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

BACKGROUND: Inflammation and oxidative stress play predominant roles in the initiation and progression of ischaemia/reperfusion (I/R) injury, with nuclear factor kappa B (NF-κB) serving as a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H(2)O(2)) by inhibiting the NF-κB pathway. METHODS: The IκBα(S32A, S36A) gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H(2)O(2) treatment. NRVMs were divided into control, H(2)O(2), GFP + H(2)O(2), IκBα+H(2)O(2), and pyrrolidine dithiocarbamate (PDTC) + H(2)O(2) groups. Nuclear translocation of the NF-κB p65 subunit was evaluated by immunofluorescence and Western blotting. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H(2)O(2)-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Western blotting was used to detect apoptosis- and autophagy-related proteins. RESULTS: IκBα transfection significantly increased cell viability and ΔΨm but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H(2)O(2). Meanwhile, IκBα overexpression decreased H(2)O(2)-induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3-II/LC3-I ratio. These effects partly accounted for the ability of IκBα to inhibit the NF-κB signalling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signalling with the specific inhibitor PDTC resembled the cardioprotective effects of IκBα during H(2)O(2) stimulation. CONCLUSION: IκBα overexpression can ameliorate H(2)O(2)-induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signalling pathway. These findings suggest that IκBα transfection can result in successful resistance to oxidative stress-induced damage by inhibiting NF-κB activation, which may provide a potential therapeutic target for the prevention of myocardial I/R injury.