Baicalein Protects Cardiomyocytes From Oxidative Stress Induced Programmed Necrosis By Stabilizing Carboxyl Terminus Of Hsc70-Interacting Protein

Objective It is reported that cardiomyocyte necrosis is the main pathogenesis of heart disease such as myocardial infarction,ischemia/reperfusion and heart failure,so that cardiomyocyte necrosis is an important therapeutic target for the prevention and treatment of heart diseases.Hsc70 carboxy-terminal reaction protein(CHIP)plays a protective role through its E3 ubiquitin ligase function,but whether CHIP can inhibit oxidative stress-induced cardiomyocyte necrosis has rarely been reported.Baicalein exerts cardioprotective effects by inhibiting oxidative stress and apoptosis of cardiomyocytes,but it has not been reported whether baicalein protects against cardiomycyte necrosis.The aim of this study was to investigate whether baicalein relieves oxidative stress-induced myocardial injury by inhibiting necrosis,and whether baicalein acts on CHIP to regulate RIPK1/RIPK3 in order to suppress Cardiomyocyte necrosis.Methods PI staining,TUNEL staining and LDH release assay were used to detect the inhibitory effect of baicalein on myocardial damage induced by hydrogen peroxide at the cellular level.Western blot,qPCR and co-immunoprecipitation were used to verify that RIPK1/RIPK3 plays an important role in hydrogen peroxide-induced myocardial necrosis.At the same time,the use of siRNA of RIPK1/RIPK3 further proved that they are important related proteins of necrosis.Western blot and co-immunoprecipitation were used to investigate whether baicalein inhibited myocardial necrosis by inhibiting the formation of RIPK1/RIPK3 necrosis complex.Western blot and qPCR experiments were used to investigate the changes in CHIP expression in hydrogen peroxide-induced myocardial necrosis.In addition,by knocking down and over-expressing the CHIP protein and detecting the level of ubiquitination,it was further explored whether the protein can modulate RIPK3 by ubiquitination to regulate hydrogen peroxide-induced myocardial necrosis.The molecular docking technique was used to predict the binding sites of baicalein and CHIP.At the same time,the level of ubiquitination was detected at the cellular level to further investigate whether baicalein promoted the function of ubiquitin ligase by CHIP protein.Finally,a mouse myocardia l infarction model was used to perform PI staining,TUNEL staining,and TTC/Evans blue double staining at the animal level to investigate whether baicalein significantly inhibited myocardial damage at the animal level.Results Necrosis and apoptosis were significantly reduced in the baicalein treatment group compared with the control group in vitro;siRNA transfection and co-immunoprecipitation experiments confirmed that the necrotic complex formed by RIPK1/RIPK3 was induced by hydrogen peroxide in the myocardium.Necrosis plays an important role,and RIPK1/RIPK3 protein expression and necrosis complex formation are significantly inhibited in the baicalein treatment group;western blot results show significant changes in CHIP protein expression in hydrogen peroxide-induced myocardial necrosis The results of knockdown of CHIP and ubiquitination showed that CHIP can exert E3 ubiquitin ligase to degrade RIPK3 protein in cardiomyocytes.Overexpression of CHIP and PI staining results demonstrate that exogenous CHI P protein can alleviate myocardial necrosis by degrading RIPK3.The molecular docking test first predicted the binding site of baicalein and CHIP protein.The results of ubiquitination experiments further confirmed that baicalein promoted the E3 ubiquitin ligase activity of CHIP protein;animal experiments further confirmed the myocardial protection of baicalein.Conclusion Baicalien alleviates hydrogen peroxide-induced myocardial necrosis in vivo;RIPK1 / RIPK3 is involved in the regulation of hydrogen peroxide-induced cardiomyocyte necrosis;baicalein reduces necrotic cell death by interfering with the necrotic complex formed by RIPK1 / RIPK3;CHIP suppresses cardiac necrosis by ubiquitylation-dependent degradation of RIPK1 and RIPK3;baicalein relieves hydrogen peroxide-induced necrosis by stabilizing CHIP;baicalein can bind to CHIP protein;baicalein attenuates myocardial damage in vitro.