DBC-1上调TNF-α信号促进心梗/再灌后心室重构的作用和机制

Heart failure is the leading cause of death after myocardial infarction. Recent study revealed that cardiomyocytes undergoing persistent apoptosis and closely related to left ventricular remodeling after MI/R, even though the haemodynamics in infarction area comes back to normal. Accumulating evidences have proved that TNF-α signals are the key factors of cardiomyocytes apoptosis. Our former research uncovered Deleted in brease cancer-1(DBC-1) promotes cell apoptosis through up-regulating TNF-α signaling to activate the p38-casepase8 signal pathway, thus accelerating apoptosis. We speculate that DBC-1 may aggravates ventricular remodeling after MI/R via up-regulating TNF-α-p38-casepase8 pathway. To prove the hypothesis, we firstly isolate myocardial cells from DBC-1 wild type and knockout mice and stimulate them with hypoxia, and evaluate the role of DBC-1 in myocardial cells apoptosis by detecting the expression of TNF-α, other inflammatory cytokines, chemokines, systolic function, and apoptosis level; Myocardial infarction/ reperfusion models will be established in each group, and some groups will be treated with p38 and/or casepase8 inhibitor. Through analyzing the degree of ventricular remodeling by histology and imaging, detecting the levles of DBC-1 and TNF-α, and evaluating the state of activation of downstream signal pathway by immunohistochemistry and western blot, we can reveal the mechanism in which DBC-1 promotes cardiomyocytes apoptosis and aggravates ventricular remodeling after MI/R via TNF-α signaling. This project would provide new evidence for the therapy and intervention of left heart faliure after MI/R in clinic setting.

心力衰竭是心肌梗死后患者主要致死原因。最新研究表明，心梗后即使血流动力学正常，心肌细胞凋亡仍持续发生并与心室重构严重程度密切相关，而TNF-α信号激活和上调是凋亡关键。本团队研究发现，乳腺癌缺失基因-1（DBC-1）通过增强TNF-α信号，激活下游p38-casepase8通路促进凋亡,因此推测DBC-1通过TNF-α-p38-casepase8通路促进心梗后心肌细胞持续凋亡及心室重构。本课题拟使用DBC-1基因敲除小鼠，不同条件下培养原代心肌细胞，体外评价DBC-1在细胞凋亡中作用；建立DBC-1基因敲除小鼠心梗/再灌注模型，使用p38及casepase8抑制剂，组织学和影像学评价心室重构程度，免疫组化及western blot检测DBC-1及TNF-α下游通路各蛋白表达水平，探讨DBC-1影响TNF-α-p38-casepase8通路参与心室重构作用和机制，为心衰防治提供新策略。

持续发生的凋亡是缺血再灌注后心室重构的主要原因之一。TNF-α信号的激活和上调是导致心肌细胞凋亡的关键，然而针对TNF-α的治疗并不能有效的抑制心室重构的进程。在本研究中，我们发现，缺血再灌注后心脏功能进行性降低，伴随高水平的细胞凋亡，同时心肌组织中TNF-a及DBC1表达水平显著上调，而且p38及caspase8信号通路被激活。进一步的研究结果显示，使用Caspase8及p38抑制剂来抑制Caspase8及p38信号通路的激活可以显著改善MI/R后小鼠的心功能，减轻心肌局部炎症反应，降低MI/R后心肌细胞的凋亡。而且，Caspase8及p38抑制剂还可显著降低MI/R后心肌组织中DBC1，TNF-a和TNFR1的表达。体外细胞实验结果显示，过氧化氢诱导心肌细胞损伤模型中DBC-1的表达显著升高，敲低DBC-1显著降低过氧化氢诱导的H9C2细胞的凋亡，及TNF-a和TNFR1的表达，DBC1-siRNA转染H9C2细胞后过氧化氢诱导的p38MAPK及caspase8 表达水平也显著降低。使用TNF-a-siRNA转染细胞敲低TNF-ɑ可以逆转过氧化氢诱导的DBC1高表达，而Caspase8及p38抑制剂也可显著降低过氧化氢诱导的细胞凋亡。因此，DBC-1 通过与TNF-α相互作用并促进 p38 和 casepase8信号通路的激活参与缺血再灌注后心肌凋亡，并最终导致心室重构。该研究结果初步揭示了 DBC1与 TNF-α相互作用促进心梗/再灌注后心肌细胞凋亡和心室重构的机制，为合理进行心梗后心室重构的治疗和预防提供依据。