Role And Mechanism Of CREG On The Regulation Of Apoptosis Induced By High Glucose And High Palmitate In Rat Cardiomyocytes

Diabetes is a metabolic disease characterized by chronic hyperglycemia,mainly classified into type 1 and type 2,in which type 2 diabetes is particularly common.The epidemiological survey of diabetes by the Diabetes Society of the Chinese Medical Association showed that in 2017,there were over 100 million patients with type 2 diabetes in China.The prevalence rate of type 2 diabetes was 11.6% among people over 30 years-old.4.9 million people died from diabetes each year,of which about 50% died from cardiovascular complications.In diabetics,left ventricular dysfunction,independent of atherosclerosis,hypertension and other underlying pathological factors,is known as diabetic cardiomyopathy(DCM).Studies have reported that the pathogenesis of DCM in patients with type 2 diabetes mellitus is mainly the dysfunction of myocardial autophagy.Persistent high glucose and high fat environment are the main factors causing myocardial autophagy dysfunction in type 2 diabetes mellitus.In high fat and high glucose environment,myocardial cells develope insulin resistance,the balance of energy metabolism of cardiomyocytes is disturbed,and the oxidation of protein and lipid in the cells increase,which cause the autophagy dysfunction.Autophagy dysfunction makes damaged organelles and harmful metabolites impossible to clear,leading to cardiomyocytes death and DCM.Although the mechanism of diabetic cardiomyopathy has been identified in type 2 diabetic patients,however,the specific mechanism of apoptosis regulation has not been clarified.Therefore,to clarify the underlying apoptosis regulation mechanism of DCM has become an urgent medical problem.Cellular repressor of E1 A stimulated genes(CREG)is a small molecular glycoprotein and is an important myocardial protective factor.Our laboratory studies have found that:(1)CREG protein is highly expressed in the in myocardial tissue.(2)Pathological injury such as myocardial infarction,angiotensin Ⅱ or ischemia-reperfusion can significantly down-regulate the expression of CREG in cardiomyocytes,and the supplementation of exogenous CREG protein can rescue the myocardial injury caused by these pathological factors.Our study also confirms that CREG is a lysosome glycoprotein that affects autophagy by regulating the genesis and function of lysosomes.Therefore,we assume that CREG may be involved in the pathogenesis of DCM as an endogenous key regulatory gene in autophagy homeostasis of cardiomyocytes.However,the mechanism of CREG involving in autophagy regulation in diabetic cardiomyopathy is not clear.The main aim of this study is to clarify the role and mechanism of CREG on the regulation of myocardial apoptosis induced by high glucose and high palmitate.Methods(1)Different concentrations of palmitate(0.2mM,0.4mM,0.6mM,0.8mM)and high glucose(10mM,15 mM,25mM,33mM)were used to stimulate rat H9C2 cardiomyocyte.Western-blot was used to detect CREG protein expression and to determine the optimal concentration of high glucose and high palmitate.In order to determine the optimal time of high glucose and high palmitate stimulation,CREG protein expression was examined at different time points(12h,24 h,36h and 48h).(2)To determine the correlation between CREG and cardiomyocytes autophagy under the action of high glucose and high palmitate,H9C2 cells with overexpression and low expression of CREG were established.Under the stimulation of high glucose and high palmitate,the expression of autophagy protein LC3BⅡ,autophagy substrate protein P62,lysosome-associated membrane protein1(LAMP1),apoptotic protein cleaved-caspase3 were detected by western-blot,and the expression of cathepsinB and cathepsinD were detected by immunofluorescence.The proportion of H9C2 cell apoptosis was examined using by flow cytometry.(3)To clarify the mechanism of CREG in the regulating H9C2 cell autophagy,autophagy inhibitor chloroquine(10mM)and autophagy agonist resveratrol(10mM)were used to interfere with CREG overexpression H9C2 cell and CREG low expression H9C2 cell.After 24 h of high glucose and high palmitate combined with autophagy intervention,the expression of CREG protein,autophage associated protein and apoptotic protein in autophagy pathway was detected using by western-blot,immunofluorescence and flow cytometry.Results(1)Using different concentrations of palmitate and glucose to stimulate H9C2 cells,results found that,CREG was significantly decreased when the high glucose concentration was 33 mM and palmitate concentration was 0.4mM.Besides,CREG protein expression was decreased when 24 h after palmitate and high glucose stimulation,and CREG expression was in a time-dependent manner.(2)H9C2 cells was stimulated with high glucose(33mM)and high palmitate(0.4mM)for 24 h,results showed that high glucose and high palmitate decreased CREG protein expression,increased the expression of LC3BⅡ and P62,decreased lysosomal enzyme cathespineB and cathespineD,and increased cell apoptosis.CREG overexpression adenovirus and CREG small interference RNA were used to construct CREG overexpression(Ad-CREG)and CREG low-expression(CREG-siRNA)cell models.In Ad-CREG group,high glucose and high palmitate was stimulated for 24 h,the expression of LC3BⅡand LAMP1 was significantly increased,while P62 and cleaved-caspase3 was significantly decreased.The expression of cathespineB and cathespineD was increased.The percentage of apoptosis in H9C2 cells was decreased.Besides,in CREG-siRNA group,high glucose and high palmitate was stimulated for 24 h,results also found that,compared with the control group,LC3BⅡ and p62 expression was increased,the expression of cathespineB and cathespineD was decreased,and cell apoptosis was increased.The above results suggested that CREG could participate in the regulation of autophagy and apoptosis in cardiomyocytes stimulated with high glucose and high palmitate.(3)In order to further clarify the mechanism of CREG on the regulation of cardiomyocytes autophagy stimulated by high glucose and high palmitate,autophagy agonist and inhibitor was used.In CREG overexpression of high glucose and high palmitate group with chloroquine intervention,CREG expression was decreased,autophagy pathway was blocked(LC3BⅡ and p62 were increased,cathepsinB and cathepsinD were decreased)and cell apoptosis was increased.Besides,after treatment with resveratrol in CREG low expression cells of high glucose and high palmitate group,autophagy function was improved,lysosomal enzyme expression was increased and cell apoptosis was reduced.It was confirmed that,in H9C2 cells stimulated by high glucose and high palmitate,CREG could participate in autophagy regulation by affecting lysosome.ConclusionsIn H9C2 cells with high glucose and high palmitate stimulation,CREG protein expression was decreased,autophage function disordered,cell apoptosis increased.CREG overexpression could improve autophage function and reduced cell apoptosis,CREG low expression could aggravate autophage dysfunction and increased cell apoptosis.Besides,CREG can participate in autophagy through the regulation of lysosomes and then affect the apoptosis of cardiomyocytes.