Protective Effects And Mechanism Of Des-aspartate-angiotensin-Ⅰ On Cardiac Microvascular Endothelial Cell Injury Induced By Ischemia/reperfusion

Background:Acute reperfusion of ischemic myocardium results in a cascade of harmful events，termed as reperfusion injury. Factors contributing to reperfusion injury includedysfunction or apoptosis of cardiomyocytes and endothelial cell. Recently，our previousfindings indicate that cardiac microvascular endothelial cells (CMECs)also play animportant role during I/R injury of the heart. In I/R myocardium，sudden increase ofoxygen free radicals and other injury can potentially induce endoplasmic reticulumstress(ERS)-initiated apoptotic signaling. Previous study showed that the cardioprotectiveeffect accorded by des-aspartate-angiotensin Ⅰ (DAA-Ⅰ) was the result of itsanti-inflammatory actions on early inflammatory processes in myocardial ischemia-reperfusion injury. However，it has not been established that whether DAA-Ⅰcould protect CMECs against I/R injury and whether ERS phenomenon is involved. In ourstudy，we explore the protective effect and possible mechanism of DAA-Ⅰ against I/RIof cardiac microvascular endothelial cell.Methods:1、CMECs were isolated from the adult Sprague-Dawley rats hearts. Build the simulatedischemia/reperfusion model:culture medium was replaced with serum-free DMEM2hbefore the start of the experiment. The cells were then treated with ischemic buffersolution with ischemia buffer and cultures were placed in an hypoxia incubator with O2concentration＜1%; following SI，the medium was replaced with glucose-containingbuffer, and cultures were placed in an incubator at~20–21%O2.The CMECs wererandomized into3groups:(1) control group;(2) simulated ischemia/reperfusiongroup(SI/R)：(3) simulated ischemia/reperfusion+DAA-Ⅰ group (SI/R+DAA-Ⅰ).2、Cell viability of CMECs was measured by MTT assay and migration ability wasdetected by Trans-well assay and cell scratch wound assay. Apoptosis of CMECs wasdetected by caspase-3activity and TUNEL method.3、Expression of ERS related proteins such as GRP78，CHOP and cleaved caspase-12were analyzed by western blot. And the expression of GRP78mRNA and CHOP mRNAwere analyzed by Real-time qPCR.Results:1、Successfully isolated CMECs from the adult SD rats hearts.And the CMECs wererandomized into three groups.Then model of simulat ischemia/reperfusion wassuccessfully established.2、Both the cell viability and migration ability were impaired after SI/R (P<0.01vscontrol)，and the apoptosis index was significantly increased compared with control group(P<0.01). While administration of DAA-I during reperfusion dramatically enhanced theviability(0.33±0.012vs0.23±0.015，P <0.01vs SI/R)，increased the migration ability,decreased the caspase-3activity， and decreased the apoptosis index(15.18%±0.40vs 24.85%±0.67，P <0.01vs SI/R).3、 Administration of DAA-Ⅰ during reperfusion significantly down-regulated theexpression of GRP78，CHOP and cleaved caspase-12. Administration of DAA-Ⅰ duringreperfusion significantly down-regulated the expression of GRP78mRNA andCHOPmRNA.Conclusion:DAA-Ⅰcould exert protective effect on CMECs against ischemia/reperfusion injury.This protection has connection with down-regulation of ERS related proteins expression.