| BackgroundCardiovascular disease is the major reason for Diabetic Patient mortality.In western countries,about 70 percents of Diabetic Patients die of diabetic cardiovascular(DCM)disease.Since the first time of DCM was proposed by Rubler et at.in 1972,all epidemiological,pathological and laboratory research results implied the existence of Diabetic Cardiomyopathy lesion.Great correlation between Diabetic Cardiomyopathy lesion and the unique metabolic disorders of diabetes exists. Typical clinical symptoms includes defect of diastolic function in early stage,and systolic in late stage.Congestive heart failure(CHF)is prone.Recent basic and clinical researches done by scholars at home and abroad have made abundant progress.However,the pathogenesis,early diagnosis and treatment of DCM is still under active investigation.It has great value for the cure of DCM to study its pathogenesis in-depth and block its prevalence.DCM prevalence and development is affected by many factors,among which are disorder of metabolism of cardiocytes,micro-angiopathy and oxidative stress.It is shown that,the apoptosis and necrosis of myocardial cells may arise in early stage of DCM.Hyperglycemia could result in oxidative stress increases and signal transduction changes,which in turn leads to abnormal gene expression and activates programmed cell death.The potential mechanism of the apoptosis of cardiocytes and signal pathways is:1.myocardial apoptosis mediated by death receptor pathway;2. Mitogen-Activated Protein Kinase(MAPK)transduction pathway and 3. Phosphatidylinositol-3 kinase/Akt signal transduetion pathway.Pathological manifestations of DCM includes hypertrophic cardiomyopathy and myocardial fibrosis etc.Cardiac fibroblasts proliferation and the changes of the type and quantity of collagen synthesis and secretion,i.e.myocardial interstitial reconstruction, myocardial apoptosis and myocardial fibrosis play important roles in DCM development.Forkhead box O transcription factors(FoxO)protein family,which has just been formally denominated in 2000,consists of 17 subfamilies that are named Fox A to Q. Among them,the FoxO subfamily plays an important part in the development, differentiation,metabolism,apoptosis,and immunity process by signal transduction pathway.According to the homology of amino acid sequence excluding the DNA binding domain,the vertebrate FoxO subunit can be further divided into three subgroups-FoxO1,FoxO3 and FoxO4.FoxO is activated in the PI3K pathway by growth factor.Phosphorylagted FoxO activates or inhibits the genes related to the apoptosis or the cell division cycle,such as Bim,P27kip1,FasL,Bc126,TRA IL,FLIP, MnSOD,GADD45(Za)etc.It has been found that FoxO may activate different genes in different cell types. Its cell type specificity,apoptosis or not,or only inducing apoptosis in stress condition may depend on suitable cofactors.The expression of apoptosis genes induced by FoxO is different in various studies,leading to different effect.Still,the apoptosis mechanism induced by FoxO has been far from clear.Literatures suggest that FoxO transcription factor can regulate the pathology and physiological changes of cell and ECM.Some scholars from abroad attest to that FoxO3a can regulate the apoptosis of endothelial cells through PI3K/Akt/ FoxO3a /Bim pathway.There is no report about the effect of FoxO3a on apoptosis of cardiocytes yet.Now,medication for diabetes includes hypoglycemic agents,can slow down the development of the disease but not decrease the process of cardiovascular events of diabetes patients.It has great value for the cure of DCM to study its pathogenesis in-depth and block its prevalence,to open up new drug intervention mechanism in pathogenesis of DCM.Statins is the inhibitor of the rate-limiting enzyme for the synthesis of cellular endogenous cholesterin:3-hydroxy-3-methylglutaryl-coenzymeA, HMG-CoA reductase,which can effectively inhibit the synthesis of cholesterin,is widely used clinically to cure hyperlipemia.These years,research reported that statins can inhibit apoptosis of endothelial cells,but the mechanism is not completely clear. Carmen Urbich et al proved that atorvastatin and mevastatin can inhibit the PI3K/Akt/ FoxO3a/Bim pathways to regulate the apoptosis of endothelial cells.Can they treat DCM by inhibiting PI3K/Akt/FoxO3a pathways and regulating the apoptosis of cardiac muscle cells?Based on systematic analysis of signal transduction pathways in DCM,we combined molecular biology,cell biology,histopathology,Echocardiography and computer image analysis,explored the effect of glucose/PI3K/Akt/FoxO3a signal transduction pathway in initiation and development of DCM.Using Resovastatin,an inhibitor of the rate limiting enzyme 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA)in endogenous synthesis,we treat DCM animal models to explore new target of DCM therapy.Objectives(1)To establish an animal model of DCM;(2)To observe the changes of histopathology and ultra structure in DCM model;(3)To discuss cardiac morphology reconstruction of DCM animal model and observing the role of crestor on the intervention of DCM with application of Echocardiography Detection Technology;(4)To confirm the existence of P13K/Akt/FoxO3a pathway in the rat DCM model through technology of RT-PCR,Western-Blot,Immunohistochemistry etc;(5)To evaluate the role of Resovastatin in DCM prevention.Materials and Methods 1.Construction of DCM animal modelMale Wistar rats,(200-400g)forty-six,after adaptive feeding one week,which were randomly divided into three groups:normal control(n=10),DCM(n=18)and DCM plus crestor group(n=18).For DCM and DCM plus crestor group,high-glucose and high-calorie diet was given.Four weeks later,giving a single intraperitoneal injection with Streptozotocin(STZ,30mg/kg,dissolve in 10mM cold citrate buffer, PH4.2).Normal control group were given standard rat feed,four weeks later,inject the same dose of citrate buffer to Intraperitoneal.Injecting STZ and measuring blood glucose using tail vein blood after 72 hours,Die-diabetic rat's standards:two consecutive fasting blood glucose≥11.1mmol/L.Those haven' treach those standards Die were removed.Those reach the standard were used to next procedure,and then, the DCM plus resovastatin group was fill stomach with Resovastatin(20mg/kg).The other two groups were given a gavage with Physiological saline,feeding another 20 weeks.2.Body weight and biochemical indicesFollowing observations on animals were performed during the whole experiments:(1)body weight(BW)was documented every week,and FBG was detected every two weeks.(2)FBG,fasting insulin,triglyceride(TG)and cholesterol(Chol)were analyzed in each of the three group before STZ injection,one week after STZ injection and at the end of the experiment.3.Echocardiogram examinationAt the beginning and at the end of the study,transthoracic echocardiogram was performed in diabetic and control animals.Rats were placed supine and the anterior chest wall was shaved.Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector equipped with a 7.5-MHz phased-array transducer.Conventional images induced 2-dimensional,M-mode,and continuous wave and pulsed Doppler images.4.Histopathologic examination of myocardialAfter the animals were killed,draw the material from tissue then fix,dehydrate, transparent,wax dip,paraffin-embedded,slice,conventional HE stain and Masson stain,observe cardiac morphology and collagen distribution then take photos.5.Detect cardiocyte apoptosis index(AI)by TUNELAmber-coloured caryon is apoptosis masculine Cell.count method:calculate masculine cells and total cellular score under×400 object glass,select campus visuals which total cellular score above 200,observe five blades each type,take mean value,calculate apoptosis index(AI).6.Real-time PCRExtract total RNA from myocardial,from which we could get cDNA through reverse transcription PCR.Taking house-keeping geneβ-actin as a reference and detect expression of the three factors' mRNA(P13K,Akt and FoxO3a)in each of the three group,which were carried out through quantitative real time RT-PCR technology.7.ImmunohistochemistryTake tissue slice and detect protein expression of P 13K,Akt and FoxO3a through immunohistochemistry.8.Western-Blot analysisTake myocardial and extract total protein.Detect ptotein expression of P13K, Akt and FoxO3a through the following procedures:SDS-PAGE segregate, membrane-trans,protein marking,DAB color development etc.Results1.General features of the experimental ratsAmong experiments,the rats in normal control group have high spirit,body weight added noticeably,react cute and have a colorful white fur.The rats in group of DCM and DCM plus Resovastatin emerge symptom of more drink,polyuria,more food and wasting.Aside from this,the slowly added body weight,spirit languish,matt fur and decreased visual acuity are also found.Three rats died in the whole experiment.Two belong to the group of DCM and the left one was from DCM plus Resovastatin group.The reason of death may relate to diabetic ketoacidosis,infection or other complications.DCM plus Resovastatin group removed a rat that hasn't reached the standard.A total of 42 completed this study,10 of them in normal control group,16 in DCM group and the rest from DCM plus Resovastatin group.2.Body weight and biochemical indicesGive four weeks' high glucoser and high calorie diet and then compare with control group.It is found that the DCM plus Resovastatin group had a higher level in aspect of body weight,insulin of limosis,triglyceride and cholesterol than other two groups(P＜0.05-0.01).There was no remarkable difference in terms of fasting blood glucose(P＞0.05).One week after STZ injection,compared with normal control group,DCM group and DCM plus Resovastatin group had no difference on fasting insulin level,but had a increasing on levels of fasting blood glucose,triglyceride and cholesterol(P＜0.01).At end,compared with normal control group,the body weight of other two groups declined noticeably(P＜0.01)and fasting insulin level declined too(P＜0.05).The level of fasting blood glucose,triglyceride and increased(P＜0.01),The level of cholesterol of DCM group increased(P＜0.01),but there was no remarkable difference in terms of cholesterol of DCM plus Resovastatin group(P＞0.05).3.Eehoeardiogram examinationAt the beginning,the Echocardiogram examination index(including LVIDs, LVIDd,EF,FS,valvular regurgitation,Max E wave speed,Max A wave speed,ratio of E/A,EDT',IVRT' and APV)of the three groups had no uncommon difference (P＞0.05).At the end of experiment,compared with normal control group,the DCM group and DCM plus Resovastatin group had uncommon increase on value of LVIDs and LVIDd(P＜0.01),as the incidence of valvular regurgitation(P＜0.05).The Max E wave speed declined,Max A wave speed increased,E/A ratio declined,IVRT' prolonged,FS declined and AVP declined too(P＜0.05).EF declined(P＜0.01),But there had no obvious difference on value of EDT'(P＞0.05).Experimental end,compared with DCM group,the group of DCM plus Resovastatin declined on value of LVIDs and LVIDd(P＜0.05),the incidence of valvular regurgitation declined(P＜0.05),Max E wave speed declined,Max A wave speed increased,E/A ratio declined,IVRT' prolonged,FS declined and AVP declined too(P＜0.05).EF increased.But there had no obvious difference on value of EDT'.4.Ultrastructural changes observed by transmission electron microscopyThe cardiocytes from the normal control group arranged regularly,a little fibroblast and collagenous fibers distributed in extra-cellular matrix.The cardiocytes from the DCM group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibrillar was disintegrated.The myofilament was distorted and interrupted,a lot of collagenous fibers distributed in extra-cellular matrix.The microvessel lumen was narrow.Compared with the DCM group,the cardiocytes in DCM plus Resovastatin were more neatly arranged,the quality of inter fiber piled up noticeably decreaseed.5.pathological observationHE staining:In normal control group,cardiocytes arranged neatly,an uniform nuclear and cytoplasmic staining;in DCM group,cardiocytes arranged disorderly, irregular nuclei size,capillary basement membrane thickening,interstitial fibring, myocardial cells overgrowth and mortify;the DCM plus Resovastatin group had a signifant improvement.Masson staining:Cardiocytes arranged neatly,the collagen tissue was appropriate arranged among cardiocytes in normal control group;however,the cardiocytes arranged irregularly,collagen tissue increased markedly,and disrupted in some area in DCM group;the DCM plus Resovastatin group had a signifant improvement.6.Detect cardiocytes apoptosis index by TUNELCompared with normal control group,the DCM group and DCM plus Resovastatin group had uncommon increase on cardiocytes apoptosis rate(P＜0.0001).Compared with DCM group,the group of DCM plus Resovastatin had obvious declined on cardiocytes apoptosis rate(P＜0.01).Correlative analysis showed that In DCM group,fasting blood glucose had positive correlation with cardiocytes apoptosis index(r=0.906,P＜0.0001).7.RT-PCRCompared with normal control group,the expression levels of P13K,Akt and FoxO3a increased evidently in DCM group and DCM plus Resovastatin group respectively(P＜0.05-0.01).Compared with the DCM group,mRNA expression levels of P13K,Akt,FoxO3a in DCM plus Resovastatin group reduced obviously respectively(P＜0.05).Correlative analysis showed thatIn DCM group,mRNA expression levels ofPI3K(r=0.396,P＜0.05),Akt(r= 0.534,P＜0.01),FoxO3a(r=0.837,P＜0.001)had positive correlation with fasting blood glucose respectively.In DCM group,mRNA expression levels ofPI3K(r=0.48,P＜0.05),Akt(r= 0.593,P＜0.01),FoxO3a(r=0.872,P＜0.001)had positive correlation with cardiocytes apoptosis rate respectively.8.ImmunohistoehemistryP13K,Akt,FoxO3a:brown granules showed the positive signal of coloration,which mainly located at cardiocytes and the Endothelial cells.In normal control group,a small quantity of light brown granules exsited in the tissue of cardiac muscle which distributed equably,sparsely.In DCM group dark brown granules exsited in the cytoplast of cardiocytes which distributed densely.Compared with the DCM group,light brown granules exsited in the DCM plus Resovastatin group which distributed much sparsely.9.Western-Blot analysisCompared with the normal control group,protein expression levels of PI3K,Akt,FoxO3a in DCM group and DCM plus Resovastatin group increased obviously respectively(P＜0.05-0.01).Compared with the DCM group,protein expression levesl of PI3K,Akt,FoxO3a in DCM plus Resovastatin group decline clearly respectively(P＜0.05).Correlative analysis showed that Protein expression levels ofPI3K(r=0.468,P＜0.01),Akt(r=0.574,P＜0.01),FoxO3a(r=0.731,P＜0.001)had obviously positive correlation with fasting blood glucose in DCM group respectively.Protein expression levels of PI3K(r=0.571,P＜0.01),Akt(r=0.645,P＜0.01),FoxO3a(r=0.891,P＜0.001)had obviously positive correlation with cardiocytes apoptosis rate in DCM group respectively.Conclusions1.An animal model of DCM with specific metabolic characteristics was established by high-calorie diet and small dose STZ injection.This model is valuable for the study of the mechanism of DCM;2.Cardiocytes apoptosis and interfibrosis are the main tissue pathology changes in DCM;3.The existence of PI3K/Akt/FoxO3a access in rat DCM model was confirmed with the technique of RT-PCR,Western-Blot,Immunohistochemistry,etc.This pathway may play important role in cardiocytes apoptosis;4.Resovastatin can treat DCM by inhibiting PI3K/Akt/FoxO3a pathway and regulating the apoptosis of cardiocyte cells. IntroductionEarly phase of diabetic cardiomyopathy will present apoptosis and necrosis of cardiocyte.The reasons may be when high blood glucose appears,oxidative stress increses,signal transduction pathway changes,which induce abnormal gene express, active cell programmed death.The signal transduction mechanism of cardiocyte apoptosis may be:①death receptor transduction pathway induced cardiocyte apoptosis;②mitogen-activated protein kinases(MAPK)transduction pathway; phosphatidylinositol-3kinase/Akt signal transduction pathway.FoxO play an important regulation role in many complicated diseases such as diabetes and cancers.FoxO is activated by growth factor activated PI3K pathway, which induce Akt dependent phosphorylation.Phosphorylated FoxO activates or inhibits cell apoptosis or cell division cycle related genes,such as Bim,P27kipl, FasL,Bcl26,TRAIL,FLIP,MnSOD,GADD45(Za),then activates or inhibits cell apoptosis.At present Carsten Skurk etc have proved Caspase8 increase by activation of PI3K/Akt/FoxO3a/FLIP pathway in human umbilical veins endothelial cells so as to promote apoptosis.The rat DCM model in the paper one has proved the present of PI3K/Akt/FoxO3a pathway.This research treats Wistar rat suckling mouse cardiocytes as experiment object, different concentration D-glucose as process factors,observes the influence of different concentration D-glucose on cardiocytes FoxO3a expression and cell apoptosis,fatherly discusses the molecular mechanism of the effect.Purpose:1.To observe the influence of high glucose on cardiocytes apoptosis.2.To explore the role of PI3K/Akt/FoxO3a signal transduction pathways on cardiocytes apoptosis when they were treated with high glucose.3.To Make sure the possible downstream molecular mechanism of PI3K/Akt/FoxO3a signal transduction pathways.Methods1.Cell cultureNeonatal rat cardiac fibroblasts were prepared by the following procedures:three to four hearts from 2- 3-day-old Wistar rats were finely minced and placed together in 0.25%trypsin.Pooled cell suspensions were centrifuged and resuspended in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.The resuspension was plated onto culture flasks for 90 min,which allowed for attachment of cardiocyte to the bottom of the culture flask.Non-adherent and weakly attached cells were removed and the medium was changed.Cell cultures were incubated at 37°C in a humidified atmosphere of 5%CO2/95%air.Studies were conducted on original passage cardiocyte that were grown to subconfluence in serum-containing media and then growth arrested for 24h in serum-free medium before treatment.2.Study designThe simulation of the diabetes mellitus in vitro,group handed with D-glucose and mannitol:To observe the effect on the myocardial cells' apoptosis of treatment by different concentration D-glucose(5,15,30 mmol/L)and mannitol(30 mmol/L) 24 hours. 3.Detection of the cardiocytes' apoptosis indexThe change of the cell cycle and the rate of the apoptosis were detected by flow cytometer,the positive cells of the apoptosis was detected by the apoptosis kit.4.Real-time RT-PCRcDNA was achieved from the complete RNA which was abstracted from the selected cells by the reverse transcription,the mRNA expression of the marker genes PI3K,Akt,Foxo3a,FLIP,Bim,Caspase8 and Caspase9 were detected by the real-time RT-PCR with the reference-β-actin,a housekeeping gene.5.Western-Blot analysisThe sum protein collected from the harvested cells was treated with isolation, trarsmembrane,protein imprinting,DAB coloration and other steps of SDS-PAGE,in order to detect the expressing level of marker proteins such as PI3K,Akt,Foxo3a,FLIP,Bim,Caspase8,Caspase9.Results1.Detection of the apoptosis index of cardiomytesCardiocytes were stimulated with different concentration D-glucose(5,15,30 mmol/L)and mannitol(30 mmol/L)24 hours,the apoptosis index are 1.38±0.32,8.41±0.99,13.12±1.14,1.69±0.35 respectively,compared with the group treated by 5mmol/L D-glucose,the apoptosis index of the groups treated by 15,30mmol/L D-glucose increase obviously(P＜0.001-0.0001);the apoptosis index of the groups treated by mannitol(30 mmol/L)had no uncommon difference(P＞0.05).Correlative analysis showed that:The cardiocytes' apoptosis index had a positive correlation with the concentration of D-glucose(r=0.871,P＜0.01).2.Detection the mRNA expressions levels of PI3K,Akt,FoxO3a,FLIP,Bim,Caspase8,Caspase9Compared with the 5mmol/L concentration D-glucose group,the mRNA expression levels of those factors PI3K,Akt,FoxO3a,Bim,Caspase8,Caspase9 in 15mmol/L, 30mmol/L concentration groups increased significantly(P＜0.05-0.01);the mRNA expression level of FLIP decreased(P＜0.05).Correlative analysis showed that:(1)The concentration of D-glucose had obvious positive correlation with the mRNA expression levels of PI3K(r=0.578,P＜0.01),Akt(r=0.369,P＜0.05),Fox03a(r=0.776,P＜0.01),Bim(r=0.417,P＜0.05),Caspase8(r=0.663, P＜0.01),Caspase9(r=0.453,P＜0.05)respectively;had negative correlation withthe mRNA expression levels of FLIP(r=-0.483,P＜0.05)(2)The cardiocytes' apoptosis index had obvious positive correlation with the mRNA expression levels ofPI3K(r=0.749,P＜0.01),Akt(r=0.423,P＜0.05),FoxO3a(r=0.822,P＜0.01),Bim(r=0.521,P＜0.05),Caspase8(r=0.698, P＜0.01),Caspase9(r=0.506,P＜0.05)respectively;had negative correlation with the mRNA expression levels of FLIP(r=-0.582,P＜0.05).3.Detection of the protein expressions levels of PI3K,Akt,FoxO3a,FLIP,Bim,Caspase8,Caspase9 by Western-Blot.Compared with the 5 mmol/L concentration D-glucose group,the protein expression levels of PI3K,Akt,FoxO3a,Bim,Caspase8,Caspase9 in the 15mmol/L,30mmol/L concentration groups increased significantly(P＜0.05-0.01); the protein expression level of.FLIP decreased(P＜0.05).Correlative analysis showed that:(1)The concentration of D-glucose had obvious positive correlation with the protein expression levels of PI3K(r=0.395,P＜0.05),Akt(r=0.436,P＜0.05),FoxO3a(r=0.804,P＜0.01),Bim(r=0.542,P＜0.05),Caspase8(r=0.658, P＜0.01),Caspase9(r=0.507,P＜0.05)respectively;had negative correlation with the protein expression level of FLIP(r=-0.696,P＜0.01).(2)The cardiocytes' apoptosis index had obvious positive correlation with the protein expression levels of PI3K(r=0.484,P＜0.05),Akt(r=0.531,P＜0.05),FoxO3a(r=0.846,P＜0.01),Bim(r=0.570,P＜0.05),Caspase8(r=0.705, P＜0.01),Caspase9(r=0.543,P＜0.05)respectively;had negative correlation with the protein expression level of FLIP(r=-0.709,P＜0.01).Conclusions1.High D-glucose concentration can result in the apoptosis of cardiocytes from neonate Wistar rat cultured in vitro,and according to the common rule of density-effect;2.FoxO3a engages in the apoptosis of cardiocytes by PI3K/Akt signal transduction pathway;3.FoxO3a engages in the apoptosis of cardiocytes by the downstream apoptosis genes: FLIP,Bim,Caspase8,Caspase9. |
PDF Full Text Request