Study Of The Protective Effect Of Adropin On High Glucose-Induced H9c2 Cardiomyocytes And Its Mechanism

ObjectivesTo investigate the protective effect of exogenous recombinant Adropin protein on myocardial injury through a diabetic cardiomyopathy(DCM)model of high glucose-induced H9c2 cardiomyocyte injury.Methods1.Rat H9c2 cardiomyocytes were routinely cultured in vitro with 25 m M glucose DMEM medium.Then they were cultured with glucose concentrations of 25 m M,55 m M,75 m M,100 m M and 150 m M high glucose medium for 24 h,48h and 72 h respectively.CCK8 assay was used to detect the changes in cardiomyocyte viability at different time points of different glucose concentrations.2.Drug concentration screening: conventionally cultured cardiomyocytes were pretreated with different drug concentrations for 24 h,the drug concentrations were: adropin 5ng/ml,10ng/ml,25ng/ml,50ng/ml,100ng/ml,200ng/ml,followed by the addition of 75 m M glucose for co-culture for 48 h to observe the effect of different adropin drug concentrations on the viability of high glucose-induced H9c2 cardiomyocytes.3.According to the results of drug concentration screening,the experimental groups were divided into: control group,high glucose group,adropin-L,M and H groups(10,50,100ng/ml),and the levels of reactive oxygen species were detected by DCFH-DA probe to investigate the effect of adropin on oxidative stress in high glucose-induced H9c2 myocardial injury.4.Annexin V-FITC/PI apoptosis kit,mitochondrial membrane potential assay kit(JC-1)and Western blot were used to explore the effect of adropin on apoptosis in high glucose-induced cardiomyocyte injury.5.adenosine triphosphate(ATP)assay kit,Mito-Tracker Red CMXRos probe;Western blot assay was used to explore the effect of adropin on mitochondrial dynamics-mediated apoptotic pathway.Results1,high glucose can reduce the cell survival rate,compared with the control group(25m M),the higher the glucose concentration,the longer the culture time,the lower the cell activity;glucose concentration of 75 m M,culture 48 h cell activity rate decreased to about 66%,cell activity significantly decreased,and maintain a certain activity,which can be used as a follow-up model group glucose concentration.2,Different concentrations of adropin increased the cellular activity of H9c2 cells in high glucose environment;compared with the high glucose group,different concentrations of adropin gradually increased the activity of cardiomyocytes induced by high glucose(p<0.05),suggesting that adropin has a protective effect on cardiomyocyte injury induced by high glucose,and has a concentration-dependent effect in a certain range.3.adropin reduced the level of reactive oxygen species(ROS)in H9c2 cells under high glucose environment.Compared with the normal group,the green fluorescence of ROS was the strongest in the high glucose group,and the intracellular ROS increased significantly(p<0.01);compared with the high glucose group,the green fluorescence of adropin in the high glucose-induced cardiomyocytes gradually decreased at different concentrations,and the intracellular ROS level gradually decreased(p < 0.01),and there was a negative correlation with the concentration in a certain range,suggesting that adropin has an antioxidant effect on the high glucose-induced This suggests that adropin has an antioxidant effect on high glucose-induced cardiomyocyte injury.4.adropin reduced apoptosis in H9c2 cardiomyocytes under high glucose environment.Compared with the normal group,the mitochondrial membrane potential of the high glucose group was significantly reduced(p<0.01),the apoptosis rate of cardiomyocytes increased by flow cytometry(p<0.01),the expression of apoptotic proteins Cytc,Bax and Cleaved-caspase 3 increased,and the expression of anti-apoptotic protein Bcl-2 decreased;compared with the high glucose group,different concentrations of adropin reduced the apoptosis rate of cardiomyocytes and increased the apoptosis rate of cardiomyocytes.At the same time,adropin up-regulated the expression of apoptotic proteins Cytc and Bax and Cleaved-caspase 3,and down-regulated the expression of anti-apoptotic protein Bcl-2,suggesting that adropin may play a protective role in myocardium through the mitochondrial apoptosis pathway.5.adropin attenuated apoptosis by regulating mitochondrial dynamics in H9C2 cardiomyocytes.Compared with the control group,the mitochondrial fluorescence probe showed a significant decrease in red fluorescence,a significant decrease in the number of active mitochondria and a disruption of the mitochondrial network structure in the high glucose group,and a significant decrease in intracellular ATP production as measured by the ATP kit(p<0.01).Compared with the high glucose group,the number of active mitochondria in cardiomyocytes gradually increased and the network structure gradually normalized in the adropin intervention group,and intracellular ATP production increased(p < 0.01),and the expression of mitochondrial division protein Drp1 was down-regulated,while the expression of fusion proteins Mfn2 and Opa1 was up-regulated.This suggests that adropin may play an anti-apoptotic role by improving mitochondrial kinetic homeostasis.ConclusionExogenous recombinant adropin may inhibit mitochondrial oxidative stress in H9c2 cardiomyocytes under high glucose environment by regulating the expression levels of mitochondrial fusion/division proteins,thereby inhibiting cardiomyocyte apoptosis and protecting cardiomyocytes.