Effects Of Penehyclidine Hydrochloride On Cardiac Microvascular Endothelial Cells Apoptosis After Hypoxia/reoxygenation Injury In Rats

After acute coronary occlusion/ reperfusion therapy,vascular injury caused by microvascular dysfunction,limits the total(or complete) of tissue perfusion, effects of treatment.Such as micro-thrombosis,calcium overload may result in microvascular dysfunction,but vascular endothelial dysfunction is one of the main reasons.To relief or reduce reperfusion injury in endothelial function to reduce reperfusion injury became the focus of the study.Therefore,cultivation cardiac microvascular endothelial cells,establishment of the cardiac microvascular endothelial cells of the H/R model,has important meanings to the study of ischemia/reperfusion injury in the way of significance and their protection.In recent years,studies have shown that scopolamine anti-cholinergic drug on myocardial ischemia/reperfusion(ischemia/reperfusion,I/R) injury has a protective effect,while penehyclidine hydrochloride(PHC) is a new type of M receptor subtype blocking agent,does have the same effect? Therefore,this experiment simulated I/RI,establish the cardiac microvascular endothelial cells H/R model,observation PHC pretreatment and to give PHC after H/R to cardiac microvascular endothelial cells has a protective effect whether or not ,understanding of their mechanisms,to provide a scientific basis for clinical treatment of the myocardial reperfusion injury.AimEstablish the I/R model at cell leve,from the ability of cell proliferation, apoptosis rate,Caspase-3 and Bc1-2 protein expression,observation Penehyclidine Hydrochloride whether microvascular endothelial cells on myocardial ischemic/reperfusion injury has a protective effect or not and its possible mechanism.Methods1.Adult SD rat microvascular endothelial cell culture and identification This experiment use two methods to carry out cardiac vascular endothelial cell primary culture what are enzyme digestion and tissue piece method,1:2 subcultured.Use fluorescent labeling acetylate Dil-Ac-LDL method to identification.2.Establish the Myocardial microvascular endothelial cells in H/R model and experiment groupCutured cells are randomly divided into 4 groups,â‘ the control group, cultured under normoxic environment and do not give any treatment;â‘¡H/R group,exchange total culture medium into D-Hank's liquid,put it into 37℃,5%CO₂,95%N₂ a close tightly jar which is hypoxia for 2h,and then retrieve the total culture medium,put it into a normoxic environment to continue to culture for 4h;â‘¢PHC pretreatment group,give PHC(final concentration is 0.1μmol/L) to the cells before hypoxia;â‘£H/R+PHC group,after the H/R,give PHC(final concentration is 0.1μmol/L) to the cells,continue to culture 2h.3.Detection index and methodâ‘ Cell viability was detected by MTT colorimetry.MTT colorimetric measured the OD value(OD value of normal minus a certain group OD values)/normal group OD value×100%,use the cell death rate reflects the situation of cell injury.â‘¡Apoptosis rates were detected by flow cytometerDetection of each experimental group 5 specimens,Cellfit software to collect 10,000 cell,CellQuest data acquisition and analysis software to record the percentage of AP area,that is,cells percentage of apoptosis.â‘¢Caspase-3 and Bc1-2 expression were detected by Western-Blot.Results1.The cardiac microvascular endothelial cells of adult rat were cultured successfully.Use fluorescent labeling acetylate Dil-Ac-LDL method to identify the cells:they all obtained showed double posistiv for Dil-Ac-LDL and DAPI,according to the source of cells,they are CMECs.2.The H/R model of cardiac microvascular endothelial cells was established successfully.Exchange total culture medium into D-Hank's liquid,put it into a close tightly jar which is on the condition of37℃,5%CO₂,95%N₂ hypoxia for 2h,and then retrieve the total culture medium,put it into a normoxic environment to continue to culture for 4h.3.H/R group,the rate of cell death and apoptosis compared with the control group significantly increased,the difference was statistically significant(P＜0.01); PHC+H/R group,the cells death and apoptosis rate were declined than the H/R group,the differences were significance(P＜0.05);H/R+PHC group compared with the control group,although the cells and the apoptosis rate of were decline, but the difference was not statistically significant(P＞0.05).4.The expression of Caspase-3 of the treated groups are all higher than the control group(P＜0.05),PHC+H/R group lower than H/R and H/R+PHC groups, the differences are significance(P＜0.05),H/R+PHC group lower than H/R a little,but the difference was not statistically significant(P＞0.05).The he expression of Bc1-2 protein of H/R group lower than the control group significantly(P＜0.01),PHC+H/R group higher than the H/R group and H/R+PHC group,the differences are significance(P＜0.05),but the H/R+PHC group higher than the H/R a little,he difference was not statistically significant (P＞0.05).ConclusionPHC pretreatment has protection function of Cardiac microvascular endothelial cells after hypoxia/reoxygenation injury in rats,the protective effect of PHC maybe associate with down regulation of Caspase-3 expression and promotion of Bc1-2 expression.