Total Sanghuangporus vaninii extract inhibits hepatocyte ferroptosis and intestinal microbiota disturbance to attenuate liver fibrosis in mice

Ethnopharmacological relevance: Sanghuangporus, the dried fruiting body of Sanghuangporus vaninii (Ljub) L.W.Zhou et Y.C.Dai. As the main species of Sanghuang, it has been well-known and used commonly as a traditional medicinal and edible macrofungi for thousands of years in many countries, including China, Korea and Japan. Although it has good hepatoprotective activity, its potential efficacy and mechanism on liver fibrosis remain elusive.

Aim of the study: Total Sanghuangporus vaninii extract (TSH) was prepared by ethanol extraction to investigate its chemical components and to conduct an initial assessment of its efficacy and underlying mechanism in a murine model of liver fibrosis.

Materials and methods: The chemical components of TSH were initially analyzed by UHPLC-Q-Orbitrap HRMS. To elucidate the effects of TSH, an in vivo model of fibrosis was established in mice using carbon tetrachloride (CCl₄), followed by assessments of serum liver function and histopathological analysis. Besides, indicators related to liver fibrosis, hepatic stellate cells (HSCs) activation, inflammation response and Ferroptosis related indicators were detected by western blotting, immunohistochemistry and real-time quantitative PCR (RT-qPCR) analysis. Additionally, the 16S rDNA Sequencing and untargeted metabolomics analysis of intestinal microbiota were employed to investigating the role of TSH in gut microbiome. In vitro, the human hepatocyte line L02 was stimulated with erastin and treated with or without TSH to elucidate its underlying mechanism.

Results: The administration of TSH significantly improved serum indicators of liver injury in CCl₄-induced fibrosis mice, reduced HSCs activation and collagen deposition, while inhibiting the expressions of transforming growth factor-β1(TGF-β1)/Smad signaling pathway. Notably, TSH treatment attenuated hepatocyte Ferroptosis and lipid peroxidation both in vivo and in vitro, as evidenced by a marked decrease in liver iron and malondialdehyde (MDA) contents. In particular, TSH was demonstrated to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-glutathione peroxidase 4 (GPX4) signaling pathway, thereby protecting hepatocytes from Ferroptosis with a particular enhancement of Nrf2 nuclear transcription. Furthermore, TSH influenced gut microbiota composition and ameliorated intestinal metabolic disorders. The increased abundance of Parasutterella and Olsenellas due to TSH treatment was significantly positively correlated with elevated phosphatidylcholines involved in linoleic acid metabolism, and negatively correlated with the reduction of fatty acyls. And the enrichment of intestinal linoleic acid metabolism presented a negative correlation in liver fibrosis biomarkers.

Conclusions: Our findings indicate that the TSH treatment exerts a significantly protective effect on CCl₄-induced mice by ameliorating hepatic injury and Ferroptosis damage, inhibiting HSCs activation and collagen deposition, and remodeling gut microbiota homeostasis and metabolic imbalance. Notably, TSH attenuated hepatocyte Ferroptosis in liver fibrosis and exhibited upregulation of the Nrf2-GPX4 signaling pathway. Furthermore, TSH could enrich the abundance of Parasutterella and Olsenellas, which may contribute to intestinal linoleic acid metabolism, thereby contributing to the reduction of liver fibrosis damage. Our study provides more effective and unreported evidence of TSH in anti-fibrosis activity.

Hepatocyte ferroptosis; Intestinal microbiota; Liver fibrosis; Nrf2-GPX4 signaling pathway; Total Sanghuangporus vaninii extract.