| Objective:To investigate the effects of glucagon-like peptide 1(GLP-1)analogue liraglutide(LRG)on cell viability,oxidative stress-related indicators and autophagy in a model of high glucose-induced cardiomyocyte(H9c2)injury and to explore the protective mechanism of LRG-regu Lated autophagy against oxidative stress injury in H9c2 cardiomyocytes based on the SIRT1/FOXO1 signalling pathway.Method:1.H9c2 cardiomyocytes were cu Ltured in vitro and given different concentrations of high sugar solution(33,50,100 and 200 mmol/L)for 24 h incubation,and low sugar medium solution was used as the normal control group(Control,glucose 5.5 mmol/L).The CCK8 method was used to detect the cell viability of each group and to screen the high sugar concentration that induced the H9c2 cardiomyocyte injury model 33 mmol/L as the next research experiment.2.H9c2 cardiomyocytes were random Ly divided into the following five groups based on the experimental resu Lts of method 1: normal control group(Control,glucose 5.5 mmol/L),high glucose group(HG,glucose 33 mmol/L),high glucose + liraglutide at different concentrations(HG + LRG,LRG concentrations of 10,100 and 1000 nmol/L respectively)and incubated H9c2 cells were incubated for 24 h.Cell viability was measured by CCK-8 and changes in cell morphology and structure were observed under inverted microscope before and after drug administration.The LRG concentration of 100nmol/L was selected as a follow-up study.3.H9c2 cardiomyocytes were random Ly divided into three groups,namely normal control group(Control),high glucose group(HG)and high glucose +liraglutide group(HG+LRG),and the oxidative stress-related kits were used to determine the levels of lactate dehydrogenase(LDH),malondialdehyde(MDA)and superoxide dismutase(SOD)in the cell supernatant.The formation of intracellu Lar autophagosomes was observed microscopically using monodansu Lfonylglutamine fluorescence staining(MDC).Western blot and RT-PCR were used to detect the expression of autophagy-related factors P62,LC3 protein and mRNA in each group of cardiomyocytes.4.After knocking down the expression of SIRT1 by small interfering RNA(siRNA)technology,the cells were random Ly divided into the following four groups,i.e.normal control group(Control),high glucose group(HG),high glucose + liraglutide + cell transfection negative control si-NC group(HG +LRG + si-NC),high glucose + liraglutide + cell transfection si-SIRT1 group(HG + LRG + si-SIRT1).LRG+si-SIRT1),and the cell viability,LDH,MDA and SOD contents of the supernatant were determined by CCK-8 assay and oxidative stress-related kits.The formation of intracellu Lar autophagosomes was observed microscopically using MDC fluorescence staining.The expression of autophagy-related factors P62,LC3 mRNA and protein in each group of cardiomyocytes was detected by RT-PCR and Western blot.The protein expression of SIRT1,Ac-FOXO1 and FOXO1 in cardiac myocytes of each group was detected by Western blot.5.3-MA autophagy inhibitor was given to inhibit intracellu Lar autophagy,and H9c2 cells were random Ly divided into the following four groups: normal control group(Control),high glucose group(HG),high glucose + liraglutide group(HG+LRG),and high glucose + liraglutide + 3-MA autophagy inhibitor group(HG+LRG+3-MA),and cell viability was measured by CCK-8 assay.Oxidative stress-related kits were used to determine the LDH,MDA and SOD contents of the supernatant.ResuLts:1.Construction of a high sugar-induced H9c2 cardiomyocyte injury modelCompared with Control,the viability of H9c2 cells decreased under high glucose incubation at 33,50,100 and 200 mmol/L.The differences were statistically significant(P<0.05),and an in vitro model of high glucose injury to H9c2 cardiomyocytes was successfu Lly established.Furthermore,the survival of H9c2 cardiomyocytes was significantly inhibited at a high glucose concentration of 33 mmol/L,and 33 mmol/L glucose incubation was used as a modeling condition for subsequent experiments.2.Effects of LRG on cell viability,oxidative stress and autophagy in high glucose environment2.1 Compared with the HG group,cell viability was significantly enhanced in the LRG100 nmol/L and LRG1000 nmol/L groups,and the difference was statistically significant(P<0.05).At a concentration of 100nmol/L,cell viability entered a plateau phase,and there was no significant change in cell viability between the 100 nmol/L and 1000 nmol/L LRG groups(P > 0.05),so 100 nmol/L LRG treatment for 24 h was chosen for the subsequent experiment.2.2 Compared with the Control group,the LDH and MDA contents in the cell supernatant of the HG group increased significantly,and the SOD content decreased significantly,and the difference was statistically significant(all P<0.05).2.3 Compared with the Control group,the yellow-green fluorescence intensity of MDC was reduced in the HG group,but enhanced in the HG + LRG group after LRG intervention.Meanwhile,compared with the Control group,the expression of autophagy-related factor P62 mRNA and protein in cardiomyocytes was increased in the HG group(P<0.05),the expression of LC3 mRNA and the protein ratio of LC3II/I was decreased in the HG + LRG group(P<0.05),and the expression of autophagy-related factor P62 mRNA and protein in cardiomyocytes was decreased in the HG + LRG group compared with the HG group(P<0.05),and the expression of LC3 mRNA and the protein ratio of LC3II/I were increased in the HG + LRG group compared with the HG group(P<0.05).3 Explicitly LRG promotes autophagy through regu Lation of SIRT1/FOXO1 pathway to ameliorate oxidative stress injury in H9c2 cardiomyocytes under high glucose environment3.1 After knocking down the expression of SIRT1 by siSIRT1 transfection technique,cell viability decreased(P<0.05),LDH and MDA contents in cell supernatant increased significantly(P < 0.05)and SOD content decreased significantly(P < 0.05)in HG+LRG+si-SIRT1 group compared with HG+LRG+si-NC group.3.2 Compared with the HG+LRG+si-NC group,the HG+LRG+si-SIRT1 group showed reduced yellow-green fluorescence intensity,increased expression of autophagy-related factor P62 mRNA and protein(P<0.05),and decreased expression of LC3 mRNA and protein ratio of LC3II/I(P<0.05).3.3 Compared with the Control group,SIRT1 protein expression was significantly lower in the HG group(P< 0.05),while Ac-FOXO1/FOXO1 protein ratio was increased and cellu Lar FOXO1 protein acetylation level was increased(P<0.05);compared with the HG group,SIRT1 protein expression was increased in the HG+LRG+si-NC group(P < 0.05)and Ac FOXO1/FOXO1 protein ratio decreased and cellu Lar FOXO1 protein acetylation level decreased(P<0.05);compared with HG+LRG+si-NC group,SIRT1 protein expression was significantly decreased in HG+LRG+si-SIRT1group(P < 0.05),while Ac-FOXO1/FOXO1 protein ratio increased and cellu Lar FOXO1 protein acetylation level increased(P < 0.05).acetylation levels were increased(P<0.05).3.4 After administration of autophagy inhibitor intervention,cell viability decreased,cell supernatant LDH and MDA levels increased significantly(P<0.05)and SOD levels decreased significantly(P<0.05)in the HG+LRG+3-MA group compared to the HG+LRG group.Conclusions:1.High glucose induces decreased myocardial viability,oxidative stress injury and abnormal expression of autophagy-related factors in H9C2 cardiomyocytes.2.Liraglutide enhanced H9C2 cell viability,reduced oxidative stress injury and restored autophagy in cardiomyocytes induced by high glucose.3.Liraglutide may promote autophagy through regu Lating the SIRT1/FOXO1 signaling pathway to reduce high-glucose-induced oxidative stress injury in H9c2 cardiomyocytes. |
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