The Protective Effects And Mechanism Of Apigenin On Diabetic Cardiomyopathy

BackgroundDiabetes mellitus(DM)can lead to multiple cardiovascular complications,including coronary artery disease(CAD),cardiac hypertrophy and fibrosis,and even heart failure(HF).These cardiovascular complications are the leading cause of death in patients with DM.In Recent years,a growing body of studies have revealed the existence of diabetic cardiomyopathy(DCM)with unique cardiac remodelling pattern when compared to other types of cardiomyopathies.DCM can be characterized by left ventricular remodeling,as well as aberrant altered myocardial energy metabolism.At present,several mechanisms have been implicated in the pathogenesis of DCM,including increased oxidative stress,cardiac chronic inflammation,mitochondrial dysfunction,cell apoptosis,interstitial fibrosis and left ventricular hypertrophy.Although a number of studies have explored the potential mechanisms in DCM,the pathogenesis of DCM has not yet been fully illuminated because of the multi-factorial pathophysiologic involvement in the development of DCM.Cardiovascular complications are the leading cause of mortality in patients with DM.Although variety confounding factors such as dyslipidaemia and hypertension are mainly implicated in this chronic process,it's now well recognized that diabetes itself can lead to a number of molecular changes in the heart.Therefore,to further explore the pathogenesis of DCM may provide a potential target and novel strategies for the clinical treatment of DCM.Apigenin is a natural flavone widely found in vegetables,fruits and herbs.Previous studies have indicated that apigenin have multitude pharmacological properties,including inhibition of tumor cell proliferation,antispasmodic,anti-inflammatory,anti-oxidant,antiatherogenic,hypolipidemic and hypoglycemia effects.Meanwhile,because of the cardiovascular protective effects,apigenin have recently gained lots of attention in the cardiovascular research field.As far as we know,there are studies revealed apigenin attenuates streptozotocin-induced pancreatic ? cell damage and endothelial dysfunction exposed to high glucose,but the role of apigenin in DCM and its specific molecular mechanism responsible for DCM remains poorly understood.In the present study,we intended to investigate the impact of apigenin on DCM in vivo and in vitro.In addition,we will compare the protective effects in H9c2 cells induced by hyperglycemia between apigenin and resveratrol,thus further clarify the protective effect and mechanism of apigenin on DCM,so as to provide theoretical basis for clinical treatment of DCM.ObjectiveExplore the protective effects and elucidate the possible mechanism of apigenin on high-fat combine STZ induced diabetic cardiomyopathy.MethodsPart one:The DCM animal model was induced by high-fat diet and low-dose STZ and then treatment with apigenin,Heart weigh(HW),body weight(BW),lung weight(LW),body weight(BW),tibial length(TL)and caculating the ratios were used to assess the nutrition and growth development of mice in each group.The picture of whole heart was used to assess the volume of heart and HE staining was used to evaluate the cross-sectional area of cardiomyocytes.Echocardiography was used to determine HR,IVSd,IVSs,LVPWd,LVPWs,LVDd,LVDs,FS,and EF for the assessment of cardiac function.Hemodynamic parameters such as LVESP,LVEDP,dp/dt max,-dp/dt min were used to evaluate left ventricular pressure-volume parameters in mice.PSR staining of the heart tissue slices was used to evaluate myocardial interstitial and perivascular fibrosis in mice.Real time-PCR was used to detect the mRNA expression of cardiac hypertrophy markers of ANP,BNP and myocardial fibrosis markers of TGF?,collagen I(Coll I),collagen III(Coll III),fibronectin(Fn)and connective tissue growth factor(CTGF),inflammatory cytokine TNFa,IL-1?,IL-6 as well.IkBa,p-IkBa,TNFa protein expression in myocardial tissue were determined by Western Blot to evaluate the myocardial inflammatory reaction and the possible mechanism.4-HEN was detected by immunohistochemistry and the determination of oxidative stress related indicators malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GPx)expression levels and activity to evaluat cardiac oxidative stress.The NF-KB/p65 protein expression both in the nucleus and cytoplasm was determined by SDS-PAGE electrophoresis and immunofluorescence staining of heart tissue slices to evaluate the NF-KB/p65 nuclear transfer in myocardial tissue.Bcl2,Bax and c-caspase3 protein expression in myocardial tissue were determinated by Western Blot and TUNEL staining to assess myocardial cell apoptosis.Part two:The H9c2 cardiac cell injury model was induced by high glucose(HG)then treatment with apigenin,the Bcl2,Bax,and c-caspase3 protein expression in H9c2 cells were determined by Western Blot to evaluate the cell apoptosis.Determination of MDA,SOD,and GPx expression levels or activity to evaluat H9c2 cell oxidative stress.Real time-PCR was used to detect the mRNA expression of inflammatory cytokines IL-1?,IL-6,and TNFa.Immunofluorescence staining of H9c2 cells to evaluate the NF-?B/p65 nuclear transfer.Part three:The H9c2 cardiac cell injury model was induced by HG then treatment with either apigenin or resveratrol,the Bcl2 and Bax protein expression in H9c2 cells were determined by Western Blot to evaluate the cell apoptosis.Determination of reactive oxygen species(ROS)by Flow cytometry to assess oxidative stress level.The NF-?B/p65 protein expression both in the nucleus and cytoplasm was determined by Western Blot to evaluate the NF-KB/p65 nuclear transfer between apigenin and resveratrol.ResultsPart one:Blood glucose in diabetic mice treatment without and with apigenin was 5-and 4.6-fold higher than the control group,respectively.Apigenin treatment significantly decreased the blood glucose,compared to the DM group.BW and HW in the DM group were decreased compared with the CON group,while DM+A group compared with DM group was increased.The cell area of cardiomyocytes enlarged accompanying with the increase of ANP and BNP in STZ-induced DM mouse heart tissue.However,treatment of apigenin significantly inhibited the enlargement of cardiomyocytes and down-regulated the mRNA expression of ANP and BNP.Echocardiography demonstrated a significant cardiac dysfunction in diabetic mice compared to the control group,while apigenin treatment obviously prevented DCM-related cardiac remodeling and improved the cardiac dysfunction.Meanwhile,hemodynamic detect have showed that diabetes mellitus significantly decreased the EF,FS dp/dt,and-dp/dt,and increased the IVSd,LVDd,and LVPWd compared with the CON group,all these parameters improved notably in the apigenin treatment group compared to the DM group.A dramatic interstitial fibrosis was observed in the DCM group by PSR staining,compared to the CON group.Apigenin treatment significantly alleviated the fibrosis in cardiac interstitium and peri-vessel.RT-PCR showed the mRNA expression levels of collagenI,collagenIII,fibronection,CTGF,and TGF? significantly increased in the heart tissue of DM mice,while apigenin improved all these parameters.Immunohistochemistry demonstrated high glucose(HG)and insulin resistance could significantly promote the accumulation of 4-HNE in the heart,while apigenin could restrain the hyperaccumulation of 4-HNE.Compared with the CON group,the MDA overproduction and the activity of anti-oxidative enzymes SOD and GPx exhibited a significantly down-regulation in the hearts of the DM group mice,treatment with apigenin could inhibit the parameters changes.In addition,compared with the CON group,the protein expression of p-IkBa and TNFa significantly increased in the DM hearts,which could be blunted dramatically by administration of apigenin.Moreover,DM induced cardiomyocyte injury accompanying with the obviously nuclear translocation of NF-KB/p655 while apigenin treatment could significantly inhibit the translocation of NF-?B/p65.To further demonstrate this,we isolated the NF-?B/p65 protein from nucleus and cytoplasm,respectively,and found out that NF-KB/p65 protein expression in the nucleus was higher in the DM group than the DM+A group,however,the NF-KB/p65 protein in the cytoplasm was inverse.What's more,the mRNA espression of inflammatory cytokines such as IL-1?,IL-6 and TNFa were significantly increased in the DM hearts compared with the CON group,treatment with apigenin could inhibit the mRNA espression of the related inflammatory cytokines.TUNEL demonstrated that DM promoted myocardial cell apoptosis while apigenin administration inhibited the process.Furthermore,the expression of c-caspase3 and the pro-apoptotic protein Bax were significantly up-regulation in the hearts of STZ-induced DM mice,however,the anti-apoptotic protein Bcl2 was down-regulation compared with the CON group.Apigenin administration could significantly improve these parameters changes.Part two:The protein expression of c-caspase3 and Bax in H9c2 cardiac cells cultured with HG were significantly up-regulation,while the Bcl2 protein was down-regulation compared with the CON group.Treatment with apigenin significantly prevented the process and down-regulated the Bax/Bcl2 ratio.The change trend of oxidative stress related parameters MDA,SOD,GPx were consistent with the experimental results in DCM model in part one.In addition,the mRNA espression of inflammatory cytokines such as IL-1?,IL-6 and TNFa were significantly increased accompanying with the obviously nuclear translocation of NF-?B/p65 in the HG group compared with the CON group,apigenin administration could significantly inhibit the mRNA espression of the inflammatory cytokines and nuclear translocation of NF-?B/p65.Part three:Both apigenin and resveratrol could increase the Bcl2 but decrease the Bax protein expression,in addition,the expression of NF-KB/p65 protein in the nucleus was significantly reduced while in the cytoplasm was obviously enhanced both in HG+A and HG+R group compared with the HG group.However,differences between the HG+A and HG+R group had no statistical significance.Futhermore,flow cytometry showed that HG significantly promoted the generation of ROS in H9c2 cells,while apigenin and resveratrol significantly inhibited the generation of ROS.Conclusions1.Apigenin could improve the cardiac remodeling and dysfunction,inhibit myocardial cell apoptosis,cardiac oxidative stress and inflammation in DCM model induced via high-fat diet in combination with small dose of STZ.2.Apigenin could alleviate the oxidative stress and inflammation,and supress the myocardial cell apoptosis in H9c2 cells cultured with HG.3.The cardioprotection effects of apigenin improve oxidative stress,cell apoptosis,and inhibit nuclear translocation of NF-KB/p65 in H9c2 cells cultured with HG is not inferior to resveratrol.4.The cardioprotection effects of DCM treatment with apigenin possibility via inhibiting activation of the NF-?B/p65 related signaling pathway.