β-hydroxybutyrate Attenuates High-glucose-induced Cardiomyocytes Injury Through Enhancing Autophagic Flux

BackgroundWith development of modern society and change of life style,the morbidity and mortality of cardiovascular disease and diabetes in China are rising rapidly.Diabetic cardiomyopathy,being the most important cardiovascular complications of diabetes,refers to the cardiomyopathy that occurs in diabetic patients without hypertension,coronary atherosclerotic cardiovascular disease or others.Diabetic cardiomyopathy is based on insulin resistance and microvascular fibrosis,which eventually develops into heart failure.Therefore,it is significant to find out appropriate treatment targets and effective drugs.β-Hydroxybutyrate(BHB)is a kind of fatty acid derived from human liver.According to related studies,exogenous supplementation of β-hydroxybutyrate can exert anti-inflammation,anti-oxidation,anti-aging and other protection.In addition,it was reported that β-hydroxybutyrate can alleviate diabetic retinopathy,atherosclerosis and Alzheimer’s disease.However,the effect of β-hydroxybutyrate on diabetic cardiomyopathy is still not clear.Autophagy is a process that transfer cellular macromolecular substances to lysosomes for degradation,in order to satisfy the metabolic need of cells,renew organelles and maintain the stability of cellular environment.The formation of autophagosome,the transport to lysosome and the process of degradation in lysosome are called autophagy flux.Studies have shown that autophagy flux is impaired in diabetic cardiomyopathy,resulting in mitochondrial dysfunction and lipid accumulation.Enhancing autophagy and promoting autophagic flux can alleviate diabetic cardiomyopathy and slow down its rate of development.Hence,we hypothesize that β-hydroxybutyrate attenuates high-glucose-induced cardiomyocytes injury through enhancing autophagic flux.Objectives1.To confirm the protective effects of β-hydroxybutyrate on diabetic cardiomyopathy at the cellular level by establishing the model of high glucose cultivation in cardiomyocytes of SD neonatal rats.2.To explore the mechanisms of β-hydroxybutyrate against high-glucose-induced cardiomyocytes injury and the relationship with autophagic flux regulation.MethodsAfter separating and extracting cardiomyocytes of SD neonatal rats by trypsin and collagenase digestion,the cardiomyocytes were identified.These cells were cultivated by high glucose,so as to simulate the internal environment of diabetic cardiomyopathy.Firstly,cardiomyocytes were divided into control group,high glucose group and high glucose+β-hydroxybutyrate group with different concentration to detect the cell viability by MTS experiment and find out the best drug concentration.Subsequently,cells were divided into control group,high glucose group,high glucose + β-hydroxybutyrate group,to assess the protection ofβ-hydroxybutyrate by the lipid peroxidation assay kit,reactive oxygen species assay kit and mitochondrial membrane potential assay kit.And then the level of autophagy-related molecules was evaluated by Western blotting.At last,adding high glucose+β-hydroxybutyrate+chloroquine group to repeat experiment above.Results1.Cardiomyocytes are successfully extracted and cultured.2.β-Hydroxybutyrate attenuates high-glucose-induced cardiomyocytes injury.3.β-Hydroxybutyrate enhances the autophagic flux of high glucose-cultured cardiomyocytes.4.The effect of β-hydroxybutyrate on attenuating high-glucose-induced cardiomyocytes injury is antagonized by the autophagy inhibitor chloroquine.5.The effect of β-hydroxybutyrate on enhancing autophagic flux is antagonized by chloroquine.Conclusionsβ-Hydroxybutyrate attenuates high-glucose-induced cardiomyocytes injury through enhancing autophagic flux and reducing oxidative stress and apoptosis.