Tongxinluo attenuates reperfusion injury in diabetic hearts by angiopoietin-like 4-mediated protection of endothelial barrier integrity via PPAR-α pathway

ObjectiveEndothelial barrier function in the onset and Tongxinluo (TXL) protection of myocardial ischemia/reperfusion (I/R) injury, and TXL can induce the secretion of Angiopoietin-like 4 (Angptl4) in human cardiac microvascular endothelial cells during hypoxia/reoxygenation. We intend to demonstrate whether TXL can attenuate myocardial I/R injury in diabetes, characterized with microvascular endothelial barrier disruption, by induction of Angptl4-mediated protection of endothelial barrier integrity.Methods and resultsI/R injury was created by coronary ligation in ZDF diabetic and non-diabetic control rats. The animals were anesthetized and randomized to sham operation or I/R injury with or without the exposure to insulin, rhAngptl4, TXL, Angptl4 siRNA, and the PPAR-α inhibitor MK886. Tongxinluo, insulin and rhAngptl4 have the similar protective effect on diabetic hearts against I/R injury. In I/R-injured diabetic hearts, TXL treatment remarkably reduced the infarct size, and protected endothelial barrier integrity demonstrated by decreased endothelial cells apoptosis, microvascular permeability, and myocardial hemorrhage, fortified tight junction, and upregulated expression of JAM-A, integrin-α5, and VE-cadherin, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with siRNA interference and inhibition of PPAR-α with MK886 partially diminished these beneficial effects of TXL and rhAngptl4. TXL induced the expression of Angptl4 in I/R-injured diabetic hearts, and was canceled by Angptl4 siRNA and MK886. TXL treatment increased myocardial PPAR-α activity, and was abolished by MK886 but not by Angptl4 siRNA.ConclusionsTXL protects diabetic hearts against I/R injury by activating Angptl4-mediated restoration of endothelial barrier integrity via the PPAR-α pathway.本研究旨在明确内皮屏障功能在心肌缺血再灌注（myocardial ischemia/reperfusion, I/R）损伤发生中的作用，以及通心络（Tongxinluo, TXL）对该损伤的保护效应；同时观察在缺氧复氧条件下，通心络能否诱导人心脏微血管内皮细胞分泌血管生成素样4（Angiopoietin-like 4, Angptl4）。本研究拟验证：在以微血管内皮屏障破坏为特征的糖尿病状态下，通心络是否可通过诱导Angptl4介导的内皮屏障完整性保护作用，减轻心肌I/R损伤。 实验方法与结果：本研究通过冠状动脉结扎法构建ZDF糖尿病大鼠及非糖尿病对照大鼠的心肌I/R损伤模型。将大鼠麻醉后随机分为假手术组，以及伴或不伴胰岛素、重组人Angptl4（rhAngptl4）、通心络、Angptl4小干扰RNA（siRNA）及过氧化物酶体增殖物激活受体α（PPAR-α）抑制剂MK886处理的I/R损伤组。结果显示，通心络、胰岛素与重组人Angptl4对糖尿病心脏的I/R损伤具有相似的保护作用。在I/R损伤的糖尿病心脏中，通心络处理可显著缩小梗死面积，并通过以下指标证实其保护内皮屏障完整性的作用：降低内皮细胞凋亡率、微血管通透性及心肌出血程度，强化紧密连接结构，上调连接黏附分子A（JAM-A）、整合素α5（integrin-α5）及血管内皮钙粘蛋白（VE-cadherin）的表达；通心络的上述保护效应与胰岛素及重组人Angptl4相当。然而，通过小干扰RNA干扰敲低Angptl4，或使用MK886抑制PPAR-α活性，可部分削弱通心络与重组人Angptl4的上述有益作用。通心络可诱导I/R损伤糖尿病心脏中Angptl4的表达，该效应可被Angptl4 siRNA及MK886阻断。通心络处理可升高心肌组织的PPAR-α活性，该作用可被MK886取消，但不受Angptl4 siRNA的影响。 结论：通心络可通过激活PPAR-α通路诱导Angptl4介导的内皮屏障完整性修复，从而对糖尿病心脏的I/R损伤发挥保护作用。