Toll样受体4调控心肌缺血再灌注损伤的细胞特异性作用及分子机制研究

Toll-like receptor 4 regulates high mobility group box 1 protein (HMGB1) release in myocardial ischemia-reperfusion injury (MIRI), resulting in the activation of inflammatory resonse and enhanced myocardial injury. TLR4 is present on various cell types of heart and vasculature, both immune and nonimmune cells, and induces HMGB1 release. However, it is unclear that effects and mechanisms of TLR4 on HMGB1 release in differrent cell types. Mitogen-activated protein kinases (MAPK) shows dynamic regulation of pro- and anti-inflammatory cytokines in TLR4-triggered innate immune responses. We hypothesized that different roles and mechanisms regarding the TLR4-regulated HMGB1 release in individual cell populations, specifically, marcophages and cardiomyocytes subsequent to MIRI, probably due to activation different MAPK through TLR4. The present study is designed to examine effects of HMGB1 and TLR4 on inflammation in vivo by using patients with coronary heart diseases undergoing percutaneous coronary intervention and TLR4-deficient mice subjected to MIRI. To further explore intracellular inflammatory signal pathways of TLR4, roles of TLR4 deficiency/silencing and different MAPK inhibtors in HMGB1 release are analyzled in vitro by using peripheral blood mononuclear cells and heparinized whole blood from patients and mice subjected to MIRI, isolated cardiomyocytes and macrophages from murine hearts suffer from MIRI, and human cardiomyocytes and macrophages. The aim of the present study is to uncover the cellular-specific role and mechanism of TLR4-regulated inflammation, and to find the new target at the cellular and molecular levels for myocardial ischemia-reperfusion injury.

Toll样受体4(TLR4)可介导高迁移率族蛋白B1 (HMGB1)释放，参与心肌缺血再灌注损伤(MIRI)的炎症反应。心肌细胞和巨噬细胞均表达TLR4并诱发HMGB1释放，丝裂原活化蛋白激酶(MAPK)可双向调节TLR4介导的炎症反应。我们假设TLR4调控MIRI过程中心肌细胞和巨噬细胞释放HMGB1的机制不同，与其活化不同MAPK分子有关。本项目拟采用再灌注治疗患者和TLR4缺陷MIRI小鼠作为研究对象，在体分析TLR4调节HMGB1释放和炎症因子表达的作用。制备外周血单核细胞和肝素化全血，培养小鼠和人的心肌细胞和巨噬细胞，沉默TLR4基因离体验证TLR4调控不同细胞中HMGB1释放的作用，应用MAPK抑制剂探索TLR4介导HMGB1释放的下游分子机制。阐明TLR4调控MIRI过程中HMGB1释放的细胞特异性分子机制，在细胞水平和分子水平寻求防治MIRI的新靶点。

项目背景：心肌缺血再灌注治疗是改善冠心病预后最有效的手段，然而再灌注本身也可造成心肌损伤，严重影响再灌注治疗效果。心肌缺血再灌注损伤(I/R)是药物溶栓、冠状动脉介入术以及冠状动脉旁路移植术后的重要病理过程，炎症反应是其特征性表现。Toll样受体4 (TLR4)可诱导高迁移率族蛋白B1 (HMGB1)释放，参与心肌I/R过程中的炎症反应。本研究拟探讨TLR4调控心肌I/R炎症反应的分子机制。研究内容：分析冠脉再灌注治疗患者血清炎症因子释放及外周血单核细胞TLR4表达。在体探讨TLR4和JAK2/STAT3调节小鼠心肌I/R炎症反应的机制。体外验证JAK2在TAK-242抑制缺氧/复氧(H/R)心肌细胞炎症反应的作用。研究结果：再灌注治疗组患者(PCI)血浆FIB、CK-MB、cTnI、TNF-α、MPO、MIF表达量显著高于冠脉造影组患者(CAG)，FIB浓度与炎症因子TNF-α、MPO、MIF表达存在一定相关性，FIB可作为心肌缺血再灌注相关炎症反应的预测因子。PCI组患者外周血单核细胞上TLR4表达明显增加。与sham组比较，I/R组小鼠心功能减弱，心梗面积增大并出现心肌炎性浸润，心肌p-JAK2/p-STAT3表达明显升高，血清IL-6、IL-10、TNF-和HMGB1水平显著升高。与I/R组比较，TAK-242给药组小鼠心功能有所改善，心肌炎症浸润减轻，心肌p-JAK2/p-STAT3表达降低，血清IL-6和TNF-水平明显下降，而IL-10和HMGB1浓度进一步升高。RNA-Seq筛查小鼠心肌差异表达基因（MAPK、TLR4、CXCL5、CLEC4D等），经验证差异基因蛋白表达与mRNA表达趋势一致。与TAK-242（TLR4抑制剂）预处理组比较，AG490（JAK2抑制剂）干预可显著加强TAK-242治疗作用，包括心肌收缩功能增强，心梗面积缩小及炎性浸润程度减轻，心肌p-JAK2/p-STAT3表达降低，血清IL-6、TNF-浓度下降而IL-10、HMGB1浓度升高。缺氧/复氧（H/R）心肌细胞的JAK2被沉默后，可抑制心肌细胞炎症反应减轻。结论及意义: TLR4抑制剂TAK-242可通过调控JAK2/STAT3-NF-kB通路抑制心肌缺血再灌注损伤炎症反应，为防治心肌I/R提供新靶点。