Protect Effects And The Underlying Mechanisms Of Total Flavonoids Extracted From Bayberry Bark Against Ischemic Heart Disease

Objective:In order to explore the protective effects and its mechanism of Total flavonoids extracted from Bayberry bark(TFB)against ischemic heart disease.Methods:The experiment was carried out both in vivo and in vitro to research and validation.First,we adopted Male Sprague-Dawley(SD)ratswere intragastrically dosed with TFB(5,10,and 20 mg/kg)for 15 d,and Subcutaneous injection of isoproterenol caused by acute myocardial ischemia,then,the serum was collected for the measurement of CK,AST,and LDH levels,myocardial homogenateswere prepared for the analysis of MDA,SOD,and CAT activities and observed the histopathological examination.The experiment consist of six groups:control group(group C);model group(group ISO);ISO+ TFB low,middle and high concentration group(5,10,and 20 mg/kg);positive controlgroup(Di-ao 80 mg/kg).Second,we adopted H9c2cells that cultured in vitro,to simulate myocardial ischemia/reperfusion injury in clinical by hypoxia/reoxygenation(H/R)in H9c2cardiocytes and observe the effect of TFB on hypoxia/reoxygenation-induced injury in H9c2cells.The experiment consists of five groups:control group(group C);model group(group H/R);H/R+TFB low,middle and high concentration group(1.5625,3.125,6.25 ?g/mL).We adopted the following methods to test the corresponding indicators:the methy thiazolyl tetrazolium(MTT)method was applied to detect the cell viability;collect the cell supernatant and measured the vitality of lactate dehydrogenase(LDH),the content of malondialdehyde(MDA),and the activity of superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-PX)in accordance with the manufacturer's instructions;using Carboxy-H2DCFDA staining to detect ROS levels in cells through flow cytometry;using Annexin V/PI double staining methods to observe apoptosis in cells with flow cytometry;with JC-1 fluorescence labeling method and flow cytometry to detect the mitochondrial membrane potential;the use of Caspase-3 staining kit to detect the activity of Caspase-3 in H9c2 cells;Western Blot was adopted to detect the expression of related protein.Results:The effect of TFB on biochemical variables in serum and myocardial homogenates:the results show that TFB decreased the levels of LDH,CK,AST,and MDA(P<0.05),as well as enhanced the activities of SOD and CAT,compared,with the ISO group(P<0.05);the effect of TFB on cardiac pathology change in mice:the results show that no abnormal changes were detected in the control and positive control groups,whereasa large number of myocardial necrosis,fibrosis,and neutrophil granulocyte infiltration of the heartwere observed in the model group;the effect of TFB on H9c2 cell viability:the cell viability of the control group was considered as 100%,whereas that of the H/R group decreased to 73.97%± 4.55%.The treatment groups exhibited a dose-dependent protective effect on the myocardial injury induced by H/R compared with the H/R group.Moreover,the cell viability increased to 89.83%± 4.07%when treated with 6.25 ?tg/mL TFB for 12 h.Hence,the optimal TFB concentration was 6.25 ?g/mL and the optimal incubation time was 12 h;the effect of TFB on LDH,MDA levels and SOD,CAT,GSH-PX activity in H9c2 cell:LDH and MDA levels significantly increasedand SOD,CAT,GSH-PX activity was significantly decreased in H/R group compared with the control group(P<0.05);however,TFB treatment notably decreased LDH,MDA levels(P<0.05)and increased SOD,CAT,GSH-PX activity(P<0.05);the influence of TFB on intracellular reactive oxygen species(ROS)levels in H9c2 cell:Compared with control group,ROS levels were significantly increased(P<0.05)in H/R group,after myricitrin incubated,ROS levels were significantly decreased(P<0.05);the effect of TFB on apoptosisin H9c2 cell:analysis showed that the apoptosis rate was 4.09%in the control group and 2.03%in normal cells combined with the TFB group.In addition,the cell apoptosis rate was 11.04%in H/R group and 4.62%in the H/R combined with TFB group;the impact of TFB on the mitochondrial membrane potentialin H9c2 cell:the presents that the positive rate of cells was 94.57%in the control group,decreased to 66.92%in the H/R group,and increased to 90.26%after TFB treatment;the influence of TFB on Caspase-3 activity:the result shows that the activity of Caspase-3 significantly increased in the H/R group but decreased in the H/R combined with TFB group(P<0.05);the effect of TFB on the expression of related proteinin H9c2 cell:TFB treatment was applied at different times(0 h to 24 h)after hypoxia.Each protein changed at different periods.The expression of Bax significantly decreased(P<0.05),whereas those of Bcl-2,Procaspase-3,P-AKT,P-GSK3?,and P-ERK increased at different periods(P<0.05).However,the total AKT,GSK3?,and ERK did not change(P>0.05);furthermore,we evaluated the effect of TFB on normal cells and hypoxic-treated cells.After hypoxia,the expression of P-AKT,P-GSK3?,and P-ERK significantly increased in the H/R combined with TFB treatment group compared with the model group(P<0.05).However,the total AKT,GSK3?,and ERK did not change in these four groups(P>0.05);after adopting inhibitor(LY294002),the expression level of Bax significantly decreased(P<0.05),whereas those of Bcl-2,Procaspase-3,P-AKT,P-GSK3?,and P-ERK increased in the H/R combined with TFB treatment groups(P<0.05).However,the expression level of Bax significantly increased when treated with 50 ?mol/L LY294002 for 2 h(P<0.05),whereas those of Bcl-2,Procaspase-3,P-AKT,P-GSK3?,and P-ERK evidently decreased(P<0.05).Conclusion:TFB had protective effect on ISO induced acute myocardial ischemia;TFB had antioxidant and anti-apoptosis effects;TFB had protective effect on hypoxia/reoxygenation induced injury in H9c2 cells,and these have releated to activate AKT protein,promote GSK-3? and ERK phosphorylation,at the same time,it can promote the expression of anti-apoptotic protein Bcl-2,and inhibit the expression of pro-apoptotic protein Bax,thereby reducing the ratio of Bax/Bcl-2,to achieve the anti-apoptotic effects.