Abstract

This study aims to investigate the protective effect of the natural phthalide compound Levistolide A (LA) against myocardial ischemia–reperfusion injury (MIRI) and to elucidate its underlying mechanisms. Utilizing network pharmacology, potential targets of LA in the treatment of MIRI were predicted. Subsequently, a hypoxia/reoxygenation (H/R) model was established using rat H9C2 cardiomyocytes to simulate MIRI, and the mechanisms of action were validated through cellular experiments. Network pharmacology analysis indicated that the potential targets of LA in treating MIRI were significantly enriched in calcium signaling pathways, with the adenosine A2B receptor (ADORA2B), a G protein-coupled receptor (GPCR), identified as a key protein. Cellular experiments demonstrated that 24 μM LA significantly alleviated H/R-induced damage in H9C2 cells, enhanced cell viability, and reduced the release of lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI). Pre-treatment with LA significantly activated the ADORA2B/Cyclic adenosine monophosphate (cAMP)/Protein kinase A (PKA) signaling axis, promoting the phosphorylation of phospholamban (PLB), enhancing the activity and protein expression of sarco/endoplasmic reticulum Ca2+-ATPase 2 alpha (SERCA2α), and effectively mitigating intracellular calcium overload induced by H/R. However, the ADORA2B antagonist MRS 1754 partially reverses the aforementioned protective effects of LA. The findings of this study reveal a novel mechanism by which LA exerts cardioprotective effects through the ADORA2B/cAMP/PKA/PLB/SERCA2α signaling axis, preventing calcium overload and improving calcium homeostasis, and identify potential candidate compounds and precise targets for the treatment of MIRI.