| Background:Diabetic cardiomyopathy(DCM)is a serious complication of DM and its mechanism remains incompletely understood.Sustained hyperglycemia is a major cause of excess reactive oxygen species(ROS)formation,disturbance of lipid metabolism,insulin resistance,and dysregulation of cytoplasmic calcium,which leads to cell death and results in left ventricular(LV)remodeling and subsequent heart failure.The phosphatidylinositol 3-kinase(PI3K)pathway is an important anti-apoptotic pathway which also serves as a new target to treat DCM.However,whether RSV ameliorates DCM by inhibiting inappropriate apoptosis via the PI3K/Akt/Fox03a pathway has not been addressed yet.Objective:To observe the protective effect of RSV on cardiac dysfunction in STZ-induced diabetic rats and in vitro and the mechanism involved.Methods and Results:Establishment of high glucose(HG)model.All cells were divided into the groups bellow:1)Control group:plus 20mmol/L of the mannitol;2)HG group:add the D-glucose to upload the concentration of glucose to 33mmol/L to establish the HG model.3)HG+RSV group:treated with resveratrol(1,10,100μM)plus high glucose for 24 h.But we used resveratrol in the concentration of 10μM/L to treat cells as the group in the experiment testing cell apoptosis.4)HG+RSV+LY294002 group:pretreated with LY294002(Abcam,USA)(10 μM)for 60 min before being treated with resveratrol(10μM)plus high glucose were incubated for 24 hours.We used the MTT(Invitrogen Grand Island,NY)assay to test the cell viability.TUNEL staining using kit(KeyGEN BioTECH,China)was used to detect the apoptosis cells in the histologic sections following the provided protocol.Western blotting(WB)was applied to value the quantity of protein:Bcl-2、Bad、Akt、p-Akt、Fox03a、p-Fox03a and so on.Establishment of type 1 diabetes mellitus(T1DM)model.Divided All diabetic rats into 3 resveratrol-treated groups(DM,DM+R5 and DM+R50 group)and rats without diabetic applied as the control group(N group):Resveratrol(Sigma;0,5 or 50mg/kg/d)was suspended in 0.9%saline containing with 0.5%carboxymethylcellulose(Sigma,USA).This solution was administered to treat rats by oral gavages every day for 8 weeks.Body weight and blood glucose were recorded every two weeks.At the end of the experiment all rats were subjected to the echocardiography and the hearts were harvested.Then percentage of left ventricular(LV)fractional shortening(FS),LV ejection fraction(EF)were measured combined with maximal velocity through left ventricular outflow tract(Vmax),Cardiac output,LV pressure halftime,LV posterior wall dimensions at both diastole and systole(LVPWd and LVPWs,respectively),interventricular septal dimensions at both diastole and systole(IVSd and IVSs,respectively),LV internal dimensions at both diastole and systole(LVIDd and LVIDs)was done at the same time,respectively.Heart weight to body weight ratio(HW/BW)and left ventricular mass to body weight ratio(LWm/BW)were measured.The observation of pathological changes was done through H&E and Masson staining.TUNEL staining using kit(KeyGEN BioTECH,China)was used to detect the apoptosis cells in the histologic section following the provided protocol.HG decreased the cell viability lowly to almost 50%(P<0.01)compared with control group while RSV(1μM,P<0.05;10μM and 100μM,P<0.01，P<0.01,respectively)increase cell viability in a dose manner.LY294002,the inhibitor of PI3K signal,blocked the protection of RSV.Compared to high glucose group,resveratrol significantly prevented myocardial cell apoptosis exposed to high glucose(P<0.01).LY294002 partly blocked the effect of RSV compared with the HG+RSV group(P<0.01).As consistent with apoptosis assay,apoptotic-related proteins Bcl-2/Bax ratio in HG group was lower compared to the control group(,P<0.01).RSV increased the Bcl-2/Bax ratio in cardiomyocytes subjected to HG(P<0.05).LY294002 abolished the protection of RSV evidenced with increasing the lever of apoptosis and reducing the Bcl-2/Bax ratio(P<0.05).HG decreased the expression of p-Akt(P<0.01)and p-FoxO3a(P<0.01)compared with Control group.RSV enhanced higher phosphorylation of Akt(/P<0.01)and Fox03a(P<0.01)in cells incubating in HG media for 24h compared to the HG group.Pretreatment with LY294002 reversed the protective effect of RSV on Akt(P<0.01)and Fox03a(P<0.01)phosphorylation indicating PI3K involved in the mechanism of RSV protection.Moreover,compared to HG group,enhancing cytoplasmic localization of Fox03a phosphorylation caused by RSV was revealed in HGH+RSV group(P<0.01)then blocking its pro-apoptotic effects.HG enhanced translocation of Fox03a to the nucleus and triggers apoptosis.RSV attended nucleus locations of Fox03a(P<0.01)and exerted its protection to against apoptosis.LY294002 abolished the protective effect of resveratrol basing on it down-regulation in cytoplasmic expression of Fox03a(P<0.01)and up-regulation of Fox03a expression in nucleus(P<0.01).Compared with the DM group,low dose RSV treated diabetic rats had markedly gained higher body weight in 4 weeks(P<0.01)post-treated,lower blood glucose(in 6 weeks)(P<0.01)while the high dose RSV treated showed the best outcome to regulate symptom of diabetes early in 2 weeks(P<0.01)and 4 weeks(P<0.01),respectively.At the end time point two RSV-treated group showed a significant difference between groups both in body weight(P<0.05)and blood glucose(P<0.01).H&E staining analysis revealed that the expanding myocyte size were detected in DM group and this phenomenon was ameliorated by treatment of RSV:DM+R5(P<0.01),DM+R50(P<0.01).Fibrosis in interstitial analyzed revealed by Masson’s staining and LVPWd,LVPWs,ⅣSd,ⅣSs,HW/BW and LVm/BW ratio show the similar outcome.RSV abrogated the increase of TUNEL-positive cells in RSV treated groups(DM+R5(P<0.05),DM+R50(p<0.01))compared to DM group in a dose dependent manner,but even treated in high dose RSV couldn’t reduce the apoptosis cell number into normal.Compared with the DM group both RSV-treated group showed great effect on up regulating the p-Akt(DM+R5(P<0.05),DM+R50(P<0.01))and p-Fox03a(DM+R5(P<0.01),DM+R50(P<0.01)).RSV boomed Fox03a expression in cytoplasm in group DM+R5(P<0.01)and group DM+R50(P<0.01)while decreased the expression in nucleus(DM+R5(P<0.01),DM+R50(P<0.01))compared with DM group.Impaired LV function evidenced by reduced left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)was observed in the DM group compared with the control group(P<0.05).LVEF and LVES were restored in the RSV treated groups:DM+R5(P<0.05),DM+R50(P<0.05)in a dose dependent manner(P<0.05)compared with the model group.LVIDd and LVIDs were also increased but Vmax was decreased in diabetic rats compared with control group and these damages were reduced by resveratrol treatment.Conclusions:1.RSV inhibits HG-induced apoptosis of neonatal rat cardiomyocytes.2.RSV alleviates the symptoms of diabetes,increasing body weight and decreasing blood glucose,cardiac hypertrophy and fibrosis,cardiomyocyte apoptosis as well as LV systolic dysfunction in diabetic heart.3.RSV servers cardiac protections by inhibiting apoptosis via the PI3K/Akt/Fox03a pathway in vitro and in vivo. |
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