| Objective:According to the guide for diagnosis and treatment of heart failure in 2014,heart failure(HF)is the final stage of various heart diseases,and the increasing incidence and mortality have caused a substantial burden to the society.It will lead to severe insufficiency of oxygen supply in the heart due to acute myocardial infarction(AMI),then resulting in systolic and diastolic dysfunction of the heart,and changes in energy metabolism(especially lipid metabolism and glucose metabolism).The aim of this study was to improve the cardiac function of AMI rats by regulating myocardial lipid and glucose metabolism,then ligation of left anterior descending coronary artery(LAD)was adopted to prepare AMI animal models of Sprague-Dawley(SD)rats,and oxygen-glucose deprivation-reperfusion(OGD/R)was used to induce H9C2 myocardial cell model.Finally the mechanism of regulating myocardial energetic disorder after AMI of DQP was explored.This research can provide new intervention steps for the prevention and treatment of HF and the development of international acknowledged drugs for heart failure.Methods:1.Male SD rats were randomly divided into 4 groups:sham operation group(Sham),model group(Model),danqi pill(DQP)treatment group and rosiglitazone(RSG)treatment group.Traditional Chinese medicine(TCM)compound danqi pill(DQP,1.5g/kg)and positive control rosiglitazone(RSG,24mg/kg)was adopted by gavage administration.Twenty eight days after surgery,two-dimensional echocardiography was used to evaluate cardiac function and hematoxylin eosin(HE)staining was applied to visualize cardiomyocyte morphological changes.High performance liquid chromatography(HPLC)was performed to detect contents of adenosine phosphates(ATP,ADP and AMP),total adenine nucleotides(TAN)and energy charge(EC)were calculated simultaneously.Based on the above results,we evaluated the efficacy of DQP on myocardial energy metabolism disorder after AMI.2.Serum levels of total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)and free fatty acid(FFA)were detected.Western blotting(WB)was applied to determine the protein expressions of carnitine palmioyl transferase 1A(CPT-1A),long chain acyl-CoA dehydrogenase(ACADL)and sterol carrier protein 2(SCP2)in fatty acid oxidation(FAO)pathway.Based on the above results,we evaluated the mechanism of DQP on regulating lipid metabolism.3.Glucose and lactate were detected by automatic biochemical analyzer.Positron emission tomography and computed tomography(PET-CT)was performed to evaluate glucose standard uptake value(SUV)and assess the glucose metabolism in the myocardium.Glucose and lactate levels in the myocardium of rats were detected by assay kits according to the manufacturer’s instructions.Immunohistochemistry(IHC)and WB were applied to detect the key protein expressions of glucose transporter protein 4(GLUT4),phosphofructokinase(PFK),glycogen synthase kinase 3β(GSK-3β),glycogen synthase(GS),phosphoinositide 3 kinase(PI3K),serine/threonine kinase(AKT)and hypoxia inducible factor-1α(HIF-1α)in glucose metabolism pathway.Based on the above results,we dynamically evaluated the mechanism of DQP on regulating glucose metabolism.4.AMI animal model and OGD/R-induced H9C2 cardiomyocyte model were used to reveal the pathway and mechanism of DQP on regulating the myocardial glucose and lipid metabolism in vivo and vitro.IHC and WB were applied to detect the protein expressions of peroxisome proliferator-activated receptor γ(PPARy),retinoic acid X receptor α(RXRα)and peroxisome proliferator-activated receptor γ coactivator 1α(PGC-1α)in cardiac tissues.Cell counting kit 8(CCK-8)was used to detect cell viability.Glucose,lactate and ATP levels in H9C2 cardiomyocyte were detected by assay kits according to the manufacturer’s instructions.WB was applied to detect the protein expression of PPARγ in H9C2 cardiomyocyte.Results:1.Effects of DQP in HF rats after AMIThe results showed that values of ejection fraction(EF)and fractional shortening(FS)in the model group were significantly reduced(vs.Sham,P<0.001),and values of left ventricular internal dimension systole(LVID;s)and left ventricular internal dimension diastole(LVID;d)were increased significantly(vs.Sham,P<0.001),indicating that the HF model was successfully induced.After the treatment of DQP,values of EF and FS were increased significantly(vs.Model,P<0.001)and LVID;d was decreased significantly(vs.Model,P<0.05),indicating that DQP could improve ventricular hypertrophy and enhance cardiac function in HF rats after AMI.The results of HE staining showed that the disorder of myocardial cells in the DQP group was obviously reduced,only the rare cells lost the normal structure and there was an infiltration of inflammatory cells.The results of HPLC showed that ATP,ADP and EC levels in model group were significantly decreased(vs.Sham,P<0.001)while AMP was increased significantly(vs.Sham,P<0.05).After the treatment of DQP,levels of ATP,ADP and EC were significantly increased(vs.Model,P<0.01)and AMP was decreased significantly(vs.Model,P<0.05),indicating that DQP could effectively improve the energy metabolism in HF rats after AMI.2.Effects of DQP on regulating cardiac lipid metabolism in HF rats after AMIThe results showed that the level of HDL-C in model group was significantly decreased(vs.Sham,P<0.01)and the levels of TG and FFA were increased significantly(vs.Sham,P<0.05;P<0.01),TC and LDL-C levels were also increased while there was no statistical differences(vs.Sham,P>0.05.DQP could significantly improve the level of HDL-C(vs.Model,P<0.05)and reduce levels of TC,TG,LDL-C and FFA(vs.Model,P<0.01;P<0.01),indicating that DQP could correct the disorders of blood lipid metabolism.WB results showed that DQP could significantly increase the expressions of CPT-1A,ACADL and SCP2 in the fatty acid β-oxidation pathway(vs.Model,P<0.01).3.Effects of DQP on regulating cardiac glucose metabolism in HF rats after AMIDQP could significantly reduce the concentrations of glucose and lactate in serum(vs.Model,P<0.01;P<0.001).Meanwhile,PET-CT results showed that DQP could significantly reduce the mean SUV in myocardium(vs.Model,P<0.001),suggesting that DQP could effectively promote the myocardial glucose metabolism.The results of glucose and lactate detections in myocardium showed that DQP could significantly reduce the level of glucose(vs.Model,P<0.05)and increase the level of lactate(vs.Model,P<0.05).WB and IHC results showed that DQP could effectively increase the protein expressions of GLUT4 and PFK(vs.Model,P<0.05)in glycolysis pathway and then promote glycolysis.Moreover,DQP could significantly reduce the expressions of phosphorylated glycogen synthetase kinase 3β(p-GSK3β)and phosphorylated glycogen synthase(p-GS)in glycogen synthesis pathway(vs.Model,P<0.001).DQP could up-regulate PI3K and phosphorylated serine/threonine kinase(p-AKT)(vs.Model.P<0.01;P<0.001)as well as down-regulate HIF-1α(vs.Model,P<0.001)in the upstream pathway of glucose metabolism.4.Effects of DQP on PPAR γ pathway in HF rats after AMIWB and IHC results showed that DQP could significantly increase the protein expressions of PPARγ,RXRα and PGC-1α(vs.Model,P<0.001)in HF rats after AMI.In the H9C2 cardiomyocytes induced by OGD/R,the morphology of cardiomyocytes results showed that the conditions of cell shrinkage and deformation were obviously improved and the number of adherent cells increased significantly after treatment of DQP.The results of detections of ATP,glucose and lactate in H9C2 cardiomyocytes showed that the contents of ATP and lactate in DQP group were increased significantly(vs.Model,P<0.05)and the glucose content was decreased significantly(vs.Model,P<0.05).WB results showed that DQP-400(DQP,400μg/ml)could significantly increase the protein expression of PPARγ(vs.Model,P<0.05),while DQP adding PPARγ inhibitor T0070907 group(DQP-400+T0070907)and T0070907 group had no statistical differences,indicating that DQP regulated cardiac glucose and lipid metabolism by regulating PPARγ.Conclusions:1.Our previous studies suggest that DQP can effectively improve the decrease of left ventricular function after AMI and reduce the infiltration of inflammatory cells in myocardium as well as enhance energy metabolism significantly,suggesting that the mechanism of DQP to improve the cardiac function after AMI is closely related to the regulation of myocardial energy metabolism.2.Regulations of DQP on lipid metabolism is mainly related to the correction of lipid metabolism disorder(TC,TG,HDL,LDL and FFA),and the expressions of CPT-1A,SCP2 and ACADL in FAO pathway.3.DQP can regulate glucose metabolism on promoting glucose uptake,glycolysis and inhibiting glycogen synthesis.DQP and RSG have significant inhibitory effects on glucose,lactate and myocardial SUV levels,briefly the regulation of DQP on GLUT4 and PFK as well as the regulation of PPARγ-PI3K/Akt pathway.4.This study provides novel therapeutic targets and intervention approaches for the treatment of HF induced by AMI through the elucidation of the mechanism of myocardial energy metabolic disorder after the treatment of DQP,which was a TCM compound.And it provides experimental basis for revealing the characteristics of TCM compound and the mechanism of pharmacological efficacy,moreover,it also provides experimental basis for new drug development and pharmacological mechanism research on cardiovascular diseases such as HF after AMI. |
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