尿病心肌病（DCM）发生发展的病理基础。受体相互作用蛋白1/受体相互作用蛋白3/混合系列蛋白激酶样结构域（RIP1/RIP3/MLKL）调控的细胞程序性细胞坏死（necroptosis）可促进心脏重构及心力衰竭。在本研究中，我们探究了RIP1/RIP3/MLKL信号通路对DCM大鼠的影响及相关机制。内容：建立DCM大鼠模型，给予DCM组NC及RIP1低表达重组腺病毒载体（AAV-RIP1-shRNA）处理以探究RIP1在DCM大鼠中的作用。用高糖刺激H9C2细胞，并分别转染RIP1高表达、RIP1低表达、RIP3低表达、MLKL低表达慢病毒载体（LV-RIP1、LV-RIP1-shRNA、RIP3-shRNA、MLKL-shRNA）以探究RIP1/RIP3/MLKL信号通路影响DCM的机制。结果：DCM大鼠心肌组织RIP1、p/t-RIP3、p/t-MLKL、PGAM5及Drp1表达升高。体内转染AAV-RIP1-shRNA后，p/t-RIP3、p/t-MLKL、PGAM5及Drp1表达降低。与Ctrl对比，DCM大鼠呈现心肌细胞坏死、心肌明显纤维化、心脏功能降低等表现，抑制RIP1表达后，上述指标有改善。高糖可诱导H9C2心肌细胞RIP1、p/t-RIP3、p-MLKL、PGAM5及Drp1表达升高。RIP1高表达可促进上述因子表达，RIP1低表达可抑制上述因子的表达。高糖可促进H9C2细胞出现坏死样超微结构表现，LDH释放增多。RIP1高表达可促进上述变化，RIP1低表达可改善上述变化。RIP3低表达可抑制p/t-MLKL、PGAM5及Drp1的表达。MLKL低表达可抑制上述因子表达，对t-RIP3无影响。RIP3或MLKL低表达可改善坏死样超微结构表现，减少LDH释放。意义：本研究结果提示RIP1/RIP3/MLKL信号通路调控的细胞程序性坏死在DCM心脏重构中发挥重要作用。RIP1是该调控通路的核心因子，可能成为DCM临床防治的潜在干预靶点。