Ma Xing Shi Gan Decoction reduces influenza A virus nucleoprotein levels in association with modulation of lactate-HMGB1 lactylation and autophagosome accumulation.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Viral pneumonia (VP), particularly that induced by influenza A virus (IAV), remains a major global health challenge and a leading cause of acute respiratory infections. Ma Xing Shi Gan Decoction (MXSG), a classical traditional Chinese medicine formula recorded in the ancient text Treatise on Cold Damage, has been widely used for centuries in China to treat febrile respiratory diseases and pneumonia. However, the molecular basis underlying its therapeutic effects remains incompletely defined. This study provides experimental evidence linking MXSG to modulation of VP and host metabolic-autophagy pathways. AIM OF THE STUDY: To investigate whether MXSG is associated with reduced IAV nucleoprotein (NP) levels and concurrent modulation of key glycolytic enzymes, HMGB1 lactylation, and autophagosome accumulation. Buddleoside was evaluated as an exploratory constituent. METHODS: A mouse model of VP was established by intranasal inoculation with IAV, while A549 cells were infected with IAV for in vitro studies. In vivo effects on glycolysis and autophagy were evaluated by assessing lung inflammatory cytokines, viral NP levels, glycolytic enzymes, and autophagy markers. Oxamate and diphenyleneiodonium chloride (DPI) were used as tools to interfere with lactate production. UPLC-MS/MS and molecular docking were performed to identify potential pathway-relevant constituents. In vitro, validation included lactate supplementation, oxamate treatment, buddleoside exposure, and HMGB1 overexpression. RESULTS: In vivo, MXSG reduced IAV NP levels and alleviated lung inflammatory injury. MXSG treatment was associated with decreased expression of glycolysis-related enzymes, reduced lactate accumulation, lower HMGB1 lactylation levels, and reduced autophagosome accumulation. Under DPI-mediated lactate accumulation, the inhibitory effect of MXSG on autophagosome accumulation was partially attenuated. Docking analysis suggested potential interaction between buddleoside and LDHA. In vitro, MXSG modulated lactate levels, HMGB1 lactylation, and autophagy-related markers, accompanied by decreased NP levels. CONCLUSION: MXSG reducing NP levels is associated with modulation of expression levels of glycolytic enzymes and reduced lactate production, and was accompanied by decreases in HMGB1 lactylation with expression and HMGB1-associated autophagosome accumulation providing a potential metabolic basis for MXSG-associated regulation of autophagy-related processes. These findings suggest a possible association between metabolic-autophagy signaling and MXSG-associated reductions in viral NP levels, but remain correlative and require further validation.