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

Abstract

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.

Downregulation of p300/CBP‐associated factor inhibits cardiomyocyte apoptosis via suppression of NF‐κB pathway in ischaemia/reperfusion injury rats

“…NF‐κB was a recognized transcription factor in modulation of inflammation, oxidative stress and cell apoptosis in the context of MIRI 17‐19 . The expression level of phospho‐NF‐κB p65 was significantly upregulated after H/R compared with the control cells.…”

“…NF-κB was a recognized transcription factor in modulation of inflammation, oxidative stress and cell apoptosis in the context of MIRI. [17][18][19] The expression level of phospho-NF-κB p65 was significantly upregulated after H/R compared with the control cells. While H9c2 cells were transfected with Ad-shRNA-PCAF, the protein level of The results of Co-IP showed that there was a direct interaction between PCAF and NF-κB p65, and after H/R induction, the interaction between PCAF and NF-κB became stronger (Figure 6C).…”

Strontium Ranelate Inhibits Osteoclastogenesis through NF-κB-Pathway-Dependent Autophagy

“…Bay 11-7082, an inhibitor of the NF-κB pathway, downregulated the autophagy-related proteins except p62 and decreased the size and number of autophagosomes, based on the analysis of monodansylcadaverine staining and transmission electron microscope observation. Similarly, there is evidence that IKK/NF-κB directly enhances autophagy by inducing the expression of related proteins such as ATG5, LC3, and Beclin1 [ 23 , 50 ]. These results demonstrate that SR attenuates osteoclastogenesis by inhibiting autophagy through the NF-κB pathway.…”

Transcription Factors Involved in the Development and Prognosis of Cardiac Remodeling

“…In rabbit I/R-injured hearts, Beclin-1 upregulation, which is induced by active p62, aggravates cardiac dysfunction, especially in the cardiac area at risk (Zeng et al, 2013). Overexpression of IκB, an inhibitor of the NF-κB signaling pathway, reduces p65 nuclear translocation and is associated with decreased Beclin-1 expression and a reduced LC3-II/LC3-I ratio in mice exposed to H 2 O 2 , suggesting that the reduced autophagy level protects the heart from H 2 O 2 -induced oxidative stress (Han et al, 2020). A similar outcome was confirmed in the diabetic mice.…”