The Protective Role And Mechanisms Of Glucagon-like Peptide-1(GLP-1) On Advanced Oxidation Protein Products (AOPPs)-induced Apoptosis Of Cardiomyocytes

BackgroundThe incidence and prevalence of diabetes mellitus(DM) are increasing globally, making diabetes the third chronic non communicable diseases harmful to human health after cardiovascular disease and carcinoma.The various kinds of acute and chronic complications caused by diabetes have severe impacts on people’s quality of life and longevity. Diabetes has become a critical public health problem threatening people’s life. Diabetic cardiomyopathy(DCM) is a special kind of chronic complications which defined as ventricular dysfunction and heart failure and becoming the leading cause of death in patients with diabetic cardiovascular complications. However,the pathogenesis of DCM remains uncertain,leading to the lack of effective means of prevention. Growing evidences have demonstrated that apoptosis of cardiomyocytes play a vital role in the development of DCM.Advanced oxidative protein products(AOPPs) are the dityrosine-containing and cross-linking protein products which formed during oxidative stress by the reaction between proteins and chlorinated oxidants. AOPPs were first discovered in the plasma of patients with uremia and undergoing dialysis and considered as a novel marker of oxidative stress.AOPPs are closely related to diabetes,coronary heart disease and obesity through increasing the generation of intracellular reactive oxygen species(ROS) and inducing the formation of proinfiammatory factor. Increased levels of plasma AOPPs were demonstrated in patients with diabetes mellitus, and were significantly higher in diabetic group with vascular complications compared to that without complications. In vitro,incubation of AOPPs with human umbilical vein endothelial cells induced superoxide generation, activation of NAD(P)H oxidase, ERK 1/2 and p38, and nuclear translocation of NF-κB by bonding to the receptor for advanced glycation end products(RAGEs). In experimental models, accumulation of AOPPs activated the p53/Bax/caspase-dependent proapoptotic pathway via RAGE and resulted in podocyte apoptosis.Thus,we speculate that AOPPs participate in the genesis and development of DCM by causing the apoptosis of cardiomyocytes.Glucagon-like peptide-1(GLP-1) is a multifunctional hormone secreted from intestinal endocrine L cells and is widely used in treating diabetes.Beside its effects on glucose control,GLP-1 reduces gastric emptying,inhibits appetite and exhibits a protect effect on cardiovascular diseases. Studies have demonstrated that the GLP-1R agonist liraglutide inhibits endothelial cell dysfunction and alleviates atherosclerosis injury.In rat with chronic heart failure, subcutaneous infusion with GLP-1 and its analogue exenatide will improve cardiac function,cardiac remodeling and survival.Furthermore,GLP-1 directly protect cardiomyocytes from hypoxia-reoxygenation injury mainly by inhibiting their apoptosis and protects vascular endothelial cells against AGEs-induced apoptosis. It has been reported that GLP-1 and its analogues inhibit cardiomyocyte apoptosis by regulating many apoptotic-related pathways,including PI3K pathway,cAMP patheway and ERK1/2 pathway.Therefore,we suppose that GLP-1 can inhibit AOPPs-induced apoptosis probably through the PI3K/Akt pathway.ObjectivesWe used the ventricular myocytes of rats,H9C2 cell line, to study if AOPPs could induce apoptosis of H9C2 cells and if GLP-1 has protect effcts on this. Further more,we verified if GLP-1 could downregulate the RAGE expression to inhibit oxidative stress and could inhibit AOPPs-induced apoptosis through PI3K/Akt pathway.We wish our findings would provide a conceivable mechanism of the development of DCM and render a new application of GLP-1 exerting favourable cardiac effects for treating diabetic cardiomyopathy.ContentThis study includes two parts:Chapter I The effects of AOPPs on the apoptosis of H9C2 cellsObjectivesWe used the H9C2 cells as the reaserch object and observed the effects of AOPPs on the survival of H9C2 cells. Methodsl.Preperation and identification of AOPPsAOPPs were prepared by dissolved RAS in phosphate-buffered saline with HOC1 for incubating for 30min in the dark.AOPPs were identified using a spectrophotometer at 340nm under acidic condition and obtained using chloramine T as the standard.2. Cell cultureThe H9c2 cells were cultured in Dulbecco’s modified eagle’s medium(high glucose) with 10%FBS plus 1% penicillin-streptomycin solution in a 5% carbon dioxide atmosphere at 37℃.Cells were subcultured every 2 to 3 days at a ratio of 1:2 or 1:3.3.Detection of cell proliferation by cck-8 assayH9c2 cells were planted in 96-well plates and treated with indicated concentrations of AOPPs for 24h or lug/ml AOPPs for indicated time.Then replaced the medium with 10% cck-8 solution,and measured the absorption at 450nm with a microplate reader according to the manufacturer’s instructions.4.Observaion of cell apoptosis by Hoechst33258 stainingAfter treatment,H9c2 cells were fixed over night at 4℃,and then permeabilized and stained with Hoechst 33258 for 5 minutes at room temperature.The morphological changes were observed under a fluorescence microscope at 350nm.5.Determination of cell apoptosis by Annein V-FITC/PI stainingEach group of cells were detected by double staining with FITC-conjugated Annexin V and propidium iodide(PI).Collected cells were washed twice with PBS and stained with 5 ul Annexin V-FITC for 15 min in dark in binding buffer.Subsequently,10 ul PI were added at room temperature.Cell apoptosis assay were performed on a FACSVerse flow cytometer using the Cell Quest program.6.Detection the protein expression of Bcl-2,Bax,Caspase-3 and active-caspase-3 by western blottingAfter treatment,cellular proteins were lysed and collected to obtain the protein-containing supernatant.The protein concentration was measured with the BCA protein assay.Proteins were separated on 12% SDS-polyacrylamide gel and subsequently transferred to a polyvinylidene fluoride membranes.The membranes were blocked for 2 hours and immunoblotted with primary antibodies for 2 hours at room temperature. Then,the membranes were incubateed with horseradish peroxidase-conjugated secondary IgG for 1 hour at room temperature.The protein bands were visualized using an ECL kit.7. Statistical analysisAll analyses were carried out with SPSS 13.0 software.Measurement data were expressed as mean ± standard deviation(SD).Differances between groups were tested by one-way ANOVA or Welch ANOVA(equal variances not assumed) once normality was verified.For pairwise comparison,we used LSD test or Dunnett’s T3 test when the equal variances were not assumed.Spearman’s rank correlation was used for correlation analysis.A two-sided P value of<0.05 was considered statistical significant.Results1.The effects of AOPPs on cell proliferation of H9C2 cells1.1 After treatment with 200,400,800,1 OOOng/ml AOPPs for 24h,the OD value of H9C2 cells were 1.121±0.032,1.099±0.07,1.041±0.131,0.928±0.066 respectively, and were significantly lower than control group (1.340±0.120) and RSA group (1.344±0.013) (P<0.01). A significant negative correlation exists between the OD value of H9C2 cells and the AOPPs concentration (r=-0.868, P=0.000).1.2 After treatment with 1μg/ml AOPPs for 24h,48h and 72h,the OD value of H9C2 cells were 0.913±0.037,0.724±0.048,0.645±0.047 respectively,and were significantly lower than control group(1.263±0.017)(P=0.000). A significant negative correlation exists between the OD value of H9C2 cells and time (r=-0.946, P=0.000).2 The effects of AOPPs on apoptosis of H9C2 cells2.1 The morphological changes in H9c2 cells were detected by a fluorescence microscope. The chromatin condensation and nuclear fragmentation represented apoptotic cells while the diffusion blue fluorescent cells represented normal one. We observed an increase of apoptotic cells in AOPPs-treated cells.2.2 The apoptotic rate of AOPPs-treated group was 15.773±3.130%,and was significantly higher than control group(P=0.000).3 The effects of AOPPs on apoptosis-related protein expression of H9C2 cells3.1 The relative expression of pro-apoptotic protein Bax of AOPPs-treated group was 1.030±0.021,and showed significant increase than control grouop(P<0.01).3.2 The relative expression of anti-apoptotic protein Bcl-2 of AOPPs-treated group was 0.955±0.001,and showed significant decrease than control grouop(P<0.01).3.3 The relative expression of active-caspase-3 of AOPPs-treated group was 0.083±0.001,and showed significant increase than control grouop(P=0.000). ConclusionOur data shows that AOPPs induce a decrease of proliferation of H9C2 cells in a dose and time dependent way,and increase the expression of pro-apoptotic protein and decrease the expression of anti-apoptotic protein,leading to H9C2 cells apoptosis.Chapter Ⅱ The mechanisms of GLP-1 on AOPPs-induced cell apoptosis ObjectivesWe observed AOPPs induced H9C2 cell apoptosis.In this part,we will further discuss the effects of GLP-1 on AOPPs-induced cell apoptosis and the mechanisms.This will present theoretical foundation and new insight on the pathogenesis and therapeutic strategy of diabetic cardiomyopathy. Methods1.Detection of cell proliferation by cck-8 assayExperimental grouping:blank control, AOPPs-treated group,GLP-1-treated group, AOPPs and GLP-1 co-treated group. Use the same method with that of Chapter I.2.Observaion of cell apoptosis by Hoechst33258 stainingExperimental grouping: blank control, RSA control, AOPPs-treated group, GLP-1-treated group, AOPPs and GLP-1-treated group, AOPPs+GLP-1+ LY294002-treated group. Use the same method with that of Chapter Ⅰ.3.Determination of cell apoptosis by Annein V-FITC/PI stainingUse the same group with above.Use the same method with that of Chapter I.4. Immunofluorescence assayAfter treatment,cells were fixed in 4% paraformaldehyde for 15min and then permeabilized with Triton for 15min.After washed twice with PBS,cells were blocked in 1% bovine serum albumin(BSA) for 30min at room temperature.They were then incubated overnight at 4℃ with primary antibody.Following 2×washes with PBS,cells were incubated with FITC-labeled anti-mouse or anti-rabbit IgG secondary antibodies for 1h at room temperature.Finally,cells were stained with DAPI for 2min.The immunofluorescent images were captured using a fluorescence microscope.5. Real-Time RT-PCR AnalysisTotal RNA was extracted using RNAiso Plus according to the manufacturer’s instructions.The purity of each RNA sample was measured by Nanodrop 2000 Spectrophotometer.To remove genomic DNA contamination,RNA samples were treated with gDNA Eraser at 42℃ for 2 minutes.We synthesized the complementary DNA in a 20-ul reaction system using the PrimeScript RT reagent Kit. Real-time PCR was performed using ABI 7300 system(ABI,CA) with a SYBR Premix EX Taq II Kit.6. Determination of intracellular reactive oxygen species(ROS) generationH9c2 cells were treated with AOPPs and/or GLP-1 for 2 hours and incubated with the probe DCFH-DA (2’,7’Dichlorodihydrofluorescin diacetate) which can be oxidized to the fluorescent DCF by intracellular ROS for 30 min at 37℃.The ROS-positive cells were visualized by fluorescence microscope and quantified by flow cytometer.7. Detection the protein expression of RAGE,GLP-1R,p-Akt, Akt,p-Bad, Bcl-2,Bax and active-caspase-3 by western blottingTo detect the expression of RAGE and GLP-1 R,cells were divided into 5 groups:blank control, RSA control,AOPPs-treated group,GLP-1-treated group, AOPPs and GLP-1-treated group.To detect the expression of p-Akt,Akt,p-Bad,Bcl-2,Bax and active-caspase-3, cells were divided into 6 groups:blank control,RSA control,AOPPs-treated group,GLP-1-treated group, AOPPs and GLP-1-treated group, AOPPs+GLP-1+ LY294002-treated group.Use the same method with that of Chapter Ⅰ.8. Statistical analysisUse the same method with that of Chapter Ⅰ. Results1 The effects of GLP-1 on cell proliferation of H9C2 cellsAfter treatment with 1μg/ml AOPPs and 10nM,50nM,100nM GLP-1 for 24h,the OD value of H9C2 cells were 1.258±0.010,1.409±0.099,1.330±0.059 respectively, and were significantly higher than positive control group(0.929±0.083)(P=0.000).Among them,50nM GLP-1 group has the highest OD value. Therefore,the subsequent experiments were performed with lug/ml AOPPs and 50nmol/l of GLP-1.2 GLP-1 protects AOPPs-induced cell apoptosis via PI3K pathway2.1 We observed an increase of apoptotic cells in AOPPs-treated cells and a decrease in AOPPs-and GLP-1-treated cells.After co-incubated with LY294002,the apoptotic cells were increased.2.2 The apoptotic rate of AOPPs and GLP-1-treated group was 9.715±0.757%,and was significantly lower than AOPPs-treated group(P=0.000).The PI3K inhibitor, LY294002, was employed to determine the mechanisms behind the anti-apoptotic effects of GLP-1.Unsurprisingly,the apoptosis rate of this group was significantly increased(p=0.000 versus AOPPs+GLP-1 group).3 GLP-1 exerts its protect function via GLP-1 receptor in H9C2 cell.We confirmed that GLP-1 receptor was exsited on the membrane of H9C2 cells. GLP-1 significantily increased GLP-1 receptor expression in both mRNA and protein level(P=0.000 versus control).4 GLP-1 downregulates the RAGE expression of AOPPs-treated H9C2 cells.4.1 H9c2 cells were immunostained with anti-rat RAGE primary antibody and FITC-conjugated secondary antibody(green) and counterstained with DAPI(blue).RAGE expression was upregulated by exposure of increasing concentrations of AOPPs for 24 h.4.2 GLP-1 significantly decreased RAGE protein expression.The relative expression of RAGE of AOPPs-treated group was 1.155±0.021,and showed significant increase than control grouop(P=0.000). The relative expression of AOPPs and GLP-1-treated group was 0.872±0.052,and was significantly lower than AOPPs-treated group(P=0.000).4.3 GLP-1 significantly decreased RAGE mRNA expression. The relative expression of RAGE mRNA of AOPPs-treated group was 1.179±0.233,and showed significant increase than control grouop(P=0.000). The relative expression of AOPPs and GLP-1-treated group was 1.144±0.152,and was significantly lower than AOPPs-treated group(P=0.000).5 The ROS generation was elevated by exposure to AOPPs for 2h as visualized by a fluorescence microscope with DCFH-DA staining.The fluorescence intensity of AOPPs-treated cells was more obvious than the control cells.While the AOPPs and GLP-1-treated cells show weaker intensity. Meanwhile,the ROS positive rate significantly increased in AOPPs-treated cells(47.817±0.878%) compared with control group(P-0.000).However,when we treated H9c2 cells with AOPPs and GLP-1,the ROS generation was almost drop to normal(P=0.011).6 GLP-1 prevents AOPPs-induced apoptosis via PI3K/Akt/BAD pathway in H9C2 cells.We investigated the role of PI3K/Akt/Bad pathway in GLP-1 prevented AOPPs-induced apoptosis in H9C2 cells.First of all,AOPPs significantly inactivated Akt phosphorylation at Ser473 and then reduced Bad phosphorylation at Ser136 compared with control and RSA group(P<0.01). Moreover,we observed a decrease of anti-apoptotic protein Bcl-2 and an increase in pro-apoptotic protein Bax in AOPPs-treated cells. In addition, we detected the elevated expression of active-caspase-3 by western blotting.However,these changes were reversed in the presence of GLP-1.Furthermore, the effect of GLP-1 was abrogated when we treated H9C2 cells together with a PI3K inhibiter LY294002.ConclusionGLP-1 downregulates RAGE expression and weakens the AOPPs-induced ROS generation.The most important:GLP-1 could change the balance between pro- and anti-apoptotic proteins and protect H9C2 cells against AOPPs-induced apoptosis via the PI3K/Akt/Bad suivival pathway.