The Effects And Its Mechanism Research Of Ilexoside E On Myocardial Ischemia-reperfusion Injury

Objective:Ilexoside E is a saponins active component of Ilex pubescens Hook.et Arn(I.pubescens).The early results of our group show that Ilexoside E can improve the survival rate of myocardial cells in hypoxia/reoxygenation,reduce the release of enzymes and ROS,decrease the apoptosis rate of myocardial cells,and its cardio-protective effect is related with inhibiting the expression of Caspase-3 apoptosis protein.However,there are no papers which report the influence of Ilexoside E against myocardial ischemia-reperfusion injury in vivo.This paper established H9c2 myocardial hypoxia/reoxygenation injury model,to compare the protective effect on myocardial cells of Ilexoside E with the listed TCM injections in vitro,and explore its protective mechanism through detecting intracellular ROS levels and the expression of Caspase-3,Cleaved caspase-3,Bcl-2,Bax and other related appoptosis gene proteins.Then verified its effects through the model of rat myocardial ischemia-reperfusion injury in vivo,which provide supports for druggability research and the new drug development of Ilexoside E.Methods:1.The comparison of Ilexoside E with listed cardiovascular TCM injections in protection against hypoxia/reoxygenation injury of H9c2 myocardial cells.H9c2 myocardial cells were randomly divided into control group(Control group),hypoxia/reoxygenation model group(H/R group),H/R+ Ilexoside E(10,50,250?g/mL)group,H/R+Salvianolate(5,10,20?g/mL)group,H/R+Maodongqing injection(0.2,2,20 L/mL)group,H/R+ Safflower yellow injection(0.2,2,20?g/mL)group,H/R+ Xueshuantong injection(4,20,100?g/mL)group,H/R+ Shenfu Injection(0.2,1,5 L/mL)group,H/R+ Danhong injection(0.2,1,5 L/mL)group,H/R+Shengmai injection(0.2,1,5 L/mL)group.Each group was given the corresponding drug to precondition for 24h before hypoxia.After hypoxia for 4h and reoxygenation for 4h,the survival rates of myocardial cells were detected by CCK-8 method.2.Research on the mechanism of Ilexoside E inhibits hypoxia/reoxygenation injury of rat H9c2 myocardial cells.H9c2 myocardial cells were randomly divided into control group(Control group),hypoxia/reoxygenation model group(H/R group),H/R+ Ilexoside E(10,50,250?g/mL)group.Given the corresponding drug to precondition for 24h before hypoxia.After hypoxia for 4h and reoxygenation for 4h,intracellular reactive oxygen species(ROS)was detected by H,DCF-DA fluorescent probe,and the expression of related apoptosis protein,such as Caspase-3,Cleaved Caspase-3,Bcl-2 and Bax,were detected by Western Blot.3.Research on the protective effects of Ilexoside E on myocardial ischemia-reperfusion injury in vivo.Male SD rats were randomly divided into sham surgery group(Sham group),ischemia-reperfusion model group(Model group),the low dose of Ilexoside E +I/R group(10mg/kg,I/R + EL group),the high dose of Ilexoside E +I/R group(40mg/kg,I/R + EH group),salvianolate group +I/R(43mg/kg,I/R+Salvianolate group).The drugs were injected into the tail vein directly before surgery.After ligation for 30min and reperfusion for 4h,the area of myocardial infarction and ischemia were measured by Evans-blue and TTC double staining.And the serum samples were collected to detect the content of LDH,AST,CK-MB,IL-6 and CRP.Results:1.The comparison of Ilexsaponin E with listed cardiovascular TCM injections in protection against hypoxia/reoxygenation injury of rat H9c2 myocardial cells.Compared with Control group,the survival rate of H/R group had significantly descended,which had a highly statistical significance[(52.57±4.33)%vs.(100.00±8.41)%,P<0.01].Compared with H/R group,the survival rates of each drug group were increased.The protective effect of Ilexoside E on hypoxia/reoxygenation injury of H9c2 myocardial cells is better than the safflower yellow hormone injection(P<0.05)on the whole,and showed no statistical significance(P>0.05).The groups of Salvianolate,Mao Dongqing injection,Xueshuantong injection,Shenfu injection,Danhong injection and Shengmai Injection showed no statistically significance(P>0.05).2.The influence of Ilexoside E on intracellular ROS and the expression of apoptosis protein in hypoxia/reoxygenation H9c2 myocardial cell.Compared with the Control group,the relative fluorescence intensity of H/R group had a highly statistical significance[(164.37±7.46)%vs.(100.00±8.26)%,P<0.01].Compared with H/R group,the relative fluorescence intensity of Ilexoside E in low dose group had no statistical significance[(151.99±9.84)%,P>0.05].The relative fluorescence intensity of Ilexoside E in medium and high dose group are(138.97±8.37)%and(140.31 ±3.21)%,which had highly statistical significance with H/R group(P/<0.01).Compared with Control group,in H/R group,the expression level of Caspase-3,Cleaved Caspase-3 and Bax proteins were significantly increased,but the expression level of Bcl-2 protein decreased significantly,which all had highly statistical significance(P<0.01).Compared with H/R group,the expression levels of Caspase-3,Cleaved caspase-3 and Bax protein in each Ilexoside E group were decreased,which all had statistical significance(P<0.05),while Bcl-2 protein expression was significantly increased,and has a highly statistical significance(P<0.01).The expression levels of Bcl-2/Bax protein in each group were higher than H/R group(P<0.05).3.The influence of Ilexoside E on myocardial infarction area and the content of LDH,AST,CK-MB,IL-6,CRP in serum of rat myocardial ischemia-reperfusion injury in vivo.Compared with Sham group,the IS/AAR values of Model group increased significantly with highly statistical significance[(41.55±8.99)%,P<0.01].The IS/AAR values of the low/high dose group of Ilexoside E and salvianolate group were(25.89±9.33)%?(20.49±6.55)%?(19.66 + 2.58)%,respectively.Compared with the Model group,the difference of IS/AAR had highly statistical significance(P<0.01)in each drug groups.And it is no significant difference(P>0.05)bewteen Ilexoside E and positive drug(salvianolate).Compared with Sham group[(8387.65±769.88)U/L?(12.88±0.99)U/L?(3.88±0.15)%?(25.20±2.12)ng/L?(1.73±0.15)mg/L],the contents of LDH,AST,CK-MB,IL-6 and CRP of Model group increased obviously[(11093.05±639.71)U/L,(60.95± 10.54)U/L,(4.85±0.49)ng/mL,(32.36±2.53)ng/L,(2.49±0.19)mg/L],the difference had a highly statistical sigrnificance(P<0.01).The contents of LDH,AST,CK-MB,IL-6 and CRP of Ilexoside E group were all lower than Model group,especially for the high dose group decreased more obviously[(8696.46±1103.49)U/L,(40.51±7.09)U/L,(3.80±0.17)ng/mL,(27.40±4.76)ng/L?(2.13±0.36)mg/L],and it was similar to salvianolate group[(9177.57±1016.95)U/L,(38.75±8.66)U/L,(3.87±0.54)ng/mL,(27.75±4.08)ng/L,(2.16±0.25)mg/L].Conclusion:1.The protective effects of Ilexoside E on myocardial cells were similar to the previous cardiovascular traditional Chinese medicine injections in vitro.2.Ilexoside E can reduce intracellular ROS,inhibit the expression of apoptosis protein,such as Caspase-3,Cleaved Caspase-3 and Bax,increase the expression of Bcl-2,thereby reducing the hypoxia/reoxygenation induced apoptosis in rat H9c2 myocardial cells.3.The precondictioning of Ilexoside E can reduce the area of myocardial infarction and the contents of LDH,AST,CK-MB,IL-6,CRP in serum,and has a certain protective effect on myocardium ischemia-reperfusion injury.Its protective effect is similar to the positive drug(salvianolate).