Glycycoumarin ameliorates pyroptosis in autoimmune hepatitis by activating autophagy and reducing NLRP3 inflammasome activation

Objective: This study explored the protective effect of glycycoumarin (GCM) against autoimmune hepatitis (AIH) and its underlying mechanisms.

Methods: An AIH mouse model was established by concanavalin A induction and treated with GCM or methylprednisolone (MP) positive control. Liver index and serum levels of alanine aminotransferase, aspartate aminotransferase, and Lactate Dehydrogenase (LDH) were measured. Histopathological changes in liver tissues were observed via H&E staining. Pyroptosis-associated proteins and inflammatory cytokines in liver tissues were assessed by Western blot, RT-qPCR, and ELISA. In vitro, AML12 cells were stimulated with lipopolysaccharide and Nigericin to construct an inflammatory Pyroptosis model. Cell viability, LDH release, autophagic flux, pyroptosis-related protein expression, Caspase-1 activity, and mitochondrial function were assessed. To assess the autophagy-dependence of GCM's effect, cells were treated with 3-methyladenine, an inhibitor of Autophagy.

Results: In AIH mice, GCM treatment reduced liver index, serum transaminases, and LDH levels while alleviating hepatic necrosis and inflammatory infiltration. GCM downregulated NLRP3, Caspase-1, and GSDMD mRNA expression, reduced NLRP3, Cleaved Caspase-1, and GSDMD-NT protein expression, and suppressed IL-1β and IL-18 release in liver tissues of mice. In vitro, GCM improved cell viability, reduced LDH release, and inhibited Caspase-1 activation and Pyroptosis in LPS+NIG-induced AML12 cells. GCM restored autophagic flux, alleviated oxidative stress, and mitigated mitochondrial damage in vivo and in vitro. Autophagy inhibition partially counteracted the effects of GCM on promoting Autophagy, protecting against mitochondrial damage, and suppressing NLRP3 inflammasome-induced Pyroptosis.

Conclusion: GCM protects against AIH by restoring autophagic flux and mitigating oxidative stress, thereby suppressing NLRP3 inflammasome-mediated Pyroptosis.