| In recent years,the incidence of diabetes increased significantly in diabetic cardiomyopathy(diabetic,cardiomyopathy,DCM)as an independent clinical disease in coronary heart disease,valvular heart disease,hypertensive heart disease,mainly for myocardial systolic and diastolic function abnormal.At present,the pathogenesis of DCM is complex,mainly related to the myocardial energy metabolism,inflammation,oxidative stress,calcium overload,cell apoptosis,and its main pathological manifestations of myocardial cell hypertrophy and extracellular matrix deposition,ventricular weight / body weight(heart weight index)increased,myocardial interstitial remodeling and myocardial fibrosis.Firodaliso study showed that streptozotocin(Streptozotocin,STZ)-induced early diabetic rats(3 days)can lead to myocardial cell apoptosis phenomenon,so that oxidative stress,myocardial apoptosis,myocardial fibrosis and heart failure ventricular remodeling in DCM plays a very important role.Like cell proliferation,apoptosis is a process of gene regulation.At present,there are two main pathways of apoptosis,activation of aspartic acid is a proteolytic enzyme by cell membrane death receptor(cysteinyl aspartate specific proteinase,caspase),and another is a way to release cytokines through the mitochondrial pathway in the cytoplasm,cell factor can activate caspase,an important protein degradation in cells,thus induction of apoptosis.Therefore,caspase-3 as a marker of apoptosis,plays a key role in apoptosis promoted by different factors,is the ultimate effect factor of caspase cascade in apoptosis,inhibiting activity or function of Caspase-3 can inhibit cell apoptosis.AKT also known as protein kinase B(protein kinase B,PKB),may induce phosphorylation of transcription factors by up regulating expression of anti apoptosis gene and down-regulation of apoptosis gene,so as to achieve the inhibition of cell apoptosis and promote cell survival.In the newly discovered anti apoptosis protein family,X linked inhibitor of apoptosis protein(X-linked inhibitor of apoptosis protein,XIAP)is the strongest apoptosis protein inhibitor,can directly inhibit caspase and protect cells from a variety of apoptotic stimuli.However,AKT is able to inhibit the phosphorylation and degradation of XIAP in Ser87,stabilize the expression in the cell,and inhibit apoptosis.Adaptor protein p66 Shc may be involved in the transduction of intracellular oxidative stress signal,regulate the production of intracellular reactive oxygen species ROS and mediate apoptosis.Many studies have shown that p66 Shc is involved in the occurrence and development of cardiovascular diseases,diabetes and other diseases.In addition,phospho-rylation of p66 Shc is regulated by activation of PKC beta in vivo.PKC beta /p66 shc signaling pathway is involved in the pathophysiolo gy of multiple organ injury mediated by small intestinal ischemia reperfusion injury.Carvedilol as the third generation of beta blockers,which can block the beta 1 and beta 2 receptor,can block alpha 1 receptors,and has antioxidant,scavenging oxygen free radicals and reduce ventricular remodeling,inhibit myocardial fibrosis and inhibit cell apoptosis.Carvedilol may have a protective effect on diabetic cardiomyopathy,and its mechanism has not been fully elucidated.To prove this hypothesis,this study 1)clinical observation on the effect of carvedilol on patients with diabetic cardiomyopathy;2)application of diabetic cardiomyopathy rats animal model,H9C2 model and myocardial cells induced by high glucose in vitro,to observe the effect of carvedilol on myocardial inflammation,apoptosis,collagen metabolism,AKT/XIAP pathway and PKC beta /p66 shc mitochondrial oxidative stress the signal pathway,and provide a theoretical basis and experimental basis for the treatment of diabetic cardiomyopathy.Part 1 Effects of carvedilol on cardiac function in patients with diabetic cardiomyopathy and mechanism.Objective: Effect of carvedilol on cardiac function in patients with diabetic cardiomyopathy and mechanism(clinical observation effect).Methods: From September 2014 to September 2016 in Tangshan City workers' Hospital Department of Cardiology and Department of Endocrino-logy were diabetic cardiomyopathy patients with a total of 90 cases were diagnosed,including 50 cases of male,female 40 cases,age 35-65 years old,the average age of 45.6 years,were randomly divided into carvedilol treatment group(n=50 cases)and control group(n=40 cases).The control group received conventional therapy,carvedilol treatment group in the control group based on the use of Carvedilol(Dudley,Roche)2 weeks 3.125 mg2/ orally,2 weeks if there is no obvious aggravation of heart failure,changed to 6.25 mg2/ orally,2 weeks of continuous oral administration,if there is still no aggravation of heart failure,then continue orally for 8 weeks.At the beginning of two groups before and after treatment for 12 weeks,respectively.The detection of serum hs CRP,TNF-alpha and NT-pro BNP by ELISA(Nterminal pro-B-type natriuretic peptide,N-terminal pro brain natriuretic peptide in type B),fasting blood glucose,total cholesterol and triglyceride levels,left ventricular end diastolic cardiac ultrasound detection(LVEDd)and end systolic the diameter(LVEDs),left ventricular ejection fraction(LVEF),the 6 minute walking distance was performed to evaluate the exercise tolerance of patients.Results:1 the changes of biochemical indicators: compared the two groups before and after treatment: there was no significant difference in fasting blood glucose,total cholesterol and triglyceride(P>0.05),and carvedilol after 12 weeks of treatment,the treatment group compared with the control group,the biochemical indexes have no significant difference(P >0.05).2 Changes of cardiac function: the two groups after treatment of left ventricular end diastolic diameter and end systolic diameter and NT-pro BNP levels were significantly lower than before treatment(P<0.05),and the treatment group compared with the control group decreased significantly(P<0.05);two groups of left ventricular ejection fraction were significantly higher(P<0.05).And the treatment group compared with the control group increased more significantly(P<0.05).Carvedilol treatment group 6 minutes walking distance(577.40 + 80.13)m,compared with before treatment(292.95 + 34.12)m improved(P<0.05),and control group(456.32 + 30.09)there is a significant difference compared to m(P<0.05),the control group before and after treatment compared to 6 minutes walking distance were significantly different(296.68 + 21.79 m VS)(456.32 + 30.09)m(P<0.05).3 the change of inflammatory index: compared with the control group,the levels of serum hs CRP and TNF-were s ignificantly decreased in the two groups before and after treatment(P<0.05),compared with the control group(P<0.05);Conclusion: In the application of carvedilol in patients with diabetic cardiomyopathy can significantly improve patients with cardiac systolic and diastolic function,improve the tolerance of patients,does not affect the blood glucose and blood lipid level of patients,the therapeutic effects of carvedilol may and can reduce the inflammatory response and oxidative stress,apoptosis,ventricular remodeling and reduce myocardial fibrosis related cells;Part 2 The expression and intervention of carvedilol on AKT/XIAP signal transduction pathway in diabetic cardiomyopathy rats.Objective: Study on the expression and intervention of carvedilol AKT/XIAP signal transduction pathway in myocardial lesion in diabetic cardiomyopathy in rats.Methods: Adult male Wistar SD rats were fed with high fat and high sugar diet and intraperitoneal injection of streptozotocin(STZ)was used to make type 2 diabetic cardiomyopathy rat model.After 12 weeks,divided into low dose carvedilol group(1mg/kg/d),high dose carvedilol group(10mg/kg/ d),using the drugs every day to rats by gavage once,saline control group and model group were given the same dose of daily gavage.After 12 weeks of continuous administration,animals were killed.Left ventricular end diastolic dimension(LVEDd),left ventricular end systolic diameter(LVEDs)and left ventricular ejection fraction(LVEF)were measured by echocardiography.The morphologic changes in healthy control group,diabetic group rats heart were observed with optical microscope and electron microscope,to determine the model of diabetic cardiomyopathy and inflammatory infiltration;by biochemical assay of serum IL-1 beta,TNF-alpha,fasting blood glucose,triglyceride(TG),total cholesterol(TC)content and NT-pro BNP in order to determine myocardial metabolism,inflammation and cardiac function changes;myocardial cell apoptosis was observed by TUNEL;using Masson and Vimentin staining to understand the situation of myocardial fibrosis;the expression of Caspase-3 was detected by immunohistochemical method;application of Western bolt technology,XIAP,p-AKT detection of AKT,p-XIAP,caspase-3 and I collagen(the expression of Collgen I).H9C2 myocardial cells cultured in vitro were divided into normal control group(NC,5.5mmol/L,D-(HG glucose),high glucose group,high glucose + 33mmol/L D-glucose),carvedilol group(HG+Carv 33mmol/L,D-+10 mol/L,carvedilol glucose)Western expression by bolt,XIAP,p-AKT detection of AKT,p-XIAP,IL-1,TNF-beta.Collgen,I and caspase-3 protein.Results:1 General condition comparison of 1 rats: control group(group NC)rats hair luster,mental state,eating water and activity are good,the diabetic model group(DM group)rats by high-fat diet feeding weight increased significantly,feeding 4 weeks,weight is significantly higher than that of the control group.At 4 weeks,given intraperitoneal injection of streptozotocin(STZ),after 2 weeks of treatment,DM group rats began to decline,but at the end of the experiment was significantly higher than group Control,but Carvedilol(Carv)rats compared with DM group treatment significantly increased(P<0.05),but was still lower than the NC group.High dose Carvedilol(CarvH)group,low dose group(CarvL)showed no significant difference(P>0.05);group DM rats 1 weeks after STZ appeared polydipsia,polyuria symptoms,and gradually increase,and appear hair becomes gray yellow,listlessness,poor activity of the.Carv group compared with DM group,the symptoms had no obvious improvement;heart mass / body weight of rats in group DM(HW/BW)was significantly higher than that of NC group(P<0.05),Carv treatment group compared with DM group(HW/BW)was significantly decreased(P<0.05),and CarvH group and CarvL group compared to the more obvious differences(P<0.05).2 Pathological change of myocardium of 2 rats: observation: group NC myocardial cells of the sarcomere structure(bright band and dark band,Z line,M line)is clearly visible,cell junction of intercalated disc structure is clear,the muscle bundle longitudinal arrangement and mitochondrial membrane integrity;DM group,sarcomere myofilament dissolution and fracture,leap disk space disappeared,mitochondria swelling,disappearance of part,pyknosis,myocardial interstitial collagen deposition,capillary endothelial swelling;changes of myocardial tissue in group Carv between DM group and NC group,the myocardial lesions significantly reduced compared with DM group,interstitial collagen deposition less capillary basement membrane thickening reduce.HE staining: s lices on cardiomyocyte hypertrophy visible DM rats and irregular,cell disorder,myocardial interstitial cells and extracellular matrix increased,fibroblasts increased;NC group: rat myocardial cells arranged in neat rows,a small amount of fibroblasts,clear structure;Carv group of myocardial cells arranged more than DM group the rules of muscle fiber degeneration was lighter,fibrous tissue hyperplasia was significantly reduced.DM and HE staining were used to observe the establishment of diabetic cardiomyopathy model in rats.3 Heart function changes in rats : at the end of the experiment,compared with the NC group,left ventricular end diastolic diameter of rats in the DM group(LVIDd),left ventricular end systolic diameter(LVIDs)were significantly increased(P<0.05),EF decreased significantly(P<0.05);while the Carv of low and high dose after pretreatment with LVIDd,LVIDs(P<0.05)were significantly decreased,EF increased significantly(P<0.05),the difference was significant;high dose of Carv group LVIDd,LVIDs value decreased compared with the low dose group,EF increased,the differences were statistically significant(P<0.05).Compared with the NC group,the levels of plasma NT-pro BNP in DM group was significantly increased(P<0.05);Carv low and high dose after pretreatment of plasma NT-pro BNP levels were significantly decreased(P<0.05)and Carv high dose compared with low dose plasma NT-pro BNP level decreased more significantly,the difference was statistically significant(P<0.05).4 The changes of biochemical indicators: compared with NC group,the expression of DM in IL-1 group,the TNF-level increased alpha beta(P<0.05)and Carv low dose and high dose treatment group after the pretreatment,the two indexes of expression levels were decreased significantly(P<0.05),and high dose group decreased more significantly,the difference is statistical significance(P<0.05).Compared with the NC group,the fasting blood glucose,triglyceride(TG),total cholesterol(TC)levels in the DM group were significantly higher(P<0.05).However,there was no significant difference in the levels of fasting blood glucose,TG and TC between the three groups of low and high dose Carv group and DM group(P>0.05).5 Myocardial cell apoptosis in rats 5 changes : normal myocardial nuclei were blue staining,TUNEL positive apoptotic myocardial nuclei were brown,counting under light microscope apoptosis 5 HPF positive myocardial nuclei number and the total number of nuclei for the proportion,are worth the apoptotic index of AI(Apoptosis Index).Compared with NC group,DM group,AI value was significantly increased,Carv low and high dose group compared with DM group,AI values were decreased obviously and Carv high dose group than in low dose group compared to Carv decreased more significantly,the differences were statistically significant(P<0.05).Western Bolt results showed that: compared with NC group,the expression level of caspase-3 protein in DM group rats increased significantly,Carv low dose and high dose drug intervention caspase-3 protein expression levels were significantly decreased,and Carv high dose group with low dose of Carv group decreased significantly,the differences were statistically s ignificant(P<0.05).Immunohistochemistry showed that caspase-3 was positive in cytoplasm with brown yellow granules,and negative control had no brown yellow reaction in cytoplasm except nucleus.Compared with NC group,DM group rats were positive staining of integral optical density was significantly higher in Carv,low and high dose drug intervention,the number of Caspase-3 positive staining cells significantly decreased,and Carv high dose group with low dose of Carv group decreased significantly,the differences were statistically significant(P<0.05).6 The changes of myocardial collagen in rats : 6 Masson staining: a small amount of collagen fibers between myocardial cells and around the blood vessels of rats in the NC group;DM group rat myocardial interstitial collagen fiber hyperplas ia,vascular disorder,obvious fibrosis,collagen fibers were red uneven distribution,connected into a mesh;low,Carv high dose after pretreatment of myocardial collagen in rats were significantly reduced,and the high dose group of Carv collagen loss is more obvious,more close to the NC group.Masson staining of myocardial collagen fraction(CVF1): compared with NC group,DM group,CVF1 value was significantly increased,Carv low and high dose after pretreatment CVF1 value decreased significantly,and Carv high dose group and low dose group compared with the CVF1 value decreased significantly,the differences were statistically significant(P<0.05).Vimentin staining and Western Blot results showed that: compared with NC group,the expression of DM in rats of type I collagen was obviously higher than that of group NC(P<0.05),type I collagen volume fraction(CVF)were significantly increased,the expression of Carv after the intervention of type I collagen significantly decreased,CVF was significantly reduced,and the high dose of Carv group low dose group decreased more significantly,the differences were statistically significant(P<0.05).7 Western Bolt results showed that: compared with NC group,the p-AKT expression of p-XIAP protein in DM group decreased significantly(P<0.05),Carv intervention both significantly increased(P<0.05),and Carv high dose group,low dose group increased more significantly,the differences were statistically significant(P<0.05).However,there was no significant difference in the expression of AKT and XIAP between the four groups(P>0.05).8 Western Bolt results showed that: compared with NC group,the expression level of HG group H9C2 myocardial cell Caspase-3,TNF-alpha,IL-1 beta and type I collagen were significantly increased(P<0.05),Carv after drug intervention the expression level of the protein were significantly decreased(P<0.05);compared with group NC,p-AKT and p-XIAP protein expression in in the HG group were significantly decreased,Carv expression levels after drug intervention both were significantly increased,the differences were statistically significant(P<0.05).However,there was no significant difference in the expression of AKT and XIAP between the three groups(P>0.05).The results are similar to those of in vivo models.Conclusion: Carvedilol can promote the phosphorylation of AKT and XIAP,relieve the inflammatory reaction,inhibit the apoptosis of myocardial cells and myocardial fibrosis,and improve the cardiac function in diabetic cardiomyopathy rats.Part 3 The expression and intervention of carvedilol on PKC beta /p66shc mitochondrial oxidative stress signal transduction pathway diabet- ic cardiomyopathy rats.Objective: Study on the expression and intervention of carvedilol PKC beta /p66 shc mitochondrial oxidative stress signal transduction pathway in the myocardium of diabetic cardiomyopathy in rats.Methods: Plasma malondialdehyde(MDA)in rats was determined by thiobarbituric acid reactive substance spectrophotometry.Superoxide dismutase(SOD)chromogenic colorimetric spectrophotometry by xanthine xanthine oxidase reaction;application of Western bolt technique to detect the expression of rat myocardial p-PKC PKC beta 1,beta 1,beta 2,p-PKC PKC 2,p-p66 shc,p66Shc,beta caspase-3 protein.In vitro highglucose cultured myocardial cells of H9C2,simulated in vivo model,with the second part,to detect the expression of p-PKC beta 1,PKC application of Western bolt technology p-PKC beta 1,beta 2,beta 2,PKC p-p66 shc,p66Shc and caspase-3 protein,using confocal laser technology to detect mitochondrial reactive oxygen species(ROS)and the expression level of p-p66 shc in the mitochondrial translocation rate.Results:1 The changes of symptoms and signs and biochemical indexes in rats : HE staining,Masson staining and electron microscope observation of the ultrastructural changes were the same as the second part.2 Malondialdehyde(MDA)and superoxide dismutase(SOD)results: in vivo experimental results show that compared with the NC group,the MDA level of DM group rats increased significantly,SOD was significantly reduced(P<0.05);compared with group DM MDA decreased significantly after treatment,carvedilol significantly elevated levels of SOD(P<0.05)CarvH,and compared with the CarvL group,the change is more obvious,the difference was statistically significant(P<0.05).The results of in vitro culture of H9C2 myocardial cells in high glucose environment are consistent with those in vivo.3 Western bolt showed: compared with NC group,DM rats p-PKC beta 2,p-p66 shc and caspase-3 protein expression levels were significantly elevated(P<0.05 Carv),low dose and high dose after pretreatment of the above indexes were significantly decreased(P<0.05),and Carv high dose group,low dose group decreased more significantly,the differences were statistically meaning(P<0.05).However,there was no significant difference in the expression of p-PKC 1 and PKC beta in the four groups()(P>0.05).The results of in vitro culture of H9C2 cardiomyocytes were similar to those in vivo.4 In vitro results: confocal results showed that compared with the NC group,HG group of mitochondrial ROS generation increased significantly,p-P66 shc mitochondrial trans location rate was significantly increased(P<0.05),however,the prognosis of H9C2 myocardial cell mitochondrial carvedilol dry ROS formation was significantly decreased in p-P66 shc mitochondrial translocation rate decreased significantly,the differences were statistically significant(P<0.05).Conclusions: Carvedilol can inhibit the activation of PKC beta 2/P66 shc signal pathway in diabetic cardiomyopathy rats,thereby reducing oxidative stress in rats with diabetic cardiomyopathy and myocardial collagen deposition and fibrosis,decrease cell apoptosis,play a protective role in diabetic cardiomyopathy. |
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