Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy through inhibiting NF-κB translocation

Background: Inflammation and oxidative stress play a predominant role in the initiation and progression of ischemia/reperfusion (I/R) injury, of which nuclear factor kappa B (NF-κB) is considered to be a crucial mediator. Inactivation of NF-κB could benefit cardiomyocytes through inhibiting apoptosis. IκBα, an inhibitor of NF-κB, is hypothesized to protects cardiomyocytes from H2O2-induced apoptosis and autophagy through inhibiting the NF-κB pathway.Methods: We designed an AAV9-delivered mutated IκBαS32A, S36A and investigated its effect on neonatal rat ventricular cardiomyocytes (NRVMs) in response to hydrogen peroxide (H2O2). NRVMs were divided into Normal (blank), Control (H2O2), GFP +H2O2, IκBα+H2O2, and Pyrrolidine dithiocarbamate (PDTC)+H2O2 groups. NF-κB p65 nuclear translocation was evaluated by immunofluorescence and western blot. Cell viability was assessed by a cell counting kit-8 kit. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD, and the mitochondrial membrane potential (△Ψm) was detected by JC-1. Western bolt was used to detect apoptosis and autophagy related proteins.Results: Consequently, H2O2-treated NRVMs showed reductions in cell viability but increased IκBα degradation and NF-κB p65 nuclear translocation in a time-dependent manner. Furthermore, LDH and MDA content, LC3-Ⅱ/LC3-Ⅰ ratio, Bax and Beclin-1 expressions, and apoptotic cells were upregulated in NRVMs exposed to H2O2, whereas △Ψm and Bcl-2 expression were downregulated. Additionally, IκBα transduction or PDTC pretreatment both attenuated the nuclear translocation of the p65 subunit and reversed the H2O2-stimulated effects in NRVMs.Conclusion: These findings suggest that IκBα could ameliorate H2O2-induced apoptosis and autophagy through targeted inhibition of NF-κB activation, which may guide strategies to prevent cardiac I/R jury.