Paeonol's protective effects on H9C2 cells against hypoxia/ reoxygenation injury through the hypoxia-inducible factor 1-alpha/B-cell lymphoma-2/adenovirus E1B 19 kDa interacting protein 3 signaling pathway.

OBJECTIVE: To elucidate the cytoprotective capacity of paeonol (PAE) against hypoxia/reoxygenation (H/R) injury in H9C2 cardiomyoblasts and to clarify its regulatory effects on the hypoxia-inducible factor 1-alpha (HIF-1α) and B-cell lymphoma-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) signaling axis governing autophagy and apoptosis. METHODS: H9C2 cells were exposed to a 12-h hypoxia condition followed by a 12-h reoxygenation regimen to simulate myocardial ischemia-reperfusion injury (MIRI). PAE pretreatment (10 μmol/L) was administered to cells before the H/R procedure. The viability of cells was determined using the cell counting kit-8 assay, whereas apoptotic rates were quantified via flow cytometry. Reactive oxygen species (ROS) levels were assayed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence staining. Western blotting was conducted to assess protein expression, including cleaved caspase-3, and the ratio of Bcl-2-associated X protein to B-cell lymphoma 2 (Bax/Bcl-2), as well as microtubule- associated protein 1 light chain 3 (LC3) and sequestosome-1 (p62), along with components of the HIF-1α/BNIP3 signaling pathway. Autophagosomes were visualized by transmission electron microscopy. To elucidate the regulatory mechanisms, siRNA was used to knockdown HIF-1α expression, while the autophagy inhibitor 3-methyladenine (3-MA) was employed as a functional intervention. RESULTS: PAE significantly improved cell viability following H/R treatment and inhibited apoptosis, as indicated by a reduction in cleaved caspase-3 levels and a decreased Bax/Bcl-2 ratio, along with diminished intracellular ROS levels. PAE treatment enhanced autophagosome formation and increased LC3-II levels, suggesting enhanced autophagic flux. Additionally, PAE upregulated the expression of HIF-1α and BNIP3. Silencing HIF-1α or inhibiting autophagy with 3-MA reversed the protective effects of PAE on autophagic flux, apoptosis, and cell viability. CONCLUSION: PAE protects H9C2 cells against H/R injury by promoting autophagy and inhibiting apoptosis, mediated through the upregulation of the HIF-1α/BNIP3 signaling pathway. These observations highlight the therapeutic implications of PAE for MIRI, particularly through targeting the HIF-1α/BNIP3 axis.