Decoding the anti-heart failure effects of Xinbao Pills: AMPKα1 activation and active compound screening

Previous studies have focused on the protective effect of Xinbao pills (XBP) on heart failure (HF) irrespective of the ejection fraction-based classification, However, there are still no reports on the mechanism and active compounds of XBP in HF with reduced ejection fraction (HFrEF).

This study aimed to elucidate the anti-HFrEF effects and mechanism of XBP and identify its active compounds.

Doxorubicin-induced HFrEF rats and H₂O₂-induced damage to H9c2 cardiomyocytes were established. Transthoracic echocardiography, HE staining, commercial assay kits, and cellular flow cytometry analysis were used to evaluate the cardioprotective effect of XBP. Subsequently, myocardial energy metabolomics and transcriptomics were used to explore the mechanism of XBP, which was verified via western blotting and commercial assay kits. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), high-throughput molecular docking, molecular dynamics simulation analysis and in vitro verification were applied to explore the active ingredients of XBP.

XBP improved cardiac function (ejection fraction: 81.1 vs DOX 55.3 %, p < 0.001, and cardiac output: 71.3 vs DOX 47.3 μl/min, p = 0.002 etc.) in HFrEF rats, and protected against myocardial H9c2 cardiomyocyte damage (cell viability: 0.56 vs DOX 0.35, p < 0.05, and LDH: 3562 vs DOX 7073 ng/ml, p < 0.05 etc.), enhanced myocardial energy metabolism levels(ATP: 2887 vs DOX 809.9 nM, p < 0.05, and Glutamine: 387.4 vs DOX 212.8 μM, p < 0.0001 etc.), and protected mitochondrial respiratory function (maximum respiratory capacity: 219.7 vs DOX 81.4 pmol/min, p < 0.05, and glycolytic function: 11.17 vs DOX 5.62 pmol/min, p < 0.001 etc.), which required the activation of AMPKα1. Inhibition of AMPKα1 blocked the cardioprotective effect of XBP. In contrast, activation of the AMPKα1 pathway can exert a similar protective effect as that of XBP. Additionally, Kaempferol-7-O-glucoside, Luteolin-7-O-glucoside, Grossamide, Isorhamnetin-3,7-O-diglucoside, and Kaempferol-7-O-rhamnoside were identified as key compounds that activating AMPKα1 and protecting against myocardial injury.

Our findings suggest that the AMPKα1 pathway is integral to the XBP's anti-HF effects, with key identified compounds likely contributing to the XBP's pharmacological protection, providing a novel mechanistic understanding and identifying the main active compounds of XBP.