In vitro and in silico analyses reveal the potential mechanisms of Liu-Wei-Di-Huang-Wan formula for tinnitus treatment

Tinnitus remains a challenging auditory disorder with limited effective treatments. Liu-Wei-Di-Huang-Wan formula (LWDHW) has shown promising effects in alleviating tinnitus symptoms. This study aims to identify the key active ingredients in LWDHW and investigate their mechanisms, with a focus on the MAPK3 and STAT3 signaling pathways, to provide a scientific foundation for its application in tinnitus treatment. We utilized ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to analyze LWDHW constituents, identified key ingredients, and explored tinnitus treatment mechanisms through network pharmacology, Mendelian randomization, molecular docking, and molecular dynamics simulations. The cochlear cell recovery model, evaluated using HEI-OC1 cells and FACS analysis, demonstrated validated protein expressions via Western blot analysis. Additionally, computational tools were employed to assess protein-ligand interactions and pathway regulation. A total of 185 compounds were identified in LWDHW by UPLC-MS/MS, with Kaempferol, Cerevisterol, and AlisolB2 detected as active ingredients. Network pharmacology and Mendelian randomization identified AKT1, MAPK3, STAT3, and MTOR as key targets, validated by molecular docking. Fluorescence activated cell sorter (FACS) analysis revealed LWDHW’s protective effect against cisplatin-induced apoptosis, enhancing cell viability. Western blot results demonstrated that LWDHW modulates tinnitus primarily through the MAPK3 and STAT3 pathways, promoting MAPK3 phosphorylation and inhibiting STAT3 activation, thereby improving cochlear cell resilience and reducing neuroinflammation. This study identifies Kaempferol, Cerevisterol, and AlisolB2 as critical active ingredients in LWDHW, influencing key biomarkers such as AKT1, MAPK3, and STAT3. These compounds effectively regulate the MAPK3 and STAT3 pathways, offering a promising approach for enhancing tinnitus management and supporting further clinical development. • The molecular mechanisms of Liu-Wei-Di-Huang-Wan (LWDHW) in treating tinnitus were uncovered. • Active compounds and tinnitus-related targets of LWDHW were identificated and constructed. • LWDHW enhanced MAPK3 phosphorylation and inhibited STAT3 to reduce neuroinflammation.