Higenamine protects against doxorubicin-induced heart failure by attenuating ferroptosis via modulating the Nrf2/GPX4 signaling pathway

Background

Higenamine (HG), a benzylisoquinoline alkaloid in Aconiti Lateralis Radix Praeparata (ALRP), has cardioprotective effects. Prior research indicated its potential anti-heart failure (HF) function, yet the molecular mechanism remained elusive.

Purpose

This study aimed to explore the underlying mechanism of HG against doxorubicin (DOX)-induced HF via an integrated approach involving gut microbiota, untargeted metabolomics, network pharmacology, and molecular biology.

Methods

DOX was employed to induce HF in rats and H9c2 cardiomyocytes injury models. Cardiac injury was assessed using hemodynamic indices, cardiac injury biomarkers, and oxidative stress markers. Cell counting kit-8 (CCK-8) method and high-content analysis were used to investigate the effects of HG on the cell proliferation, morphology and mitochondrial function of H9c2 cardiomyocytes. 16S rDNA sequencing analysis, untargeted metabolomics, and network pharmacology were performed to identify the multi-target and multi-pathway mechanisms of HG in treating HF. Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, and Western Blotting was used to investigate its intervention on the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) ferroptosis pathway.

Results

HG alleviated DOX-mediated myocardial injury by enhancing cardiac and mitochondrial function, reducing oxidative stress levels, and promoting cell proliferation. Effects of HG on changes in the gut microbiota of rats is characterized by a low abundance of Firmicutes and Proteobacteria, along with a high abundance of Bacteroidetes and Actinobacteria, indicating an improvement in DOX-induced dysbiosis. Untargeted metabolomics combined with network pharmacology showed that HG exerted anti-HF effects by regulating eight metabolites, eight pathways, and interacting with ferroptosis-related targets. Molecular biology studies revealed its cardioprotective effects via regulating the Nrf2/GPX4 ferroptosis pathway.

Conclusion

HG could inhibit ferroptosis and protect against HF by regulating the Nrf2/GPX4-mediated "mitochondrial-ferroptosis" pathway, offering a potential treatment strategy for HF.