| Background and purpose:Cardiovascular disease is a major public health problem,which threatens to people's health.According to the Global Burden of Disease(GBD)Study 2016,ischemic heart disease(IHD)has become the second leading cause of death in China,especially myocardial infarction(MI)is the main cause of sudden death.Although the methods such as thrombolysis,percutaneous coronary intervention(PCI),and coronary artery bypass grafting to rapid restoration of coronary artery blood flood can limit infarct size and eventually improve MI patients' survival,these methodsare not adequately prevented deterioration of cardiac function and ischemia-reperfusion injuiy.It is a pressing need to find novel therapeutic options for the clinical.With the concept of "energy deficit",people began to realize that supporting the endogenous energy of myocardium,optimizing myocardial metabolism patterns and enhancing the ability of myocardial cells to resist injury as a new therapeutic way.Yiqihuoxue decoction(YQHX),a Traditional Chinese Medicine(TCM)prescription re-developed based on the well-known formula Danggui Buxue decoction(DBD),has been widely used to prevent and treat ischemic heart disease.YQHX is widely used in the prevention and treatment of cardiovascular disease,including promoting angiogenesis,inhibiting inflammatory response,and regulating left ventricular functionand protecting mitochondrial function.However,the effects and mechanisms of YQHX on energy metabolism in the ischemic heart are unclear,especially in the cardiomyocytes.Based on the model of rat myocardial infarction in vivo and the hypoxia-induced cardiomyocyte injury in vitro,this study investigatedthat the effectsof YQHX on cardiomyocytes in myocardial ischemia,and then explored the potential mechanisms of YQHX on AMPK/PGC-1?pathway in myocardial ischemia.Part ? The effect and underling mechanism of YQHX in post-myocardial infarction injury.Aim of the study:This study was designed to investigate the effects and potential mechanisms of YQHX on AMPK/PGC-1? expression in myocardial ischemia.Materials and methods:The experiment was performed both in vivo and in vitro.1.Animal experiments:The rat MI model was conducted by left anterior descending coronary artery ligation.The 8-week-old male SD rats were randomly divided into 4 groups:?Sham group(Sham),?myocardial infarction model group(MI),?Yiqihuoxue decoction group(YQHX),?Trimetazidine group(TMZ).The changes of corresponding indicators at 7 days and 28days two time points were observed.Cardiac functions were detected by Echocardiography,and cardiomyocytes morphology were observed by hematoxylin-eosin(HE)staining.The ATP levels were detected by ATP Assay Kits and PGC-1? expression was evaluated by western blot in vivo.2.Cell experiments:In order to determine the optimal dose of YQHX for H9c2 myocardial cells,H9c2 cardiomyocytes were divided into 5 groups:?Control group(C),?Ischemia/Hypoxia group(I/H),?Low-dose YQHX group(Y1),?Middle-dose YQHX group(Y2),?High-dose YQHX group(Y4).In vitro,the effects of YQHX on H9c2 cell viability were evaluated by CCK-8 assay.LDH,MDA and ROS levels in serum and cells were measured by biochemical kits.The levels of mitochondrial membrane potential(??m)and intracellular reactive oxygen species(ROS)in hypoxia-induced H9c2 cells were evaluated by confocal microscopy and fluorescence,respectively.The mitochondrial ultrastructural details of H9c2 cells and myocardial tissues were observed by transmission electron microscopy(TEM).PGC-la,p-AMPK,NRF-1 and Tfam relevant gene and proteins after hypoxia were evaluated by RT-PCR and Western blot.In order to further explore the underling mechanism of YQHX on PGC-la expression in post-myocardial infarction injury,H9c2 cardiomyocytes were further divided into 4 groups:?Control group(Control),?Ischemia/Hypoxia group(I/H),?Middle-dose YQHX group(Y2),?Middle-dose YQHX+Compound c group(Y2CC),PGC-1?p-AMPK,NRF-1 and Tfam proteins after-hypoxia were evaluated by Western blot.Results:1.Echocardiogram results On the 7th day,cardiac function assessment:the left ventricular ejection fraction(LVEF)and short axis rate(LVFS)were significantly decreased in MI group(P<0.001).Compared with MI group,YQHX group and TMZ group significantly increased LVEF and LVFS(P<0.01).cardiac structural assessment:Compared with Sham group,MI group was significantly increased left ventricular internal dimension-diastole/systole(LVIDd/s)(P<0.01).Compared with MI group,YQHX group and TMZ group significantly decreased LVIDs(P<0.05),but LVIDd was no significant difference(P>0.05).On the 28th day,cardiac function assessment:LVEF and LVFS were significantly decreased in MI group(P<0.001).Compared with MI group,YQHX group significantly increased LVEF and LVFS(P<0.05).LVEF in TMZ group was significantly higher(P<0.05),but not in LVFS(P>0.05).cardiac structural assessment:Compared with Sham group,MI group was significantly increased left ventricular internal dimension-diastole/systole(LVIDd/s)(P<0.001).Compared with MI group,YQHX group and TMZ group significantly decreased LVIDd/s(P<0.05).2.HE stain results On the 7th day,the myocardial cells in the Sham group were neatly arranged.Compared with Sham group,MI group showed that the number of myocardial cells in the infarct marginal area was reduced,the structure was disordered,a large number of muscle fibers were broken,swelling,and a large number of inflammatory cells infiltrated in the interstitium.The arrangement of myocardial cells in the YQHX and TMZ groups was decreased inflammatory cell infiltration compared with the MI group.On the 28th day,the structure of myocardial cells in the Sham group was neat and clear.Compared with Sham group,MI group showed that abundant inflammatory cell infiltrates,intercellular space enlarged and extensive edema of cardiomyocytes.After treatment with YQHX and TMZ,there were numerous changes of the myocardium,including slight inflammatory cell infiltrates and mild edema of cardiomyocytes.3.Effect of YQHX on mitochondrial struction and function in post-myocardial infarction injury.On the 7th day,myocardium ultrastructure and myocardial fiber arranged neatly in the Sham group.Compared with Sham group,MI group showed myocardial fibers were disordered and the mitochondrial ultrastructure was significantly damaged.Compared with MI group,myocardial ultrastructural damage was significantly improved in YQHX group,such as complete mitochondrial structure,slightly enlarged and clustered.On the 28th day,the ultrastructure of myocardium in Sham group were normal and cardiac muscle fibers arranged regularly.After MI injury,myocardial ultrastructure did not displayregularly and mitochondrial ultrastructure disruption such as swelling,rupture,vacuoles and loss of cristae.Interesting,YQHX treatment improved MI-induced myocardial ultrastructure injury and induced the relative proliferation and accumulation of mitochondria.The 7 and 28 days ATP results showed that myocardial infarction decreased the production of ATP in rats(P<0.001),but YQHX increased ATP production to rescue mitochondrial function(P<0.001).4.Effects of YQHX on PGC-la expression in post-myocardial infarction injury.After 7 days of MI,PGC-la protein expression in MI group was significantly lower compared with Sham group(P<0.05),while YQHX group and TMZ group markedly enhanced PGC-1?expression(P<0.05).After 28 days of MI,the expression of PGC-la protein in MI group was significantly decreased(P<0.05).Compared with MI group,YQHX group was significantly increased PGC-la protein expression(P<0.05),but TMZ group failed to increase PGC-la activity.5.Effects of YQHX on H9c2 cardiomyocytes induced by hypoxia.H9c2 cells were cultured under ischemic/hypoxic conditions for 12h,24h,36h or 48h.CCK8 results showed that the percentage of survival cells was markedly decreased in the I/H group,which had a significant time-dependent reduction compared to the control group.YQHX had no cytotoxicity in normal H9c2 cells at the concentration range of 50-800?g/mL(P>0.05).Compared with the I/H group,YQHX at concentrations of 100 ?g/mL,200 ?g/mL,and 400 ?g/mL reversed I/H-induced cell death and significantly increased cell viability(P<0.05).In order to further evaluate the effects of I/H model and YQHX on H9c2 cardiomyocytes,the results showed that at 12h and 24h of hypoxia,the release of LDH in the hypoxia group was extremely aggravated in comparison to the control group for 12h,whereas YQHX treatment significantly reduced the level of LDH.The SOD activity was extremely diminished in comparison to the control group,whereas the level of MDA and ROS was increased in H9c2 cells after hypoxia for 12 or 24h(P<0.01).YQHX treatment markedly reduced this decrease of MDA and ROS,and increase of SOD activity(P<0.01).6.Effects of YQHX on H9c2 cardiomyocytes mitochondrial struction and mitochondrial membrane potential induced by hypoxia.TEM results showed that the changes in the hypoxia-induced mitochondrial morphology,including swelling,black matrix and loss of cristae compared to the control.However,YQHX treatment improved the mitochondrial ultrastructures in I/H-induced H9c2 cells.??mm of hypoxia group was lower than that of the control group(P<0.001).,whereas 100-400?g/ml YQHX treatment reversed the mitochondrial depolarization,especially 200?g/ml(P<0.05).These data indicate that YQHX at the concentration of 200?g/ml,as the best dose for mitochondria,significantly protected the mitochondrial function in hypoxia-induced H9c2 cells.Based on these results,we next used the 200?g/ml concentration of YQHX in the present study.7.Effects of YQHX on PGC-la expression and related induced by hypoxia.Western Blot results showed that:PGC-1?,p-AMPK,NRF-1 and Tfam expression was significantly decreased in I/H-induced H9c2 cells(P<0.05),YQHX treatment remarkably increased PGC-la,p-AMPK,NRF-1 expression(P<0.05),whereas AMPK inhibitors Compound c reversed the above-mentioned effects of YQHX(P<0.05).We further extended hypoxia to 24h and measured PGC-la protein expression.Interestingly,compared to control group,PGC-la protein expression remarkably decreased after 12h of hypoxia,whereas at 24h of hypoxia PGC-1? and p-AMPK protein expression nearly returned to the normal level or even increased,but there were no significant differences(P>0.05).Interesting,treatment with YQHX enhanced PGC-la expression and AMPK phosphorylation in hypoxia-induced H9c2 cells for 12h and 24h(P<0.05),but these effects were completely blocked by 5?M Compound c(P<0.05).These results further indicate that PGC-la expression is partially through AMPK phosphoiylation,and the YQHX-induced activation of PGC-la expression may be attributable to the activation of AMPK phosphorylation in hypoxia-induced H9c2 cells.Part ? The effect and underling mechanism of YQHX on Lipid Metabolism in post-myocardial infarction injury.Aim of the study:This section uses a rat model of myocardial infarction to investigate the changes in lipid metabolism on the 7 and 28 days after myocardial infarction.Taking the AMPK/PGC-1? pathway as an entry point,and relying on the hypoxic model of myocardial cells,the regulatory effect and mechanisms of YQHX on lipid metabolism disorders were further clarified.Materials and methods:The experiment was performed both in vivo and in vitro.1.Animal experiments:Serum lipid levels in each group were detected using an automatic biochemical analyzer;p-AMPK,PPARa,and CPT-1? protein expression were evaluated by western blot in vivo.2.Cell experiments:PPARaand CPT-1? proteins after hypoxia were evaluated by Western blot.Results:1.Effect of YQHX on plasma lipid indicators in post-myocardial infarction injury.At 7 days after MI,compared with the Sham group,the level of TC in the MI group was increased(P<0.05),whereas YQHX group and TMZ group were slightly decreased(P>0.05).Compared with Sham group,the level of FFA,TG,LDL-c in the MI group were slightly increased(P>0.05),whereas YQHX group were significantly decreased the level of FFA and TG(P<0.05),but there was no significant difference between TMZ group and MI group.At 28 days after MI,compared with the Sham group,the level of TG,GLU and LD in the MI group was significantly increased(P<0.05).Compared with MI group,YQHX group were significantly decreased TG,GLU and LD levels(P<0.05),and TMZ group were significantly decreased TG and GLU level(P<0.01).Compared with MI group,YQHX group and TMZ group remarkedly decreased the level of TC(P<0.05).2.Effects of YQHX on p-AMPK,CPT-la and PPAR? expression in post-myocardial infarction injury.At 7 days after MI,compared with Sham group,p-AMPK protein expression in MI group was significantly enhanced(P<0.05),CPT-1? and PPARa expression were significantly increased(P<0.05).Compared with MI group,YQHX group was significantly increased p-AMPK and CPT-1? protein expressions(P<0.05),but increased PPARa activity,which was no significant difference(P>0.05).At 28 days after MI,compared with Sham group,p-AMPK protein expression in MI group was significantly enhanced(P<0.05),and YQHX group was further significantly increased p-AMPK expression(P<0.05),but failed to increase CPT-1? and PPARa activity(P>0.05).3.Effects of YQHX on CPT-la,PGC-laand PPARa expression induced by hypoxia.Western Blot results showed that:CPT-1? and PPARa expression were significantly decreased in I/H-induced H9c2 cells(P<0.05),YQHX treatment remarkably increased CPT-1 a,PGC-la and PPARa expression(P<0.05),whereas AMPK inhibitors Compound c reversed the above-mentioned effects of YQHX(P<0.05).These results further indicate that the effect of YQHX in regulating lipid metabolism is related to AMPK phosphorylation.Conclusion1.YQHXcan improve the left heart function,including protecting mitochondria structure and function,regulating lipid metabolism,decreasing oxidative stress and reducing myocardial cell injury.2.YQHX can up-regulate p-AMPK,PGC-la,NRF-land related mitochondrila biosynthesis proteins,improve mitochondrial ultrastructural destruction and mitochondrial membrane potential depolarzation,and increase mitochondrial ATP production,indicating that YQHXprotects mitochondria structure and function in ischemia heart.3.YQHX can up-regulate the expression of p-AMPK,PGC-1?,CPT-1?and PPARalipid metabolism related proteins,reduce blood lipid content,indicating that YQHX regulates lipid metabolism after myocardial infarction.4.YQHX treatment could improve mitochondrial structure and biosynthesis by enhancing p-AMPK,PGC-1?,NRF-1 and Tfam protein expressions in hypoxia-induced H9c2 cells.The upregulation of PGC-1?,NRF-1 and AMPK expression were completely blocked by AMPK inhibitor Compound C,indicating that protecting mitochondrial structure ofYQHX may be through activation of AMPK/PGC-1? pathway.5.YQHX treatment could regulate lipid metabolism by enhancing p-AMPK,PGC-1?,CPT-1?and PPAR? protein expressions in hypoxia-induced H9c2 cells.The upregulation of PGC-1?,CPT-1?and PPAR? expression were completely blocked by AMPK inhibitor Compound C,indicating that regulatinglipid metabolism of YQHX may be by activation of AMPK/PGC-1?pathway.In summary,the experimental results show that the AMPK/PGC-1? pathway,a key pathway,protects mitochondrial structure and function,regulates lipid metabolism,which plays an important role in myocardial energy metabolism after myocardial infarction. |
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