The E3 Ubiquitin Ligase Cul4a Regulates Parp-1 Through Binding Against Oxidative Stress-induced Injury In Rat Myocardial Cells

Objective:Acute myocardial infarction is one of the important causes of cardiovascular disease death,and reperfusion is the best treatment for it.Even if the blood supply is restored,there is still a progressive increase in damage to the myocardial tissue,which is called ischemia-reperfusion injury.Among many factors of ischemia-reperfusion injury,oxidative stress injury of active oxygen accumulation is the core.Therefore,by exploring the regulatory mechanism of oxidative stress injury,we can provide a new therapeutic strategy for the treatment of ischemia-reperfusion injury and other cardiovascular diseases caused by oxidative stress.In recent years,many studies have confirmed that the ubiquitin-proteasome system,especially E3 ubiquitin ligase,plays a key role in the regulation of oxidative stress.cul4a is a recently discovered E3 ubiquitin ligase,is closely related cancer and widely involved in cell physiology and pathology.However,there are no studies that indicate whether cul4a is involved in cardiovascular diseases and what physiological and pathological effects it plays.Therefore,this study intends to explore the role and molecular mechanism of cul4a in oxidative stress injury of cardiomyocytes by vitro cardiomyocyte experiments.Methods:H₂O₂ was selected as the inducer of oxidative stress injury in H9c2cardiomyocytes.Firstly,treated with 50μM,100μM,200μM,and 400μM H₂O₂ for 2hours,and then determined its cell viability using CCK-8-kit.Using Annexin V-FITC/PI dual staining method Flow cytometry was used to detect apoptosis.Cellular immunofluorescence was used to detect the expression level and location of cul4a after oxidative stress injury.Western blot was used to detect the expression of cul4a and apoptosis-related proteins cleaved PARP-1 and cleaved caspase3.ROS fluorescence was used.Detection of ROS production in cardiomyocytes.Next,cul4a was overexpressed in cardiomyocytes,and treated with H₂O₂ at 50μM,100μM,200μM,and 400μM for 2 hours.The cell viability was also measured using CCK-8-kit,and Annexin V-FITC/PI dual staining method-flow cytometry was used.Apoptosis was detected.The expression of Flag-cul4a and apoptosis-related proteins cleaved PARP-1 and cleaved caspase3 were detected by western blot,and ROS production in cardiomyocytes was detected by ROS fluorescence.Next,the RNA interference of cardiomyocytes using three cul4a-siRNA fragments was used to knock down the protein,and the knockdown efficiency of cul4a was detected.The fragment with the highest knockdown efficiency was selected and treated with 50μM,100μM,200μM,400μM H₂O₂ for 2 hours.Later,CCK-8-kit was also used to determine its cell viability,and Annexin V-FITC/PI dual staining-flow cytometry was used to detect apoptosis,and Western blot was used to detect Flag-cul4a and apoptosis-related proteins cleaved PARP-1,cleaved caspase3 expression was measured by ROS fluorescence in cardiomyocytes.Finally,the co-immunoprecipitation experiment was used to detect the endogenous and semi-exogenous interactions of ubiquitin ligase cul4a and apoptosis-related protein PARP-1 in physiological states and H₂O₂-induced oxidative stress injury,and it was overexpressed in gradients After knocking down cul4a,the expression level of apoptosis-related protein PARP-1 was detected.Results:1.H₂O₂ stimulated H9c2 cells,with the gradual increase in concentration,the cell viability decreased significantly.After 200μM H₂O₂ treated cells for 2 h,the cell viability decreased compared with the blank control group,and the apoptosis significantly increased,intracellular ROS production increased and cul4a expression was up-regulated.The expression level of cul4a in the 200μM H₂O₂ treatment group increased compared with the control group.The expressions of apoptosis-related proteins cleaved PARP-1 and cleaved Caspase 3 were significantly increased.2.After over-expressing cul4a,the cell viability of the over-expression group treated with 200μM H₂O₂ increased compared with the no-load control group,and the apoptosis rate decreased significantly.The expression levels of apoptosis-related proteins cleaved PARP-1 and cleaved caspase3 significantly reduced,respectively,and intracellular ROS production was reduced either.3.After knocking down cul4a,the cell viability of the cul4a siRNA group interfered with 200μM H₂O₂ decreased,the apoptosis rate and the expression levels of apoptosis-related proteins cleaved PARP-1 and cleaved caspase3increased significantly,respectively,and intracellular ROS production increased either.4.Co-immunoprecipitation experiments confirmed that cul4a can interact with PARP-1 in H9c2 cardiomyocytes,and this effect will be enhanced in oxidative stress injury.And cul4a acts as an E3 ubiquitinated ligase to reduce PARP-1 expression.Conclusion:In the oxidative stress injury of myocardial cells,the increased expression of cul4a and the increase in the binding of apoptosis-related protein PARP-1reduce the expression of PARP-1 and the generation of ROS,thereby inhibiting cell damage.It is suggested that cul4a can provide a new potential treatment strategy for oxidative stress-induced reperfusion injury and other cardiovascular diseases.