TXL Promotes Human Cardiac Microvascular Endothelial Cells’ Function Partly By Neuregulin-1/ErbB Signaling Pathway

Backgrounds and Objectives:The integrity of cardiac microvascular endothelial cells’structure and function plays a key role in effective myocardial perfusion and normal function sustain. Neuregulin-1 (NRG-1) is a member of the epidermal growth factor (EGF) gene family, which specifically expressed in endocardium and cardiac microvascular endothelial cells. It can activate downstream pathways via binding to ErbB receptor on myocardium and play a key regulatory role in cardiac development, metabolism and other processes. However, in recent years, the tissue specificity of its expression is controversial and its autocrine effect on cardiac microvascular has not been elucidated. TXL, a compound chinese medicine, is widely used to treat cardiovascular and cerebrovascular diseases which has endothelium protection. This study was aimed to explore the autocrine effects of NRG-1 on cardiac microvascular endothelial cells and the regulatory effects of TXLMethods:We validated the tissue expression specificity of NRG-1 via NRG-1 and vWF (endothelial marker) immunofluorescent co-staining in the heart samples of rat and human. Transfected NRG-1 shRNA via lentivirus to inhibit NRG-1 expression of.human cardiac microvascular endothelial cells (HCMECs). CCK-8 assay detected cell proliferation viability in vitro. Scratch test detected cell migration ability in vitro. Subjected to hypoxia 4 h/reoxygenation 2 h, cell survival assay detected cell anti-injury ability. TUNEL staining detected cell apoptosis. Treated by TXL solution (800 μg/ml) and detected its effects on HCMECs’proliferation, migration and anti-injury ability. Detected the change of these effects when NRG-1 expression was inhibited.Results:Neuregulin-1 was specifically expressed in cardiac microvascular endothelial cells and endocardium. NRG-1 was not expressed in endothelial cells of big coronary arteries and veins. NRG-1 autocrine significantly promoted HCMECs proliferation, migration and anti-injury ability. When supplied with exogenous rhNRG-1, these effects could be further strengthened. TXL could significantly promoted HCMECs proliferation, migration and anti-injury ability (p<0.05). These effects were partly eliminated when NRG-1 gene expression was suppressed.Conclusions:These results suggest that NRG-1 is specifically expressed in endocardium and cardiac microvascular endothelial cells. TXL promotes human cardiac micro vascular endothelial cells’ function partly by neuregulin-1/ErbB signaling pathway.Backgrounds and objectives:Myocardial no-reflow and reperfusion injury are the two major problems in the reperfusion era of acute myocardial infarction (AMI) treatment. Previous studies have showed that compound chinese medicine Tongxinluo (TXL), a microvascular endothelial protective agent, could effectively reduce the myocardial no-reflow and reperfusion injury after reperfusion of AMI, while the mechanism is still unclear. Neuregulin-1, which is specifically expressed in cardiac microvascular endothelial cells and endocardium, plays a clear protective role in myocardial ischemia-reperfusion injury by binding to the ErbB receptors on the surface of myocardial cells and activating the downstream signal pathways. Thus, this study is aimed to explore whether the cardioprotection of TXL against myocardial no-reflow and ischemia-reperfusion injury after reperfusion of AMI in rats is mediated by neuregulin-1/ErbB signaling pathway or not, and further to explore the pathological mechanism of myocardial no-reflow.Methods:The heart of rats is processed by ligating the left anterior descending artery (LAD) for 1 hour and then releasing for 24 hours or 4 weeks respectively to establish acute and chronic AMI reperfusion models. Neuregulin-1 neutralizing antibody was injected through jugular vein to inhibit neuregulin-1/ErbB signaling pathway 2 hours before LAD ligation and TXL was given by gavage 15 minutes before LAD ligation. Pathological staining was used to determine the myocardial ischemic area at risk (AAR), the myocardial area of no-reflow (ANR), and the myocardial area of necrosis (AN). Echocardiography and hemodynamics were applied to evaluate the cardiac function. Pathological staining and transmission electron microscope were used to observe pathological changes of myocardial tissues. ELISA and Real-time PCR were applied to detect the expression of neuregulin-1, VE-cadherin and etc. in each part of heart tissue.Results:TXL could promote the secretion of neuregulin-1 significantly (p<0.05), reduce myocardial no-reflow and reperfusion injury in rats after reperfusion of AMI, reduce the infarct size and improve heart function in acute and long stages. The protective effect of TXL was partly eliminated by neuregulin-1 neutralizing antibody which can inhibit neuregulin-1/ErbB signaling pathway (p<0.05). The microvascular endothelial junction was severely damaged in myocardial no-reflow area and a large number of red blood cells infiltrated into tissue space.Conclusion:The cardioprotection of TXL against myocardial no-reflow and ischemia-reperfusion injury after reperfusion of AMI in rats is partly mediated by neuregulin-1/ErbB signaling. The damage of microvascular endothelial barrier is an important mechanism of myocardial no-reflow.