Protective Effects Of The Total Flavone From Ginako Biloba Leaf Capsule Against Acute Myocardial Ischemia-reperfusion Injury(MIMR) In Rat

Aim: The purpose of this study was to establish the pathological model of left anterior descending?LAD?coronary artery ligation and to investigate the protective effects of the total flavone from Ginkgo biloba leaf capsule?TFGC?against acute myocardial ischemia-reperfusion injury?MIRI?in rat.Methods: 1.The MIRI model was established by ligation of the rat LAD coronary for 30 minute and reperfused for another 120 minute.The limb lead II electrocardiography of normal and ischemia-reperfusion phase was recorded.2)60 male Sprague Dawley?SD?rats were randomly assigned into sham operation group?saline?,ischemic-reperfusion damage group?saline?,TFGC pretreatment groups(100,50 and 25 mg·kg^-1·d^-1)and positive control group(Danshen droppill 270 mg·kg^-1·d^-1).Indicated treatments were administered via gavage with a dose of 5mg·kg^-1·d^-1for seven days continuously.The operations were performed at the eighth day.3)Colorimetric methods were used to assess the activities of lactate dehydrogenase?LDH?,creatine kinase?CK?,creatine phosphokinase MB isoenzyme?CK-MB?and aspartate aminotransferase?AST?in serum and cardiac tissue.4)Enzyme-based colorimetric methods were used to measure the activities of antioxidant enzymes,including superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?and xanthine oxidase?XOD?in rat serum and cardiac tissue,the levels of malondialdehyde?MDA?,and the activities of crucial metabolic enzyme Na⁺-K⁺-ATPase and Ca²⁺-ATPase.5)Hematoxylin and eosin?HE?staining was used to assess the morphological changes in cardiac tissue,and triphenyl tetrazolium chloride?TTC?staining was used to determine the degree of myocardial infraction.6)Statistics were performed with SPSS 11.0.Results: 1)The success rate of MIRI model?calculated by successful cases/total cases?was90%.10 rats from same batch were used to replenish lost animals.2)Comparing to sham group,the activities of LDH,CK,CK-MB and AST in the serum of ischemic-reperfusion damage group?model group?were found significantly higher?P<0.01?,while the pretreatment with TFGC dose-dependently decreased these activities in rat serum comparing to the model group?P<0.05?,suggesting cardioprotective effects.3)Remarkably lower activities of LDH,CK,CK-MB and AST in the myocardium from rats in the model group relative to control?P<0.01?,while TFGC pretreatment dose-dependently elevated the activities of all these enzymes in myocardium comparing to model group?P<0.05?,suggesting membrane-stabilizing effects in myocardium.4)Significantly lower activities of serum SOD and GSH-Px,higher activity of XOD and concentration of MDA were observed in rats from model group relative to those from control group?P<0.01?.Except for the 25mg·kg^-1·d^-1TFGC treatment,all other treatments significantly improved the activities of serum SOD and GSH-Px,while remarkably decreased the activity of XOD and concentration of MDA?P<0.05?.Identical change patterns were observed in the antioxidant endpoints?SOD,GSH-Px,XOD and MDA?in the myocardium as the serum ones.6)Comparing to the sham group,activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase were found to be significantly lower?P<0.01?,while 100 mg·kg^-1·d^-1 TFGC treatment remarkably increased the activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase relative to model group?P<0.01?.A trend of enzymatic activities elevation was observed following 50 and 25mg·kg^-1·d^-1TFGC treatment,but not statistically significant?P>0.05?.7)As indicated by HE staining and light-microscope observation,normal myofibril morphology without cell swelling or necrosis were observed in tissues from sham group;on the other hand,widespread fusion lesions,cellular swelling,disrupted myofibril and neutrophil granulocyte filtration were present in tissues from model group.Meanwhile,relatively normal myofibril and cellular morphology were observed in tissues from 50 or 25 mg·kg^-1·d^-1 TFGC treatment group,but some cellular swelling and necrosis were still present.Additionally,relatively normal myocardial fibers and cellular morphology without apparent cellular swelling and necrosis were observed in tissues from 100 mg·kg^-1·d^-1TFGC treatment group,indicating dose-dependent protective effects against MIRI.8)TTC staining results indicated normal myocardium for rats in sham group?no white area?,while the rats in model group had over50 % myocardial infarction area?P<0.01?,large white areas were present,especially for those area below the ligation.The infraction area in rats treated with 100,50 or 25mg·kg^-1·d^-1 TFGC was dose-dependently decreased to approximately 25%,35% and 45%,respectively?P<0.05?.Conclusion: MIRI model was successfully established,and the tested doses of TFGC had protective effects against MIRI,which is associated with antioxidant effects,myocardium membrane stabilizing effects,and improvement of Na⁺-K⁺-ATPase/Ca²⁺-ATPase activities thus less calcium overload in myocardium.