| BackgroudAccording to the current statistics,the number of world’s diabetic patients is about 0.18 billion,and by 2025,the number will increase to 0.3 billion.Diabetes is an independent risk factor for heart failure.Studies have shown that the incidence of heart failure in normal people is about 1%-4%.but the incidence of heart failure in diabetes can reach to 12%.Diabetic cardiomyopathy(DCM)is a specific metabolic abnormalities caused by diabetes mellitus.The main features of DCM are left ventricular enlargement and ventricular wall motion reduction;the early feature is diastolic dysfunction,while the late performance is systolic dysfunction.DCM is one of the major cardiovascular complications of diabetic patients and is an important cause of high morbidity and mortality in cardiovascular disease in diabetic patients.The causes of DCM mainly include hyperglycemia,oxidative stress,myocardial autonomic neuropathy and insulin resistance,and hyperglycemia plays an important role in the metabolic disorders of cardiomyocytes.The major pathological changes of DCM are including myocardial interstitial fibrosis,cardiomyocyte apoptosis and autophagy.Myocardial interstitial fibrosis is the secretion of collagen in the myocardial interstitial,which led to reduced cardiac relaxation and increased stiffness,thereby affecting cardiac function.Cardiomyocyte apoptosis is programmed cell death,the main reason for the loss of DCM cardiomyocytes.High glucose state leads to cell metabolism disorders,endoplasmic reticulum and mitochondria damage,leading to apoptosis,because the myocardium is can be not proliferation,so myocardial apoptosis can lead to reduced myocardial contractility.Autophagy is a major metabolic pathway that regulates the degradation and recycling of macromolecules and organelles in cells.Autophagic impairment can lead to the accumulation of misfolding proteins and damaged organelles,resulting in cell death.Normal adult heart metabolism is mainly from two parts:60%to 70%of fatty acid beta oxidation and 20%to 25%of glucose oxidation.Under the state of diabetes,studies have shown that the ratio of fatty acid beta oxidation and glucose oxidation is further widened,fatty acid beta oxidation is significantly increased and glucose oxidation is further reduced.Fatty acid oxidation can produce a large number of lipid intermediates,which accumulate in the cardiomyocytes eventually lead to impaired cardiac function.Fatty acids can also damage the calcium regulation of cardiomyocytes,leading to impaired myocardial contractility.Another study confirmed that fatty acid oxidation will increase the production of reactive oxygen species,excessive fatty acid oxidation will cause the accumulation of reactive oxygen species,leading to oxidative stress,and thus damage myocardial cells.Studies have confirmed that the normal proportion of metabolic recovery can restore cardiac function,but the specific mechanism of action is unknown,whether it can regulate myocardial cell apoptosis,cardiomyocyte autophagy and myocardial interstitial fibrosis remains to be explored.In this study,we will use proteomics techniques to administer metabolic regulators trimetazidine(TMZ)to analyze the changes in energy metabolism in high glucose-induced cardiomyocytes,as well as changes of secretory proteins in cardiomyocytes and cardiac fibroblasts induced by high glucose,To explore the relationship between differential protein and cardiomyocyte apoptosis and autophagy after energy metabolism transformation,and to analyze the relationship between fibrin collagen secretion and its possible mechanism,and to provide a theoretical basis for further experiments.Objective1.To investigate the alteration of differential proteins between cardiomyocyte protein and cardiomyocytes and fibroblasts secreted proteins by high glucose after energy metabolism transformation.2.To explore the relationship between differential proteins and energy metabolism,myocardial apoptosis,myocardial autophagy and myocardial interstitial fibrosis after the alteration of energy metabolism,and to provide a basis for subsequent experiments.3.To explore the relationship between the three types of differential proteins and key pathways which are involved in apoptisis,autophagy and fibrosis after the alteration of energy metabolism,and to provide a basis for subsequent experiments.Methods1.The establishment of experimental modelIn this study,The subjects are primary cultured neonatal rat ventricular myocytes(NRVM),H9c2 rat cardiomyocytes and cardiomyobular fibroblasts(CF),and divided into three groups,including the control group,high glucose group and The control group are stimulated by low glucose(5 mmol/L)with mannitol(27.5 mmol/L);the high glucose group are stimulated by,and TMZ group are stimulated by TMZ(10μmol/L)and high glucose(33 mmol/L),and then incubated the three kinds of cells,and then collected medium of H9c2 cells.NRVM medium and CF.2.Collection of proteinsIn serum-free medium,high glucose was used to stimulate NRVM and CF 24h,and the supernatant was collected.Centrifuge the centrifuge tube and prepare for the next steps.High glucose stimulates H9c2 cells for 24 h,and after washing with PBS,the cells are scraped and centrifuged to remove serum.3.Sample processing,mass spectrometry and data analysisThe cell concentration was determined by Bradford protein quantification,followed by TCEP reduction,IDA alkylation,trypsin digestion,C18 desalting,nanodrop assay protein concentration,TMT marker protein Peptides or unlabeled proteins.Finally,the samples were analyzed by Q-Exactive mass spectrometer.Proteome Discoverer 1.4 was used for database retrieval.The GO classification was used to classify the identified proteins and classify the biological processes.Path analysis was performed with Ingenuity Pathways Analysis(IPA)software.Results1.Mass spectrometry resultsA total of 1235 proteins were identified by ESI-MS/MS and identified by SEQUEST.Among them,there are 161 differential expressed proteins were identified between the control and high glucose groups,and the protein was up-regulated by 123 and 38 down-regulated proteins.After TMZ intervention,there were 84 up-regulated protein and 29 down-regulated proteins are back.A total of 121 proteins were identified in the culture medium.There were 26 different proteins between the control group and the high glucose group,of which 14 were up-regulated and 12 were down-regulated.After TMZ intervention,7 up-regulated proteins and 2 down-regulated proteins were reversed.A total of 1415 proteins were identified in mouse fibroblast culture medium.Among them,there were 218 different proteins between the control and high glucose groups,of which 47 were up-regulated and 171 were down-regulated.After TMZ intervention,17 up-regulated proteins and 163 down-regulated proteins were reversed.2.The analysis of differential protein expression profile of rat cardiomyocytes induced by high glucose in TMZ interventionAfter treatment with TMZ,the localization of differential proteins mainly includes cytoplasmic protein(46.9%),macromolecule complex(14.3%)and organelle protein(26.55).The function of differential protein mainly includes the activity of catalyzed protein(45.3%),active protein(32.6%),structural active protein(14.0%),and so on.After analyzing the differential proteins,the different proteins in rat cardiomyocytes induced by hyperglycemia after TMZ intervene related with energy metabolism are mainly including fructose-bisphosphate(Aldoa),6-phosphogluconate dehydrogenase,G6pd,cytochrome b-cl complex subunit 1(mitochondrial,Uqcrc1)and so on.Apoptosis related proteins mainly include arginine-tRNA ligase(cytoplasmic,Rars),serine protease(HTRA1)and so on.Proteins associated with autophagy are including Hsp90ab1 and stat 1,Ubiquitin-like modifier-activating enzyme 1,Ubal,and so on.Proteins associated with the pi3k/AKT pathway are mainly including adhesion protein(Vinculin,Vcl),depolymerized protein(Destrin,Dstn),ADP ribosylation factor 4(Arf4),Arhgdia,Statl,Gdi2.The proteins related to AMPK protein are including Trifunctional enzyme subunit alpha,mitochondrial,Hadha,Fumarate hydratase(Fh),Aspartate aminotransferase(mitotondria,Got2).3.Analysis of differential protein expression profile of cardiomyocytes after TMZ interventionAfter high glucose induction,the differential protein was mainly divided into bioregulatory protein(5.7%),cell component regulatory protein(8.6%),cell process regulatory protein(31.4%),developmental regulatory protein(8.6%),metabolic regulatory protein(22.9%),multicellular biological process regulatory protein(8.6%),stimulated response protein(5.7%);After TMZ intervention,the differential protein involved in the biological process of biological regulation of protein(12.5%),regulatory proteins(6.3%),metabolic regulatory proteins(25%),multicellular organisms Process-regulated protein(6.3%),stimulated response protein(6.3%).According to the function of differentiation proteins of secretory proteins in cardiomyocytes,It can be divided into catalytically active protein(47.6%),binding active protein(28.6%),structural active protein(23.8%),structural active protein(23.8%).After TMZ intervention,compared with the high glucose group,the activity of the differential proteins secreted by cardiomyocyte can be divided into catalytically active proteins(37.5%),bound to active protein(37.5%),and constitutively active protein(25%).After protein-protein interaction analysis,among the differential proteins in rat cardiomyocytes induced with high glucose by TMZ interfered,the apoptosis-related proteins are including annexin A7(AnXa7)and four and a half LIM domains protein 2(Fhl2).The protein associated with the pi3k/AKT signaling pathway is the rho GDP-dissociation inhibitor 2(Gdi2).4.Analysis of secreted differential protein expression profile of fibroblasts after TMZ interventionAfter induction of high glucose,the differential protein was divided into cell-binding active protein 39.70%,recipient activity protein 5.80%,signal transduction active protein 3.30%,antioxidant activity protein 0.80%,transport active protein 5.80%;The differential protein was changed according to the functional classification to 38.80%of the bound active protein,7.10%of the receptor active protein,4.30%of the structural active protein,4.10%of the signal transduction active egg,38.80%of the catalytic activity,1.00%of the antioxidant activity protein,and 5.10%of transshipment active protein.After high glucose induction,compared with the control group,the differential proteins secreted by fibroblasts were divided into 4.90%of biologically regulated proteins,5.30%of regulatory proteins,1.50%of regulatory proteins,and multicellular biological process regulatory proteins,6.00%of stimulated protein,6.00%of bio adhesive regulatory protein.6.40%of multicellular or synthetic regulatory protein,6.80%of immune response protein,7.90%of developmental regulatory protein,20.00%of metabolizing protein and 29.40%of cell process regulatory protein.After treatment with TMZ,the difference protein of fibroblasts secreted mainly consisted of 6.10%of biologically regulated proteins,5.70%of localized regulatory protein,1.90%of regulatory protein,5.70%of multicellular or synthetic protein,7.10%of stimulated Protein,5.20%of bio adhesive regulatory protein,6.10%of multicellular biological process regulatory protein,5.20%of immunoregulatory protein.8.00%of developmental regulatory protein,19.80%of metabolic regulator protein,29.20%of cell process regulatory protein.After analyzing the differential proteins,the different proteins in rat cardiomyocytes induced by hyperglycemia after TMZ intervene related with energy metabolism are mainly including creatine kinase(mitochondrial 2,Ckmt2),chromosome-desorption-DNA-binding 7(Chromodomain-helicase-DNA-binding protein 7,Chd7)and Aldoa and so on.The proteins associated with apoptosis are including the genes of the axonemal dynein heavy chain 8,Dnahc8,Trafl,Sox5 and so on.The proteins associated with autophagy are including Dapk1 and Zc3h12d,whereas those associated with mTOR include Dnahc8 and Smad5.The proteins associated with collagen secretion are including non-muscle alpha-actinin 4,Acton 4,p58/GTA protein kinase(Cdk 11 b),etc.Nucleoside diphosphate kinase(Gm20390)and Ppard are related with AMPK signaling pathway;The proteins that related to pi3K/AKT signaling pathway are phospholipase C betal(Plcb1),Myofubularin homologous protein 2(Mtmr2)and so on;The proteins that related to AKT signaling pathway are Fanconi anemia protein(Fanca),connective tissue growth factor(Ctgf),and so on;Protein interaction with MAPK are including Smad5,Mapkapk5,parp1,Traf1,Ppard,Dapk1,Plcb1,Grik2,Dnahc8 and Kcnh8,which have been analyzed by protein interaction.5.Effects of TMZ on Inflammatory Related Proteins in High Glucose Induced CardiomyocytesCompared with the high glucose group,the differential protein in the TMZ intervention group was associated with the validation,including Annexin A3,Adenine phosphoribosyltransferase(APRTase),RNA Dexamethasone p68(DEAD(Asp-Glu-Ala-Asp),filamin alpha(FLNA),and so on.6.Effect of TMZ on ROS-related proteins in high glucose-induced cardiomyocytes after TMZ interventionCompared with the high glucose group,12 kinds of differential proteins in the TMZ intervention group were involved in the regulation of oxidative stress,including G6PD,17β-Hydroxysteroid dehydrogenase(HSD10)Heat shock protein 9090(HSP90beta),thioredoxin,thioredoxin-dependent peroxidase(Peroxiredoxin 3,PRX3)and so on.7.Effect of TMZ intervention in cardiomyocytes induced by high glucose and cardiomyocyte hypertrophy-related regulatory proteinsCompared with the high glucose group,there were 9 differential proteins in the TMZ intervention group related to the regulation of cardiac hypertrophy,including Adenylyl cyclase-associated protein 1(CAP 1)Dihydropyrimidinase-related protein 3,DPYL3,Lysosome membrane protein 2,LIMP-2,Moesin and so on.Conclusions1.After metabolic regulation,84 different proteins in the high glucose-induced cardiomyocyte,26 in high glucose-induced myocardial and 218 kinds of secretion differential proteins in high glucose-induced cardiomyocyte fibroblasts are reversed,suggesting that metabolism regulation of high glucose stimulation is very extensive intervention;2.The reversed differential proteins are closely related to metabolism,myocardial apoptosis,myocardial autophagy and cardiac fibrosis,suggesting that metabolic regulation may regulate the above process under high glucose conditions and provide a basis for further research.3.The reversed differential protein is closely related to metabolic pathways PI3K/AKT,fibrosis pathway MAPKs,apoptosis and autophagy key protein AMPK,suggesting that the pathway may be a key pathway for metabolic regulation of cardiomyocytes and cardiac fibroblasts,Providing a basis for a study this year.BackgroudWith the social economic development and people’s living standards are rising,unhealthy lifestyles and diet custom have become the main incentives of diabetes.According to the World Health Organization’s statistics,the global diabetes population is expected to increase to 300 million in 2025,about half of the diabetic patients will die of cardiovascular complications.Diabetes has become one of the most important noncommunicable diseases that threaten global human health.The social and economic burden and the impact on human health are serious problems that are common to the world.Diabetic Cardiomyopathy(DCM)is the main cause of heart failure and sudden death in diabetic patients,characterized by early asymptomatic impaired diastolic function,which in turn is a damaged systolic function and eventually develops into symptomatic congestive heart failure.At present,there are many studies on the mechanism of diabetic cardiomyopathy,but drug therapy is not very effective.So the need to screen for the treatment of diabetic cardiomyopathy effective drugs becomes urgent.Diabetic cardiomyopathy(DCM)is a kind of metabolic cardiomyopathy,whose incidence does not depend on coronary heart disease,hypertension,vascular disease,and other heart diseases that can lead to heart failure.At present,the drug treatments of DCM mainly include sulfonylureas,metformin,thiazolidinediones(TZDs),insulin,dipeptidyl peptidase-4(DPP-4)inhibitors,Renin-angiotensin-aldosterone System(RAAS)inhibitors,Angiotensin Receptor Blockers(ARBs)and beta-blockers,but the retrospective study shows that many types of Diabetes treatment drugs,such as sulfonylureas,TZDs and insulin,have no significant effect in improving heart failure.Recent studies have found that intensive blood glucose treatment has no effect on DCM mortality.Epidemiological studies have shown that the incidence of DCM is still increasing,suggesting that the current treatments for DCM are ineffective.The development of DCM has a long subclinical phase,so the diagnosis of DCM is easily overlooked and delayed.The prevention of DCM is particularly important.The above studies show a need for a new approach to the prevention and treatment of DCM.Diabetes-related cardiac energy metabolic disorders play an important role in the pathogenesis of DCM,characterized by increased fatty acid beta oxidation and reduced glucose metabolism.Previous studies have confirmed that fatty acid beta oxidation can increase the generation of free radicals,and its further accumulation causes oxidative stress,leading to the development of DCM.Excessive fatty acid metabolism can also reduce the calcium flow across the sarcoplasmic reticulum,damage myocardium contraction,and thus affect heart function;excessive fatty acid metabolism can also inhibit the use of sugar,further damage to glucose metabolism.Studies have shown that changes in myocardial metabolic substrates are much earlier than DCM,suggesting that metabolic substrates may be a promoter of DCM.Based on the above study,the recovery of fatty acid beta oxidation and the normal proportion of glucose metabolism,may direct a new direction of DCM treatment.The main pathogenesis of DCM includes myocardial interstitial fibrosis,cardiomyocyte apoptosis and cardiomyocyte autophagy.Myocardial interstitial fibrosis is the over-deposition of extracellular matrix in the myocardial interstitial,which led to reduced cardiac relaxation and increased stiffness,thereby affecting cardiac function.Apoptosis of cardiomyocytes is the programmed cell death.Studies have shown that cardiomyocyte apoptosis is 85 times higher than that of non-diabetic state in diabetic state.Because myocardial cells can not proliferation,and the decrease of cardiomyocyte count will lead to the decrease of effective shrinkage unit,further affect the heart function.Autophagy is the mechanisms for cell degradation and recycling of protein,lipids and organelles.Autophagic dysfunction can affect myocardial function,which can also affect cardiac function.The results of our first part of proteomics show that metabolic regulation have a significant effect on the secreted protein of cardiac fibroblasts,cardiomyocyte protein and its secreted protein,and these differential proteins are closely associated with cardiomyocyte apoptosis,myocardium cell autophagy and myocardial interstitial fibrosis,suggesting that metabolic regulation may regulate the development of DCM through the three processes.In summary,we propose the following hypothesis:the early promotion of fatty acid β oxidation to glucose oxidation changes,can partly prevent the development of DCM.The mechanism may be mainly through the regulation of myocardial cell apoptosis,cardiomyocyte autophagy and myocardial interstitial fibrosis.In this study,we established a model of type 2 diabetes mellitus and administered a metabolic regulator of trimetazidine to investigate the effect of early metabolic substrate transformation on DCM.Objectives1.To establish the DCM model of type 2 diabetic rats.To study the effect of fatty acid oxidation transform to glucose oxidative oxidation to the development of DCM;2.To explore the effect of energy metabolism regulation on cardiac structure and cardiac function in DCM rats;3.To study the effect of energy metabolism regulation on myocardial interstitial fibrosis,cardiomyocyte apoptosis and autophagy in DCM rats;to explore whether it plays the heart Protective effects.through the PI3K/AKT,MAPKs and AMPK/Bcl-2-Beclinl signaling pathway.Methods1.Establishment of type 2 diabetic rats model18 male SD rats(about 5 weeks old)weighing about 120g-140g were randomly divided into two groups:control group(control,n = 6)and diabetic group(DM,n ＝12)after intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed.Rats in the control group were fed with normal chow and DM rats were fed with a high-fat(HF)diet.After four weeks,IPITT and IPGTT were repeated.DM rats with insulin resistance were given a single intraperitoneal injection of STZ(27.5 mg/kg).Fasting blood glucose(FBG)and fasting membrane insulin(FINS)were measured in the rats of DM group,and the insulin sensitivity index(ISI)was calculated(formula:In[(FINSxFBG)-1)]).Rats with plasma glucose levels>11.1 mmol/L were considered a diabetic rat model.DM group was randomly divided into two groups:DM group(n = 6)and DM + TMZ group(n = 6).After 8 weeks of TMZ,rats were sacrificed and hearts were taken.2.Peritoneal glucose tolerance test(IPGTT)and insulin tolerance test(IPITT)IPGTT:rats were fasted for 12 hours and blood glucose was measured by tail vein.20%glucose solution(1g/kg)was injected intraperitoneally and blood samples were collected at 15min,30min,60min and 120min.Plasma glucose was measured with a one-touch glucometer.The mean area under the receiver operating characteristic curve(AUC)was calculated for glucose.IPITT:rats were fasted for 4 hours and blood glucose was measured by tail vein.Subsequently,insulin was injected intraperitoneally(1unit/kg),and AUC was calculated as described above.3.Blood analysesBlood samples were collected at 0 weeks,4 weeks,5 weeks,and 13 weeks,respectively.FBG,total cholesterol(TC)and triglyceride(TG)were measured by Bayer1650 blood biochemical analyzer after centrifugation.Fasting insulin levels(FINS)were measured by enzyme-linked immunosorbent assay.4.Measurement of blood pressureThe systolic blood pressure(SBP),diastolic blood pressure(DBP),mean arterial pressure(MAP)and heart rate(HR)were measured using a noninvasive tail-cuff system.5.Hemodynamic examinationRats under deep anesthesia underwent hemodynamic measurement.LV end-diastolic pressure(LVEDP)and systolic blood pressure(LVSP)were recorded by inserting the catheter into left ventricle.6.Histological specimensThe rat hearts were fixed neutral formaldehyde(4%).After 1-2 weeks,paraffin was embedded and produce into tissue sections of 4.5μm thick.Part of the left ventricular myocardium was frozen at-80℃ for Western blotting.7.Morphometric analysisThe sections were stained with HE,Masson-Trichrome,and Picrosirius red,showing the general morphology of the heart and the degree of fibrosis,respectively.Pictures were analyzed by Image-Pro Plus 5.0 software.8.Detection of myocardial cell apoptosisTUNEL(terminal deoxynucleotidyl transferase dUTP nick end labeling)was used to detect the apoptosis of left ventricular cardiomyocytes.9.Immunohistochemical stainingSamples sections were incubated with primary polyclonal anti-collagen I,and anti-collagen Ⅲ overnight at 4℃,and incubated with secondary antibody for 1 h at 37.The reaction was visualized with DAB solution and sections were stained with hematoxylin.The results showed the distribution and expression of Collagen I,III in myocardium.Pictures were analyzed by Image-Pro Plus 5.0 software.10.Western blot analysisProteins were extracted from hearts,and separated by SDS-PAGE.Proteins are then transferred to polyvinylidene difluoride membrane.The membranes were incubated overnight with antibodies for p-AMPK,t-AMPK,Caspase 3,PARP,p-ERK,t-ERK,p-P38,t-P38,p-JNK,t-JNK,p-PI3 K,t-PI3K,p-AKT,t-AKT,ATG5 and ATG7 and then secondary antibody.Immunoreactive bands were visualized by an enhanced chemiluminescence reagent.’11.Statistical analysisValues are presented as mean±SEM.SPSS 16.0(SPSS,Chicago,IL)was used for statistical analysis.Differences between experimental groups were determined by one-way ANOVA or Student’s t test.p<0.05 was considered statistically significant.Results1.TMZ improve the glucose-lipid metabolism disorder in DCM ratsThe results showed that FBG and FINS began to rise at 4 weeks and the elevated of TG appeared at 5 weeks.At the end of experiment,TC,TG,FBG and FINS increased markedly in the DM group while TC,TG,FBG and FINS in DM + TMZ group were significantly lower than those in DM group(p<0.05.2.TMZ improved glucose tolerance and insulin sensitivityAfter 4 weeks HF diet,the results of IPGTT showed a significant increase in blood glucose levels(p<0.05)in the DM group at 0 min and 30 min compared with the baseline level,while IPITT results showed a significant increase at all time points(p<0.05).The areas under the curve(AUC)of IPGTT and IPITT were also significantly higher at week 4 than at baseline.At the end of the experiment,blood glucose the levels of and AUC on both IPGTT and IPITT in the DM group were higher than in the control group.After the TMZ treatment,the IPGTT and IPITT showed that levels of blood glucose in the DM+TMZ group were lower than those of the DM group.The DM+TMZ group revealed lower AUC on both IPGTT and IPITT than the DM group.3.TMZ ameliorated cardiac remodelingThe heart weight/body weight ratio(HW/BW)was significantly increased(3.39±0.07 vs.2.51±0.05,p<0.05)in DM group,and HE staining showed thickened left ventricular wall.The cardiomyocytes in DM group were disorganized and twisty,while control group was compact and well-organized.The heart mass of the DM +TMZ group was slightly smaller than that of the DM group,and the HW/BW decreased significantly(2.88 ± 0.09 vs.3.39 ±0.07,P<0.05).The cardiomyocytes arrangement is relatively dense and orderly.4.TMZ reduced myocardial interstitial fibrosisMasson staining and Sirius red staining showed that the fibril arranged more random and loose in DM group than the control group,and there were lots of collagen in the interstitial and perivascular areas.Collagen volume fraction(CVF%)and vascular fibrosis index PVCA/LA)increased significantly in DM group(p<0.05).Compared with DM group,the fibroblasts arranged in DM + TMZ group were more dense,and the collagen decreased significantly in interstitial and perivascular areas,and CVF%and PVCA/LA decreased significantly(P<0.05).Immunohistochemical semi-quantitative results showed that the expression of collagen Ⅰ/Ⅲ in DM group was significantly increasedcompared with control group(p<0.05),and the expression of collagen Ⅰ/Ⅲ in myocardium was significantly decreased after TMZ treatment.5.TMZ inhibits cardiomyocyte apoptosisTunel staining showed that Tunel positive cells in DM group were significantly higher than those in control group(p<0.05).Western blot showed that cleaved Caspase and Parp expression also increased significantly in DM group(p<0.05).After TMZ treatment,Tunel-positive cells decreased significantly(p<0.05)and the expression of cleaved Caspase and Parp was significantly lower(P<0.05).6.TMZ increases cardiomyocyte autophagy in type 2 diabetic ratsWestern blot showed that the ratio of LC3-Ⅱ/LC3-Ⅰ in DM group was significantly lower than that in Control group(p<0.05),and the expression of ATG5 and ATG7 decreased significantly(p<0.05).After TMZ treatment,the ratio of LC3-II/LC3-I in DM group was significantly increased,and the expression of ATG5 and ATG7 increased significantly(p<0.05).7.TMZ improves cardiac function in diabetic ratsAt the end of the experiment,the results of invasive heart catheterization showed that LV end-diastolic pressure(LVEDP)in the DM group were higher than those in the control group(p<0.05).After the TMZ treatment,LV end diastolic pressure was significantly lower in the DM+TMZ group than in the DM group(p<0.05).It showed that TMZ can improve the left ventricular diastolic function.8.TMZ regulated PI3 K/AKT,MAPK and AMPK/Beclinl-Bcl2 pathwaysThe levels of p-PI3 K,p-AKT and p-AMPK in DM group were significantly lower than those in the control group.However,p-ERK,p-P38 and p-JNK were significantly elevated in the DM group.Meanwhile,enhanced interaction between Bcl-2 and Beclin1 was enhanced in the DM group.After TMZ treatment,p-PI3 K,p-AKT,p-JNK and p-AMPK activity were enhanced and the complex of Bcl-2 and Beclinl in the DM+TMZ group was disrupted compared with DM group.However,p-ERK and p-P38 activity in the DM+TMZ group were inhibited compared with DM group.Conclusion1.Early metabolic regulation can partially prevent the occurrence and development of DCM;2.Early metabolic regulation can improve the ventricular remodeling of DCM by reducing myocardial interstitial fibrosis,cardiomyocyte apoptosis and enhancing cardiomyocyte autophagy in type 2 diabetic rats.3.Early metabolic regulation Improves insulin resistance by activating PI3 K/AKT,selectively activates MAPK to reduce myocardial interstitial fibrosis,and inhibits cardiomyocyte apoptosis and promotes autophagy by AMPK/Beclinl-Bcl2 signaling pathway. |
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