The Protective Effect And Mechanism Of Statins On Myocardial Microvascular Endothelial Cells In High Glucose Enviroment

Part 1 Effects of statins on patients with acute myocardial infarction and diabetes before primary PCIBackground:The efficacy and safety of pre-operative high-dose statins for primary PCI in patients with acute myocardial infarction and diabetes are not yet clear now.Objective:To investigate the efficacy and safety of statins on patients with acute myocardial infarction and diabetes undergoing primary PCI.Methods:140 patients with STEMI and diabetes undergoing primary PCI in Department of Cardiology Fengxian Central Hospital were selected.They were randomly divided into four groups,loading-dosage group(atorvastatin 80 mg,35 cases),loading-dose group(rosuvastatin 20 mg,35cases),routine dose group(atorvastatin 20 mg,35 cases),routine dose group(rosuvastatin 5 mg,35 cases).The levels of high sensitive C-reactive protein(hs-CRP),Serum amyloid protein(SAA),ALT,BNP,cTnI,Endothelin(ET),Nitric oxide(NO),Tissue type plasminogen activator(t-PA),Plasmino-gen activator inhibitor(PAI),MDA and SOD were detected before and at 2 hours after PCI.18 lead electrocardiogram was recorded after two hours of PCI.Calculate ST-segment resolution(STR).Corrected TIMI frame count(CTFC)after PCI were compared.LVEF,Hepatorenal function and the incidence of major adverse cardiovascular events(MACE)were followed up 1 month later afer PCI.Results:The levels of hs-CRP,SAA,cTnl,NO,t-PA increased after two hours of PCI(P<0.05),but the levels of hs-CRP,SAA,cTnI in loading dose group were all lower than in routine dose group(P<0.05);the levels of NO?t-PA in loading dose group were all higher than in routine dose group(p<0.05);the levels of ET-1?PAI-1 deereased after two hours of PCI(P<0.05),but in loading dose group were all lower than in routine dose group(P<0.05),all indicators were not statistically different among the loading dose groups(P>0.05).It was not significantly statistical different between the loading dose groups and routine dose groups in the level of MDA increased and the levels of SOD before PCI.After 2 hours of reperfusion,the level of MDA increased and the levels of SOD decreased in each group.However,the extent of increasing of MDA in loading dose group was lower than that in conventional-dose group,the extent of decreasing of SOD in loading dose group was higher than conventional-dose group,which was statistically different(P<0.05);STR,CTFC,LVEF,Hepatorenal function and MACE were better in the loading dose groups after PCI(P<0.05),but it was not statistically different among the loading dose groups(p>0.05),There was no significant statistical difference between the conventional dose group and the loading dose group.Conclusion:Loading-dose statin in patients with STEMI and diabetes before undergoing primary PCI can improve myocardial microcirculation perfusion,reduce oxidative stress,decrease the necrosis of myocardial cells and improve the short-term prognosis of the patients.There was no significant difference in clinical effects between atorvastatin and rosuvastatin.Part 2 Isolation,Culture and Identification of Myocardial Microvascular Endothelial CellsObjective:To explore the feasibility of separating adult rat myocardial microvascular endothelial cells by enzymatic digestion to obtain cells of high purity and activity.Methods:S-D Rat myocardial microvascular endothelial cells were isolated by enzyme digestion,cultured and passaged.Dil-acetylated-low density lipoprotein(Dil-Ac-LDL)was used to identify passaged cells.Results:Myocardial microvascular endothelial cells can be obtained by enzyme digestion method,which shows the characteristics of relatively short time,stable and reliable quality,and high cell purity.Conclusion:Enzyme digestion method can be used to obtain high purity and good condition of myocardial microvascular endothelial cells,which can be used for subsequent experiments.Part 3 The protective effect and mechanism of statins on myocardial microvascular endothelial cells injury in high glucose environmentObjective:To study the protective effect and mechanism of statins on myocardial microvascular endothelial cells injury in high glucose environment.Methods:The S-D rats were administrated with atorvastatin(10 mg/kg/day),rosuvastatin(5 mg/kg/day)or placebo for one week.The expression of SmgGDS was analyzed by western blot.S-D rat myocardial microvascular endothelial cells were cultured in vitro and divided into control group,hyperglycemia group,atorvastatin group and atorvastatin?high glucose group.The generation of reactive oxygen species(ROS)were detected using Superoxide Assay Kit.Apoptosis of cells were detected by TUNEL.The levels of SmgGDS expression were measured using western blot and measured again after the expression of Aktl and ?1-Integrin were down-regulation using short-interfering RNA(siRNA)technique.Results(1)The level of ROS was higher in the high glucose group than in the control group(t=4.154,P<0.01),lower in the atorvastatin group and the atorvastatin+high glucose group than in the high glucose group(t=4.233 and 2.893,both P<0.05).(2)The TUNEL results showed that the proportion of apoptotic cells was higher in high glucose group than in control group(t=4.058,P<0.01),lower in atorvastatin group and atorvastatin+high glucose group than in the high glucose group(t=4.157 and 2.601,both P<0.05).(3)The expression level of Aktl was lower in the high glucose group and the high glucose+atorvastatin group after transfection of Aktl-siRNA than in the mock control group(t=4.058 and 4.167,both P<0.01).The expression levels of pl-integrin was lower in the high glucose group and the high glucose+atorvastatin group after transfection of ?1-integrin-siRNA than in the mock control group(t=4.073 and 4.215,both P<0.01).(4)Western blot analysis showed that the relative expression level of SmgGDS in the low dose of atorvastatin group(lumol/L)and atorvastatin high dose group(10umol/L)was higher than in the control group(t=2.671 and 2.832,bothP<0.05)The relative expression level of SmgGDS in high dose group was higher than that in low dose group(t=2.612,P<0.05).The expression level of SmgGDS was lower in high glucose+atorvastatin group and in the high glucose group after transfection of Aktl-siRNA than in the high glucose+atorvastatin+mock group,higher in the high glucose+atorvastatin+mock group than in the high glucose+mock group(t=4.051,P<0.01).The expression level of SmgGDS was lower in high glucose+atorvastatin group and the high glucose group after transfection of pl-integrin-siRNA than in the high glucose+atorvastatin+mock group,higher in the high glucose+atorvastatin+mock group than in the high glucose+mock group(t=4.068,P<0.01).The expression level of Akt phosphorylation in high glucose+atorvastatin group was higher at 10 minutes than at 5 minutes(t=2.608,P<0.05),higher at 15 minutes than at 10 minutes(t=3.127,P<0.05).The expression level of p-Akt/t-Akt was lower in the high glucose group after transfection of ?1-integrin-siRNA than in high glucose+mock group(t=3.371,P<0.05).Conclusions Statin could protect myocardial microvascular endothelial cells in high glucose environment from oxidative stress and apoptosis possibly by up-regulating SmgGDS partly through ?1-integrin/Akt1 pathway.