| BackgroundDiabetic cardiomyopathy(DCM)is an important complication characterized mainly by structural and functional disorders of the heart in diabetes,which has been the leading cause of death in the diabetic patients.A series of changes can occur in cardiomyocytes and fibroblasts under diabetic conditions.In the cardiomyocyte,the main characteristics are cell hypertrophy which has a significant increase of the surface area of cells,and in the later stages the cell can be be induced to damage and death.The high glucose can also promote dysfunction of the fibroblast.Excessive accumulation of fiber components,and imbalance of various types of collagen can happen in the extracellular matrix(ECM).Myocardial fibrosis can increase myocardial stiffness and reduce ventricular compliance.In addition,studies have found that total coronary resistance increases and maximum coronary blood flow decreases in diabetic patients,suggesting that there is a microcirculation disorder in myocardial tissue in diabetic patients.A variety of mechanisms are involved in the development of DCM.The increase of ROS is one of them.Hyperglycemia-induced generation of reactive oxygen species(ROS)stimulate various cytokines and inflammatory factors including nuclear factor-κB(NF-κB)and inflammasome.Sterile inflammatory response plays an important role in the development of diabetic cardiomyopathy Previous studies have found that inflammasome are closely related to diabetes-related complications,but the role and regulatory mechanisms of AIM2 inflammasome in DCM have not been elucidated.Inflammasome are protein platforms that interact with multiple physiological and pathological reactions,can sense a variety of signals closely related to cellular immunity and death.Inflammsome which have been indentified include multiple members,including the NLRs family and the AIM2 family.Absent in melanoma 2(AIM2)was first reported in 1997 and belongs to the interferon-induced HIN-200 family.Studies have found that AIM2 protein is missing in melanoma,but it is widely distributed in many other tissues and cells,and plays an important role in regulating cell death,inflammatory response,migration and proliferation.AIM2 is distributed in the cytoplasm of cells and can recognize double-stranded DNA(dsDNA).The main source of dsDNA is foreign bacteria and viruses,so AIM2 plays a vital role in the immune process.After binding to dsDNA,AIM2 can recruit ASC protein(apoptosis associated speck like protein containing a card)to form an inflammatory platform which can further recruit and activate caspasel protein to transform to it active forms(Caspasel p10 and Caspasel p20)As a cysteine protease,caspase1 can activate IL-1(3 and IL-18 in the cytoplasm,and can also participate in GSDMD-N mediated pyroptosisPyroptosis is a form of programmed cell death that is dependent on caspasel.The morphological characteristics of pyroptosis include mainly two parts.The first one is DNA fragmentation.The second one is the deconstruction of the cell membrane and the formation of membrane pores,which accelerates cell swelling and damage to organelles,eventually leading to cell ruptureElectron microscopy results of myocardial tissue in diabetes indicate that myocardial cells can exhibit cellular morphological features similar to the pyroptosis,such as cell swelling,formation of cell membrane channels,and mitochondrial swelling and vacuolation.Not only that,we found that high glucose can the expression of AIM2 inflammasome in H9c2 cardiomyocytes in previous experiments.Based on this,we propose the hypothesis:Hyperglycemia induced ROS in myocardial tissue promotes excessive activation of AIM2 inflammasome,and abnormal activation of AIM2 inflammasome can promote IL-1βmediated inflammatory response and GSDMD-N mediated pyroptosis process,which lead to abnormal heart structure and function finally.Objective(1)To establish the DCM model of type 2 and investigate the expression of AIM2 inflammasome in DCM.(2)To study whether the inhibition of AIM2 by shAIM2 lentivirus in DCM model can alleviate the DCM.(3)To explore the mechanism of AIM2 inflammasome in regulating the development of DCM.Methods1.Animal model of type 2 diabetesAfter one week of feeding of experimental animals to adapt the enviroment,60 male Sprague-Dawley(SD)rats of 100-120g were randomly divided into 4 groups(15 in each group):control group(control group),diabetes group(DM group),Diabetes+AIM2-shRNA lentivirus group(DM+shAIM2 group)and diabetes+negative control lentivirus group(DM+shNC group).The rats in the control group were given a basic diet.The main ingredients included:5%lipids and no cholesterol.The other three groups were fed with a high-fat diet.The main components included:16%lipids and 0.3%cholesterol.After 4 weeks,the intraperitoneal insulin tolerance test(IPITT)and intraperitoneal glucose tolerance test(IPGTT)were performed on the four groups of rats.Streptozocin(STZ,40mg/kg)was injected intraperitoneally in rats with insulin resistance after high-fat feeding.Fasting blood glucose(FBG)was performed 1 week later.DM group,DM+ AIM2-shRNA group and DM+ shNC group rats with FBG≥11.1mmol/L twice and the presence of insulin resistance are considered successful model 2 diabetes.After successful establishment of the diabetes model,high-fat feeding was continued for 12 weeks,and heart function tests were performed after 12 weeks.Echocardiography revealed left ventricular systolic or diastolic dysfunction in rats,suggesting the creation of a DCM model.2.EchocardiographyAfter anesthesia with isoflurane and removal the hair on the chest,the rats were subjected to transthoracic cardiac ultrasound examination,and the values of LVEDd,LVEF,FS,E/A,and E’/A’ were collected for evaluation the cardiac function of rat.3.Lentivirus interventionEight weeks after intraperitoneal injection of STZ,rats in the DM+shAIM2 group and DM+shNC group were injected with lentivirus through tail vein.Hearts of rats were randomly selected 2 weeks after virus injection,and frozen sections were used to detect myocardial virus transfection.The sequence of shAIM2 is GGTCACCAGTTCCTCAGTT.Four weeks after the lentivirus intervention,rats in the four groups were euthanized.4.Serum measurementsRats were fasted one night before euthanasia,and peripheral venous blood was drawn.Serum blood glucose(BG),triglyceride(TG),and total cholesterol(TC)were measured5.Histological examinationAfter the rats were euthanized,the weight of the rats and the weight of the heart were measured,and the image of the whole heart was obtained by camera.Subsequently,tissue fixation was performed and heart tissue was cut along the coronal plane at the level of the papillary muscle for paraffin section.The sections were stained with hematoxylin and eosin after dewaxing and the images were obtained by camera.6.Measurement of myocardial fibrosisMasson trichrome staining and Sirius red staining were performed to detect the collagen.The staining results were analyzed using Image-proplus to evaluate left ventricular myocardial fibrotic area fraction.7.ImmunohistochemistryImmunohistochemistry of myocardial paraffin sections was performed to observe the distribution of IL-I β in tissues.Data were analyzed by Image-Pro Plus.8.Western blotTissue of left ventricle in the model and H9c2 cells were cut up and proteins were extracted.The expressions of AIM2,ASC,pro-caspasel,caspasel,IL-1β and GSDMD-n were detected by Western blot9.TUNEL examinationThe nuclear DNA damage of cardiomyocytes in myocardial tissue of rat was detected by TUNEL.10.Cell culture and treatmentH9c2 cardiomyocytes were cultured with DMEM(1g/L)+5%fetal bovine serum(FBS)+penicillin/streptomycin.Prior to intervention,cells were cultured with serum-free medium overnight,followed by sugar stimulation at different concentrations(11.1mmol,22.2mmol,33.3mmol)and times(Oh,6h,12h,24h,48h).In the ROS inhibition test,10mmol/L N-acetylcysteine(NAC)was used before HG stimulation to inhibit cardiomyocyte ROS in advance.11.Lentivirus transfectionIn order to investigate the role of AIM2 in cardiomyocytes and its regulatory mechanism,AIM2 siRNA was used in cardiomyocytes.The sequence of siAIM2 is GGUACCAGUUCCUCAGUUTT.12.ROS detectionPeroxide-sensitive fluorescent probe 2’,7’-dicholorofluorescein diacetate(DCFH-DA)was used to detect the ROS production in cardiomyocytes under high glucose stimulation.13.Cell death assayTUNEL was used to detect the level of nuclear DNA damage in cardiomyocytes,and EthD-Ⅲ was used to detect the level of cell membrane damage in cardiomyocytes.14.Statisticlal analysisSPSS v20.0 and Graphpad 6.0 processes the data.Continuous variables are expressed as mean ± SEM.Independent sample T-tests were used to compare continuous variables between the two groups.One-way ANOVA test was used to compare continuous variables between multiple groups.Significance was defined as p<0.05.Result1.Basic characteristics of type 2 diabetic ratsAfter 4 weeks of high-fat diet(HF;34.5%fat,17.5%protein,and 48%carbohydrate),the mean area under the receiver operating characteristic curve(AUC)for intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)was higher for HF diet rats than the controls.After 20 weeks of diabetes,diabetic rats showed significantly higher fasting blood glucose(FBG),total cholesterol(TC),triglycerides(TG)levels,insulin tested at the end of the experiment(INS)and insulin sensitivity index(ISI).AIM2 inhibition had no effect on the TC,TG,FBG,ISI and INS.The AUC for IPITT and IPGTT was not lower significantly in rats with the inhibition of AIM2 than in vehicle treated animals.2.Diabetes activated the expression of myocardial AIM2 and AIM2 inhibition attenuated myocardial remodelling in diabetic ratsWe found that AIM2 protein levels were significantly increased in rats in the DM group compared with those in the control group.We also found that AIM2-shRNA inhibition downregulated the diabetes-induced increase in expression of AIM2.Additionally,the ratio of heart weight to body weight increased in the DM group compared with the control group.The DM group also showed eccentric ventricular hypertrophy and higher cardiomyocyte widths compared with those in the control group,but were alleviated by AIM2-shRNA treatment.These results indicate that AIM2 inhibition attenuates cardiac remodelling in DM rats.3.AIM2 inhibition improved the cardiac dysfunction caused by diabetesEchocardiography assessment was performed 4 weeks after AIM2-shRNA transfection.Compared with the control group,DM rats revealed cardiac dysfunction,which manifested as lower LVEF,FS,E/A ratio,and e’/a’ratio.In addition,LVEDd and the E/e ratio were significantly increased.The results showed that AIM2-shRNA improves cardiac dysfunction compared with the DM and DM+NC groups.4.AIM2 inhibition prevented myocardial fibrosis induced by diabetesMasson’s trichome and Sirius red staining of heart tissue indicated that diabetic myocardium appeared to have higher deposits of extracellular matrix compared with controls,but was alleviated by shRNA treatment.Additionally,diabetic animals exhibited overexpression of collagen I and collagen III compared with the control,and AIM2 inhibition significantly reduced the levels of both proteins.Also,the elevated levels of MMP2 and MMP9 were inhibited through shRNA-AIM2 transfection in DM rats compared with the control group.These results demonstrate that AIM2 inhibition prevents diabetic-induced myocardial fibrosis.5.AIM2 gene silencing protects cardial cells from deathThe proportion of TUNEL-positive cells was significantly increased in DM cardial tissue,but AIM2-shRNA treatment alleviated this phenomenon.Additionally,the protein levels of AIM2 inflammasome,caspase-1,and GSDMD were all increased in the DM group,but were downregulated by AIM2 inhibition 6.AIM2 expression was increased after HG treatment in H9c2 cardiomyo-blastsThe AIM2 protein levels of H9c2 cells treated in DMEM with 5.5mM glucose(control group,NG)and DMEM with 25mM glucose(high glucose control,HG)for 6,12,24,and 48 h presented with significant differences.Also,H9c2 cardiomyoblasts were cultured with DMEM media containing 5.5,11.1,22.2,or 33.3mM glucose for 24 h.The protein expression of AIM2 significantly increased after being treated with HG concentrations.The results demonstrate that HG stimulation promotes the expression of AIM2.7.ROS mediated AIM2 expression with HG stimulationBecause ROS production was increased by HG,we investigated the potential mechanism of HG stimulation of AIM2 in H9c2 cells.For this,we used NAC,a ROS inhibitor to inhibit the production of ROS.We found that the expression of AIM2 in the HG+NAC group was significantly downregulated compared with that in the HG group.8.AIM2 was involved in HG-induced pyroptotic cell deathFinally,we investigated whether AIM2 is involved in HG-induced cell death.After inhibiting AIM2 expression using AIM2-shRNA lentivirus,the protein level of AIM2 was decreased in HG-stimulated H9c2 cardiomyoblasts.The efficiently inhibited AIM2 also decreased the levels of activated caspase-1 and mature IL-1βcompared with the HG group.In addition,the expression of GSDMD decreased significantly inthe AIM2-siRNA transfected HG group compared with the HG control group.TUNEL and EthD-III staining indicated that HG promoted DNA fragmentation and pyroptotic cell death in H9c2 cardiomyoblasts.From the results,AIM2 inhibition reduced the proportion of TUNEL positive cells and EthD-Ⅲ positive cells compared with the HG group.Conclusion(1)In the state of diabetes,myocardial tissue has fibrosis and myocardial cell pyroptosis,which promotes abnormal heart structure and dysfunction.(2)The expression of AIM2 inflammasome in myocardial tissue of diabetic rats was significantly increased.(3)AIM2-shRNA lentivirus was used for in vivo,and the expression of AIM2 in myocardial tissue was effectively inhibited.(4)AIM2 gene silencing significantly improved the myocardial structure and dysfunction induced by diabetes.(5)High glucose stimulation can promote the increase of ROS expression in cardiomyocytes.The increase of ROS can promote the expression of AIM2 inflammasome,and further induce caspase-1/GSDMD-N-mediated pyroptosis.(6)AIM2 gene silencing in cells can relieve cardiomyocyte death.Background As one of the major chronic diseases in the worldwide,diabetes has become the top priority of clinical prevention and treatment.Diabetic cardiomyopathy is a major complication of diabetic patients and one of the main causes of death.Different from the secondary heart disease caused by atherosclerosis,hypertension,virus and other factors,diabetic cardiomyopathy(DCM)is a type of primary myocardial disease caused by diabetes.Ventricular remodeling caused by diabetes can lead to myocardial diastolic and systolic dysfunction,and ultimately lead to heart failure and even death.Previous studies have shown that a variety of factors has jointly promoted the occurrence and development of DCM,including hyperglycemia,insulin resistance,metabolic abnormalities,oxidative stress,and calcium overload,which have led to the occurrence of fibrosis and apoptosis in myocardial tissue.Several studies have proved that the PI3 K / Akt pathway,as an important pro-survival signalling pathway in the human body,has an inhibitory effect on myocardial cell apoptosis.At present,effective control of the blood glucose is still the basic method in DCM.With the deepening of related research,new treatment methods are constantly being explored.In addition to myocardial protective drugs,new technologies such as ultrasound-targeted microbubble destruction(UTMD)and cell transplantation that may play an important role in repairing or even replacing damaged myocardium have also become hotspots in DCM.Nicorandil,as an anti-angina pectoris drag,has a nitrate-like effect and can release nitric oxide(NO);it can also be used as an ATP-dependent K channel opener to open mitochondrial K +-ATP channels.Studies have shown that nicorandil plays a protective role in a variety of cardiovascular diseases.In the rat model of myocardial infarction,nicorandil can inhibit PKC activation by opening K+-ATP channels,prevent Ca2 + overload,reduce ventricular preload and postload,and increase myocardial perfusion.But whether nicorandil can delay the course of diabetic cardiomyopathy and its related mechanisms is still not clear.A number of studies have proven that the PI3 K / Akt pathway,as an important pro-survival signal pathway in the human body,can protect the occurrence and development of diabetic cardiomyopathy.The PI3 K / Akt pathway can enhance cardiac function in diabetes by regulating blood glucose levels,regulating lipid metabolism,protecting endothelial cells,reducing inflammation,resisting fibrosis,and alleviating myocardial cell apoptosis.The activation of PI3 K / Akt pathway downstream factors mTOR and eNOS helps to alleviate apoptosis.Previous studies have found that the PI3 K / Akt pathway is inhibited and the level of apoptosis is increased in diabetes.However,whether nicorandil can activate mTOR and eNOS through the PI3 K / Akt pathway and thus improve myocardial apoptosis has not been reported.In summary,our research group intends to explore the role of nicorandil in the occurrence and development of DCM by constructing a rat model of DCM,and to explore whether nicorandil can exert myocardial protection by regulating the PI3K/ Akt pathway,and further explore Potential role of PI3 K / Akt in DCM.Based on this,this study proposes the following hypothesis: Nicorandil can alleviate apoptosis of cardiomyocyte in diabetic cardiomyopathy,delay fibrosis,and improve DCM disease progression through factors such as the PI3 K / Akt pathway and NO release.Objective(1)To establish a model of type 2 diabetes and give nicorandil intervention,and explore the effect of nicorandil on the DCM.(2)To explore the effects of nicorandil treatment on apoptosis and fibrosis in the myocardial tissue of type 2 diabetic rats.(3)To investigate the effect of nicorandil on the apoptosis of cardiomyocytes under stimulation of the high glucose and related molecular and biological mechanisms in vitro.Method1.Animal model The animal model of type 2 diabetes is the same as the Part I.Briefly,60Sprague-Dawley rats of 100-120 g were selected and randomly divided into four groups: Control group,DM group,DM + N7.5 group,DM + N15 group.The control group was given a basic diet,and the other three groups received high-fat feeding for 4 weeks for IPITT and IPGTT.Insulin-resistant rats were selected and injected with STZ(40mg / kg)intraperitoneally.Fasting blood glucose > 11.1mmol / L is considered successful model.Eight weeks after the formation of the type 2 diabetes model,nicorandil was given by drinking water at a concentration of 7.5 mg / kg day and 15 mg / kg day.After 4 weeks of drug intervention,IPITT and IPGTT were tested,and euthanasia was performed after all tests were completed.All experimental procedures met the requirements of the Animal Ethics Committee of Shandong University.2.Echocardiography Cardiac function tests were performed in rats 4 weeks after nicorandil treatment.After isoflurane anesthesia,the chests of the rats were depilated.The Vevo-770 animal cardiac ultrasound system collects LVEF,FS,LVEDd,E / A,e V a!5 E / e ’and other indicators.3.Serological test The animals were fasted one night before euthanasia,and fasting blood glucose was measured next day;blood was collected from the apex of the heart and serum was measured for triglyceride(TG),total cholesterol(TC)and other indicators.4.Histological test Body weight and heart weight of rats were retained,heart size was measured and heart images were taken.The tissue of heart was fixed and then the heart at the level of the papillary muscle was cut to take 3-5mm paraffin sections.Sections can be dewaxed for histological staining such as HE and other stainings.5.Myocardial fibrosis Myocardial tissue sections were stained by Masson staining and Sirius-red staining.Image-proplus was used to assess fibrosis levels.6.Immunohistochemistry Immunohistochemical staining of myocardial paraffin sections with Collagen I3 Collagen III and other indicators was performed to assess the level of fiber deposition in myocardial tissue.7.Cell culture H9c2 cardiomyocytes were cultured with low-glucose DMEM(lg / L)+ 10 fetal bovine serum + antibiotics.Cells were starved overnight in serum-free medium before treatment.The various interventions and conditions performed on the cells were:(l)Nicorandil gradient treatment: Nl: lOumol;N2: 50umol;N3:lOOumol.(2)As a receptor competitor of nicorandil,5-hydroxydecanoate(5-HD)was added to inhibit the binding of nicorandil to the receptor.(3)Mitifosin(MTF,100 |imol),rapamycin(Rapa,100 |imol)were used to inhibit the PI3 K / Akt pathway.8.Western blot The myocardial tissue proteins and cellular proteins were extracted,and Bcl-2,Bax,cleaved caspase3,caspase3,p-eNOS? eNOS,p-PI3 K,PI3K,p-Akt,Akt,pmTOR in myocardial tissue were detected by Western blot.9.TUNEL assay Apoptosis level of rat myocardial tissue and H9c2 cardiomyocytes was detected by TUNEL.10.Data analysis The data were processed by SPSS v20.0 and Graphpad 6.Continuous variables are expressed as mean ± standard error.One way ANOVA was used to compare continuous variables between multiple groups.Independent sample T-tests were used to compare continuous variables between the two groups.Statistical differences were considered when p <0.05.Result1.Nicorandil treatment delays ventricular remodeling in diabetic rats Compared with the control group,the heart volume of the rats in the DM group was significantly increased,especially in the left ventricle;the heart cavity was enlarged,and the left ventricular wall was hypertrophic.HE staining showed that compared with the control group,the myocardial tissue of the DM group was disorderly arranged and the number of inflammatory cells was increased,also,the diameter of the myocardial cells was significantly increased;the cardiac weight was significantly increased,and the cardiac weight/ body weight ratio was increased.After treatment with nicorandil,the ventricular remodeling process of the rats in the DM + N7.5 group and the DM + N15 group was alleviated compared with the DM group: the heart volume decreased,the degree of hypertrophy decreased,and the diameter of myocardial cells decreased,the ratio of heart weight to body weight decreases significantly.These results indicate that nicorandil treatment can delay ventricular remodeling in DM rats.2.Nicorandil treatment improves heart dysfunction in diabetic rats After 4 weeks of nicorandil treatment,rats in each group were examined by echocardiography.Compared with the control group,the left ventricular short axis shortening rate(FS),left ventricular ejection fraction(LVEF)of the DM rats were significantly reduced,and the E / A ratio was significantly reduced.At the same time,the e V a* ratio of the DM rats was significantly reduced.Left ventricular enddiastolic diameter(LVEDd),E / e ’ ratio decreased significantly.It is suggested that rats with type 2 diabetes can develop systolic and diastolic dysfunction at 16 weeks.Compared with the DM group,the LVEF,FS,E / A,and e 7 a1 in the DM+ N7.5 group and the DM + N15 group were significantly increased,and the LVEDd and E / e’ were significantly decreased,suggesting that cardiac dysfunction caused by diabetes can be improved by nicorandil after 4 weeks of treatment.3.Nicorandil treatment improves myocardial fibrosis in diabetic rats Masson staining and Sirius-red staining showed that the extracellular matrix of myocardial tissue in the diabetic rats increased compared with the control group,but the content of extracellular matrix in the heart decreased after treatment with nicorandil.At the same time,compared with the control group,myocardial collagen I,collagen III,matrix metalloproteinase 2(MMP2)and matrix metalloproteinase 9(MMP9)increased in the diabetic rats.Nicorandil treatment significantly reduced these protein levels.These results demonstrate that nicorandil treatment prevents myocardial fibrosis in diabetic rats.4.Nicorandil treatment reduces apoptosis in the heart of diabetic rats TUNEL staining was performed on myocardial tissue sections from 4 groups of rats.Compared with the control group,the proportion of TUNEL positive cells in the heart tissue of the DM group was significantly increased.Compared with the DM group,the proportion of TUNEL positive cells in the myocardial tissue of the DM + N7.5 group and the DM + N15 group was significantly reduced.Compared with the control group,the protein levels of Bax,cleaved caspase-3 in the myocardial tissue of the DM group were significantly increased,and the protein levels of bcl-2 and eNOS were significantly reduced in the DM group.Compared with DM group,the levels of Bax,cleaved caspase-3 in myocardial tissue of DM + N7.5 group and DM + N15 group were significantly reduced,while those of bcl-2 and eNOS were significantly increased.5.Nicorandil reduces high glucose-induced apoptosis in H9c2 cardiomyocyte H9c2 cells were randomly divided into 5 groups: NG group(normal sugar group,5.5 mM),HG group(high glucose group,33.3 mM),HG + N1(high glucose group + 10 nmol nicorandil),HG + N2(High glucose group +50(xmol nicorandil),HG + N3(high sugar group +100 jamol nicorandil),and osmotic pressure was balanced with mannitol.Western blot showed that compared with the NG group,Bax,cleaved caspase-3 in the HG group were significantly increased,while bcl-2and eNOS protein levels were significantly reduced.Apoptosis levels were significantly improved after treatment with nicorandil,and the 100 ^mol of nicorandil met the best effect of improvements(Figure 11 A).6.Protective effect of nicorandil on apoptosis can be blocked by 5-HD 5-HD is a selective inhibitor of mitochondrial K+-ATP channels and can competitively bind K+-ATP channels with nicorandil.The cells were randomly divided into four groups: NG group;HG group;HG + N group;HG + N + 5-HD group.The HG + N + 5-HD group were co-treated with 5-HD and nicorandil.TUNEL staining showed that the proportion of TUNEL-positive cells was significantly reduced after nicorandil(100(xmol)treatment compared with the HG group alone,and co-treatment with 5-HD and nicorandil could partially reverse this protective effect.Western blot showed that the levels of Bax,cleaved caspase-3 and other proteins were significantly reduced,and the levels of bcl-2 and eNOS were significantly increased after nicorandil(100 jamol)compared with the HG group.Compared with the nicorandil group,the expression level of apoptosisrelated proteins in the 5-HD co-treatment group was partially reversed,indicating that 5-HD can partially reverse the protective effect of nicorandil on apoptosis.In addition,Western blot showed that p-PI3 K,p-Akt,p-eNOS,and p-mTOR protein levels in the nicorandil group increased significantly,and 5-HD co?treatment could partially reverse this change.7.Nicorandil can alleviate high glucose-induced cardiomyocyte apoptosis through the PI3 K / Akt pathway Mitifosin(MTF,100 |imol)is an Akt inhibitor,and rapamycin(Rapa,100(irnol)is an mTOR inhibitor.The cells were randomly divided into five groups:NG group;HG group;HG + N group;HG + N + MTF group;HG + N + Rapa group.TUNEL staining showed that the proportion of TUNEL-positive cells in the HG group increased significantly compared with the NG group,and the proportion of TUNEL-positive cells decreased significantly after nicorandil treatment,while reatment with MTF or Rapa could partially reverse this protective effects.Western blot showed that the levels of Bax,cleaved caspase-3 were significantly reduced, and the levels of bcl-2 and eNOS were significantly increased after nicorandil(100jxmol)compared with the HG group.Compared with the nicorandil group,the levels of Bax,cleaved caspase-3 were increased in H9c2 cardiomyocytes of the MTF or Rapa group,while the protein levels of bcl-2 and eNOS were significantly reduced,indicating that high glucose-induced H9c2 cardiomyocyte apoptosis can be regulated by nicorandil through the PI3 K / Akt pathway.Conclusion(1)Diabetic rats can improve their cardiac dysfunction after treatment with nicorandil.(2)Diabetic rats can improve myocardial fibrosis and apoptosis after treatment with nicorandil.(3)Nicorandil can reduce the apoptosis of H9c2 cardiomyocytes induced by high glucose,while 5-HD can block its effect.(4)As a NO donor,Nicorandil can also alleviate high glucose-induced apoptosis in cardiomyocyte through the PI3 K / Akt pathway. |
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