Qushi Huoxue ointment ameliorates metabolic associated steatotic liver disease through autophagy activation and ferroptosis inhibition

BACKGROUND: Metabolic associated steatotic liver disease (MASLD) has become a growing global health burden, with its potential to progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Qushi Huoxue ointment (QSHXO), a traditional Chinese medicine formula, has demonstrated efficacy in the management of MASLD. However, its underlying mechanisms remain incompletely elucidated. AIM: To investigate the mechanism by which QSHXO alleviated hepatic lipid deposition and inflammatory injury in MASLD, with a focus on its role in activating hepatocyte autophagy and inhibiting ferroptosis. METHODS: This study employed a comprehensive research strategy. First, a methionine-choline-deficient diet-triggered MASLD mouse model was established and treated with different doses of QSHXO. The therapeutic effects of QSHXO were comprehensively evaluated using histological analysis, serum biochemical assays, and inflammatory cytokine measurements. Subsequently, bioactive components of QSHXO in serum were identified utilizing liquid chromatography-tandem mass spectrometry. Network pharmacology was then applied to predict potential targets of QSHXO in treating MASLD related to autophagy and ferroptosis. These predicted targets were validated through western blotting, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and transmission electron microscopy. RESULTS: QSHXO significantly ameliorated liver lipid deposition and inflammation in MASLD mice. Specifically, QSHXO promoted autophagic flux, as indicated by upregulation of Beclin1, an increased light chain 3 II/light chain 3 I ratio, and downregulation of P62. Concurrently, QSHXO activated the nuclear factor erythroid 2-related factor 2 pathway, promoting its nuclear translocation and enhancing the expression of downstream targets (SLC7A11 and glutathione peroxidase 4), while reducing hepatic iron deposition; these collectively suggested suppression of ferroptosis. Ultrastructural analysis further confirmed improved mitochondrial morphology and increased autophagic vesicles in QSHXO-treated groups. CONCLUSION: QSHXO ameliorates MASLD by reducing lipid accumulation, mitigating inflammation, and suppressing hepatocyte damage, which is mediated through the activation of autophagy and inhibition of ferroptosis.