Mechanism of Inonotus hispidus in treating melasma: integrated in vivo, in vitro, network pharmacology and untargeted metabolomics investigation.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Inonotus hispidus (Bull.) P. Karst. is a traditional Chinese medicine (TCM) with a long history. Although its use in treating melasma was documented in the Bencaogangmu, its therapeutic mechanism remains unexplored and is poorly understood in modern research. AIM OF THE STUDY: To investigate the mechanism of Inonotus hispidus extract in treating melasma. MATERIALS AND METHODS: By establishing a melasma mouse model and integrating Network Pharmacology, Untargeted Metabolomics, and in vitro cellular experiments using drug-containing serum, we systematically investigated the therapeutic mechanisms of its extracts. RESULTS: Both ethanol extract and water extract of Inonotus hispidus effectively ameliorated skin hyperpigmentation in model mice and demonstrated positive regulatory effects on key indicators related to oxidative stress, inflammatory response, and melanin metabolism, with the ethanol extract exhibiting more pronounced efficacy. Network Pharmacology screening identified the MAPK signaling pathway as a crucial pathway of action. Molecular docking revealed stable binding between MAPK1 and active components such as Hispidin, with both Hispidin and Ergosterol showing strong binding affinity to multiple critical targets, suggesting their potential roles as core therapeutic substances in the ethanol extract. Untargeted Metabolomics analysis indicated that the ethanol extract effectively modulated endogenous metabolic disorders in model animals, primarily influencing pathways related to lipid metabolism and amino acid metabolism. Further in vitro cellular experiments confirmed that the drug-containing serum derived from the ethanol extract could inhibit melanin synthesis and alleviate inflammation and oxidative stress at the cellular level. Western blot and RT - qPCR analyses ultimately demonstrated that the ethanol extract, by regulating the p38 MAPK/ERK signaling pathway and simultaneously activating the Nrf2/HO-1 pathway, significantly downregulated the gene and protein expression of key melanogenesis factors such as MITF and TYR. CONCLUSION: The ethanol extract of Inonotus hispidus exerts its therapeutic effects against melasma through a multi-target and multi-pathway mechanism. This mechanism involves the inhibition of melanogenesis, the exertion of antioxidant and anti-inflammatory effects, and the synergistic amelioration of metabolic disorders.