Qing Hua Yu Du formula ameliorates alcoholic hepatic fibrosis by regulating MAPK/TLR4-MyD88 inflammatory pathways, restoring hepatic metabolism and modulating gut microbiota.

Alcohol-induced liver injury (ALI) and subsequent hepatic fibrosis pose significant global health burdens, with limited effective therapeutic options. Traditional Chinese Medicine (TCM) formulas, such as Qing Hua Yu Du (QHYD) formula, have shown potential in treating alcoholic hepatic fibrosis in clinical, but their therapeutic effects, and underlying mechanisms remain incompletely characterized. Additionally, the interplay between hepatic inflammation, metabolic disorders, and gut-liver axis dysregulation in ALI-related fibrosis necessitates comprehensive validation across multiple models. This study aimed to evaluate the therapeutic effects of QHYD formula on alcohol-CCl₄-induced alcoholic hepatic fibrosis in diverse models (alcohol-CCl₄, acute binge alcohol, and cell models), explore its mechanisms involving inflammatory signaling, hepatic metabolism, and gut microbiota, validate the key metabolite l-histidine's role, and assess its safety profile. The QHYD formula's chemical composition was characterized using advanced high-performance liquid chromatography (HPLC) fingerprinting for quality control and ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) for constituent identification. Male C57BL/6 mice were assigned to normal, model, QHYD (2.7, 4.05, 5.4 g/kg/day), and fecal microbiota transplantation (FMT) groups. Acute binge alcohol and AML-12 cell models were used for supplementary validation. Liver injury was evaluated by plasma biochemical markers (ALT/AST/GGT/TC/TG) and histological staining (H&E/Masson). Inflammatory pathways (MAPK/TLR4-MyD88), metabolic changes, and gut microbiota were analyzed via Western blot, ELISA, transcriptomics, metabolomics, and 16S rRNA sequencing. l-histidine's mechanism was validated in LX-2 cells using qRT-PCR and immunofluorescence. Acute/chronic toxicity assays were conducted to assess safety. HPLC confirmed QHYD's batch consistency, and UHPLC-Q/TOF-MS identified 82 constituents. QHYD significantly ameliorated liver injury and fibrosis in alcohol-CCl₄ and acute binge alcohol models, reduced plasma TC/TG, and inhibited Col1a1/α-SMA expression. It suppressed MAPK/TLR4-MyD88 signaling, restored protein digestion/absorption pathway (upregulating l-histidine), and modulated gut microbiota richness/composition. FMT experiments confirmed QHYD-modulated gut microbiota directly mediated anti-fibrotic effects. l-histidine dose-dependently inhibited HSC activation via the NF-κB-TIMP1 axis. QHYD ameliorates alcoholic hepatic fibrosis through multi-targeted mechanisms: inhibiting MAPK/TLR4-MyD88 inflammatory pathways, restoring hepatic metabolism via l-histidine, and modulating gut microbiota. Its favorable safety profile and efficacy across diverse models support QHYD as a promising therapeutic candidate, with l-histidine serving as a key mediating metabolite.