Effect Of ARC On High Glucose Induced Rat Cardiomyocyte Pyroptosis And Its Mechanism

Objective: The incidence of diabetes in China has reached 10.4%,and chronic hyperglycemia will lead to chronic damage and dysfunction of various tissues.Diabetic Cardiomyopathy(DCM)is one of the main complications of diabetes.To reveal the molecular mechanism of diabetic cardiomyopathy is beneficial for targeted treatment.Cardiomyocytes are terminally differentiated cells,and various injury factors lead to the death of cardiomyocytes,which leads to the occurrence of cardiovascular diseases such as myocardial infarction and heart failure.Many studies have shown that high glucose can induce the death of cardiomyocytes and hyperglycemia is considered to be the cytological basis for the occurrence of diabetic cardiomyopathy.Pyroptosis is a newly discovered form of programmed cell death in recent years,which involves a large number of inflammatory factors and mediators.Relevant studies have proved that the occurrence and development of diabetic cardiomyopathy is related to cell pyrotosis,which involves the activation of inflammatory bodies and the release of cytokines in the process of pyrotosis.It is of great theoretical significance and practical application value to further study the molecular mechanism of cardiomyocyte pyrotosis in diabetic cardiomyopathy.ARC(Apoptosis repressor with caspase recruitment domain)is an endogenous protein highly expressed in the heart and skeletal muscle,which can inhibit the apoptosis and necrosis of cardiomyocytes.ARC plays a protective role by inhibiting apoptosis of cardiomyocytes through various mechanisms.In recent years,it has been found that ARC can also play a protective role by inhibiting programmed necrosis of cardiomyocytes.Many studies have shown that ARC plays an important role in cardiovascular diseases such as myocardial infarction and heart failure.However,there are few studies on ARC in diabetic cardiomyopathy,and whether ARC can inhibit pyroptosis in diabetic cardiomyopathy is not clear.This study revealed the effect and molecular mechanism of ARC on rat cardiomyocyte pyroptosis induced by high glucose.Methods: The H9c2 cells were treated with 50mmol/L glucose medium to construst the high-glucose-induced pyroptosis model at the cell level;Western blot was used to detect the expression of gasdermin D(GSDMD)-N,caspase-1,cleaved-caspase-1,IL-1β,cleaved-IL-1β,apoptosis-associated speck-like protein containing CARD(ASC),ARC and p53;ARC was overexpressed and knocked down by adenovirus transfection;Whether ARC and ASC bind to each other was detected by immunoprecipitation reaction;PI staining was used to detect cell death;Mitochondrial membrane potential was detected by JC-1 staining;ROS was labeled with fluorescent probe DCFH-DA,and the levels of ROS were detected by enzyme plate analyzer and fluorescence microscope.The diabetic mouse model was induced by intraperitoneal injection of STZ,and the heart function of the mice was detected by ultrasound.Results: Compared with the control group,the expression of ARC in the heart tissue of diabetic mice and H9c2 cells treated with high concentration of glucose decreased significantly,suggesting that ARC may be involved in the development of diabetic cardiomyopathy;Under the condition of high glucose treatment,the cell death increased with the extension of treatment time,and the expression of GSDMD-N,cleaved-caspase-1 and cleaved-IL-1β increased continuously,indicating that high glucose can induce cardiomyocyte pyroptosis;Endogenous inhibition of ARC can promote the occurrence of cardiomyocyte pyroptosis induced by high glucose,while overexpression of ARC can inhibit pyroptosis,suggesting that ARC is involved in cardiomyocyte pyroptosis induced by high glucose;Pyroptosis increased after ASC knockdown,suggesting that the pyroptosis induced by high glucose was dependent on the activation of inflammasomes;Overexpression of ARC can inhibit the increase of ROS induced by high glucose;Mechanically,ARC inhibits the activation of inflammasomes by interacting with ASC,thereby inhibiting the pyroptosis of H9c2 cells induced by high glucose.It was further verified that p53 can regulate the expression of ARC under high glucose conditions;Compared with the control group,the heart function of diabetic mice was impaired,and overexpression of ARC could alleviate the impaired heart function of diabetic mice.Conclusion: ARC inhibits cardiomyocyte pyroptosis induced by high glucose and the development of diabetic cardiomyopathy;ARC can inhibit inflammasome activation by binding to ASC,and then inhibits the pyroptosis of H9c2 induced by high glucose;Under the condition of high glucose,p53 can inhibit the expression of ARC.Our study reveals a new molecular mechanism of diabetic myocardial injury,which may provide new ideas and targets for the prevention and treatment of diabetic cardiomyopathy.