The Protective Effects And Mechanisms Of Glucogon Like Peptide-1 On AGEs-induced H9C2 Cardiomyocytes Injury

Part ?:The protective effects and mechanisms of GLP-1 on AGEs-induced apoptosis in H9C2 cardiomyocytesExperiment ?:The model establishment of AGEs-induced H9C2 cardiomyocytes injury ObjectiveTo observe whether Advanced Glycation end products(AGEs)results in H9C2 cardiomyocytes injury for studying the related molecular mechanisms.MethodsThe model of AGEs-induced cardiomyocytes injury was established by receiving AGEs-BSA(50?100?200mg·L-1)for 24 h in H9C2 cardiomyocytes.The activity of lactate dehydrogenase(LDH)was assayed through spectrophotometric procedures,cell viability was quantified by CCK-8 assay,and apoptosis was detected by Hoechst 33258 assay.Results1)AGEs resulted in H9C2 cardiomyocytes injury in a dose-dependent manner.2)There was not significant different between 100mg·L-1 group and 200mg·L-1group in apoptosis(P>0.05).Conclusion1)AGEs induces apoptosis and injury in a dose-dependent manner in H9C2 cardiomyocytes.2)100mg·L-1AGEs-BSA is chosen in the following experiment.Experiment ?:The protective effects and mechanisms of GLP-1 on AGEs-induced by inhibiting apoptosis in H9C2 cardiomyocytes injury ObjectiveTo investigate the effects and mechanisms of Glucogon like peptide-1(GLP-1)on AGEs-induced apoptosis in H9C2 cardiomyocytes.MethodsH9C2 cardiomyocytes were randomly divided into the following five groups: Control group(1mg·L-1BSA),AGEs group,AGEs+5nmol·L-1GLP-1group,AGEs+10nmol·L-1 GLP-1 group,AGEs+20nmol·L-1 GLP-1 group.Cell viability rate was measured by CCK-8 assay,activities of lactate dehydrogenase(LDH)was assayed through spectrophotometric procedures;Flow cytometry and Honchest33258 assay were performed to detect apoptosis;the m RNA of Bax,Bcl-2 were measured by RT-PCR,western blotting was applied to assess the protein of Bax and Bcl-2.Results1)There were lower cell viability,higher LDH and more apoptotic rate in AGEs group than Control group(P<0.05).2)Compared with AGEs group,GLP-1(10nmol·L-1?20nmol·L-1)ameliorated significantly the adverse effects caused by AGEs(100mg·L-1)in cell viability,apoptosis and LDH(P<0.05).3)Compared with Control group,AGEs(100mg·L-1)resulted in significantly a higher expression of Bax,and a lower expression of Bcl-2 in m RNA and protein(P<0.05).4)GLP-1(10nmol·L-1)could reverse significantly the adverse effects caused by AGEs(100mg·L-1)in expression of Bas/Bcl-2(P<0.05).Conclusion1)GLP-1 has a protective effect on AGEs-induced injury by inhibiting apoptosis in H9C2 cardiomyocytes.2)GLP-1 inhibits AGEs-induced apoptosis through regulating Bax/Bcl-2 in H9C2 cardiomyocytes.Part ?:The protective effects and mechanisms of GLP-1 on AGEs-induced autophagy in H9C2 cardiomyocytesExperiment ?:The effects of AGEs on autophagy in H9C2 cardiomyocytes ObjectiveTo investigate effects of different doses of AGEs on autophagy in H9C2 cardiomyocytes.MethodsH9C2 cardiomyocytes were randomly divided into the following groups: Control group(1mg·L-1%BSA),AGEs-BSA(50?100?200mg·L-1)group.After incubating 24 h,Western blotting was applied to assess the autophagy associated protein(Beclin1 and LC3B).Results1)In AGEs groups,there were higher and higher expressions of LC3B?Beclin1 in a dose-dependent manner.2)Compared with Control group,AGEs groups of 100mg·L-1?200mg·L-1had a significantly different expression of LC3?Beclin1(P<0.05)respectively.3)There was not a significantly different between 100mg·L-1AGEs group and 100mg·L-1AGEs group in expression of LC3B?Beclin1(P>0.05).Conclusion1)AGEs activates autophagy in H9C2 cardiomyocytes in a dose-dependent manner.2)100mg·L-1AGEs is chosen in following experiment.Experiment ?:The protective effect of GLP-1 on H9C2 cardimyocytes against AGEs-induced injury by inhibiting excessive autophagy ObjectiveTo investigate the effect of GLP-1 on H9C2 cardiomyocytes against AGEs-induced injury and potential protective mechanisms of autophagy.MethodsCultured H9C2 cardiomyocytes were randomly divided into the following four groups: Control group(1mg·L-1BSA),AGEs group,AGEs+10nmol·L-1GLP-1 group,AGEs+10nmol·L-1GLP-1+5umol·L-1rapamycin group.After 24 h,Intracellular ROS production was measured by DCFH-DA fluorescent probe;Cell viability was quantified by CCK-8 assay;The formations of autolysosomes were detected by flow cytometry using the PH-sensitive fluorescent dye acridine orange(OA);The m RNA of LC3,Beclin-1were measured by RT-PCR;Western blotting was applied to assess the protein expression of LC3 B,Beclin-1;The autophagy flux was detected by Ad-m Cherry-GFP-LC3 B.Results1)AGEs impaired cell viability,increased Intracellular ROS production,enhanced formation of autolysosomes and up-regulated the m RNA and protein expressions of LC3 B and Beclin-1(P<0.05).2)GLP-1 reversed remarkably the adverse effects caused by AGEs(100mg·L-1)in cell viability,ROS production and autophagy.3)Effects of GLP-1 on AGEs were inhibited by rapamycin(autophagy agonist)in cell viability,amount of autolysosomes,and expressions of autophagy-associated genes but not in Intracellular ROS production.Conclusion1)AGEs results in a higher level of ROS which may be related to activation of autophagy.2)GLP-1 may protect H9C2 cardiomyocytes against AGEs-induced injury partly by inhibiting excessive autophagy.