Wendan Decoction-Derived TMF Alleviates Endolymphatic Hydrops by Modulating the DUSP3/p38 MAPK/AQP2 Axis

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.

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.

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.

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.

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.