Wendan decoction-derived TMF alleviates endolymphatic hydrops by modulating the DUSP3/p38 MAPK/AQP2 axis

TMF was prioritized from Wendan Decoction by network analysis. • TMF reduced cochlear inflammation and injury in experimental EH. • TMF suppressed p38 MAPK/AQP2 signaling and restored DUSP3/ZO-1. • DUSP3 knockdown weakened TMF regulation in vitro and in vivo. Ethnopharmacological relevance Wendan Decoction is a classical traditional Chinese medicine formula historically used for phlegm-related dizziness and vertigo. Recent studies have shown that Wendan Decoction can ameliorate experimental endolymphatic hydrops (EH), a characteristic pathological correlate of Ménière's disease. However, the bioactive constituents responsible for these effects and their molecular targets remain incompletely defined. Aim of the study This study aimed to identify and quantify a pharmacologically relevant constituent of Wendan Decoction and to investigate its molecular target and mechanism of action in experimental EH. Materials and methods Candidate constituents and regulatory targets were prioritized through ADME-guided screening, target prediction, MD-associated transcriptomic analysis, protein–protein interaction network analysis, pathway enrichment, MAPK pathway mapping, and molecular docking. Targeted UPLC-ESI-MS/MS was used to verify and quantify 5,6,7,4′-tetramethoxyflavone (TMF) in Wendan Decoction granules. The interaction between TMF and DUSP3 was examined using surface plasmon resonance (SPR) and a cell lysate-based cellular thermal shift assay (CETSA). The effects of TMF were evaluated in LPS-stimulated HEI-OC1 cells and a DDAVP-induced guinea pig model of EH. DUSP3 knockdown and pharmacological inhibition of p38 MAPK with SB203580 were used to investigate the functional relationships among DUSP3, p38 MAPK, AQP2, and ZO-1. Results TMF was detected in Wendan Decoction granules at 0.0379% (w/w). SPR showed micromolar binding between TMF and recombinant DUSP3, while CETSA showed that TMF increased the thermal stability of DUSP3. In LPS-stimulated HEI-OC1 cells, TMF reduced inflammatory mediator production, increased DUSP3 and ZO-1 expression, inhibited p38 MAPK activation, and decreased AQP2 expression. SB203580 attenuated AQP2 upregulation, ZO-1 reduction, and pro-inflammatory cytokine production, including after DUSP3 silencing, supporting p38 MAPK as a downstream component of DUSP3-associated signaling. In DDAVP-treated guinea pigs, TMF reduced morphometrically quantified cochlear hydrops, alleviated cochlear ultrastructural injury and vestibular-associated behavioral impairment, and decreased cochlear inflammatory cytokine levels. Cochlear DUSP3 knockdown was confirmed at both the mRNA and protein levels and partially weakened TMF-mediated regulation of the p38 MAPK/AQP2 axis. Conclusions TMF is a quantified Wendan Decoction-derived constituent with protective activity against experimental EH. The findings support a direct interaction between TMF and DUSP3 and suggest that TMF alleviates EH-associated pathological changes, at least in part, through DUSP3-associated regulation of p38 MAPK activation and downstream AQP2 and ZO-1 expression. Download: Download high-res image (263KB) Download: Download full-size image