Qijia Rougan Formula alleviates liver fibrosis by inhibiting NLRP3-mediated pyroptosis and regulating macrophage polarization.

BACKGROUND: Stage F3 liver fibrosis represents a critical phase for intercepting the progression toward irreversible cirrhosis. Despite its clinical significance, effective interventions remain scarce. Clinical observations indicate that the traditional Chinese medicine (TCM) formula Qijia Rougan Formula (QRF) exhibits pronounced anti-fibrotic efficacy specifically at Stage F3. Preliminary data suggest that QRF may exert immunomodulatory and anti-fibrotic effects by targeting NLRP3 inflammasome/GSDMD axis. Its precise mechanistic underpinnings warrant further elucidation.

METHODS: C57BL/6 mice were administered CCl4 to establish stage F3 model. Following a 6-week therapeutic regimen, hepatic structural integrity and functional recovery were assessed. A multi-tiered investigative strategy was employed: molecular docking predicted ligand-protein interactions, while transcriptomic profiling identified differentially expressed genes (DEGs). In vitro, LPS/ATP-primed macrophages were treated with QRF-medicated serum. The ultrastructural hallmarks of pyroptosis were visualized using transmission electron microscopy (TEM). A functional rescue experiment was performed using NLRP3-specific agonist Nigericin (NIG) to establish causality.

RESULTS: High-dose QRF significantly ameliorated hepatic dysfunction and histopathological injury in F3 model mice. Integrated in molecular docking and transcriptomic analysis identified the NLRP3-mediated pyroptotic pathway as QRF's primary therapeutic target. Specifically, QRF suppressed NLRP3, Caspase-1, and GSDMD expression, thereby curtailing pro-inflammatory cytokines (IL-1β, IL-18) and LDH release. Concurrently, QRF facilitated a macrophage phenotypic switch from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. TEM confirmed that QRF preserved membrane integrity and mitigated organelle damage. Notably, the anti-pyroptotic efficacy of QRF was abolished by NIG, confirming its dependence on NLRP3 inhibition.

CONCLUSIONS: QRF exerts significant anti-fibrotic activity in stage F3 fibrosis by inhibiting NLRP3/Caspase-1/GSDMD mediated macrophage pyroptosis. By quenching this pathway, QRF curtails the inflammatory cascade and promotes M1-to-M2 macrophage polarization, thereby optimizing intrahepatic immune microenvironment and retarding fibrotic progression. Utilizing the reliable established F3 animal model, these findings systematically elucidate the QRF's key targets and provide experimental foundation for developing stage-specific therapeutic strategies.