Saikosaponin A targets HDAC6 to inhibit Mycobacterium tuberculosis-induced macrophage Pyroptosis via autophagy-mediated NLRP3 inflammasome inactivation

BACKGROUND: Mycobacterium tuberculosis (Mtb) is among the oldest and most resilient human pathogens, remaining a major global public health threat. Its characteristic pathological features include granuloma formation and a systemic inflammatory response, primarily resulting from dysregulated host immune reactions. Therefore, host-directed therapy (HDT) is considered an important complement to conventional anti-TB treatment. PURPOSE: This study sought to examine the inhibitory effects of Saikosaponin A (SSA), an active compound extracted from Bupleurum, on Mtb-induced macrophage pyroptosis, as well as the underlying molecular mechanisms. METHODS: The effects of SSA on key molecules involved in pyroptosis and autophagy were examined in an in vitro model of Mtb-infected macrophages using Western blotting, ELISA, co-immunoprecipitation, and immunofluorescence assays. The function of histone deacetylase 6 (HDAC6) in modulating autophagy and pyroptosis in Mtb-infected macrophages was elucidated using gene silencing techniques. The SSA-HDAC6 interaction was validated using drug target identification methods such as molecular docking and site-directed mutagenesis. Furthermore, we established an in vivo model of lipopolysaccharide-induced pulmonary inflammation via intraperitoneal injection to assess whether SSA exerts a protective effect by inhibiting pyroptosis. RESULTS: In vitro experiments demonstrated that SSA enhanced autophagy to inactivate the NLRP3 inflammasome, thereby inhibiting Mtb-induced pyroptosis. Mechanistically, SSA interacted with HDAC6 and effectively suppressed its enzymatic activity. This interaction enabled SSA to target HDAC6, thereby modulating autophagy via the AMPK/mTOR/ULK1 axis, ultimately attenuating Mtb-induced pyroptosis in macrophages. Furthermore, in vivo experiments revealed that SSA regulated the acetylation of α-tubulin (Lys40), alleviating inflammatory lung injury in mice. CONCLUSION: SSA targets HDAC6 and exerts an immunomodulatory effect, highlighting its potential as a promising novel host-directed anti-tuberculosis agent.