Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia

Background Hepatic encephalopathy (HE) is a severe syndrome characterized by profound cognitive and neurological deficits and an increased risk of mortality. Unfortunately, targeted therapeutic interventions for this condition are lacking. ANXA1 is a pleiotropic cytokine that exhibits either proinflammatory or anti-inflammatory properties depending on the context; however, its involvement in the pathogenesis of HE has yet to be elucidated. Methods RNA sequencing data from both murine and human samples were used to investigate ANXA1 expression in HE. HE cell models were developed using the mouse microglial cell line BV2 and the endothelial cell line bEnd.3, and the model cells were cultured in the presence of ammonium chloride. A murine model of HE was established via intraperitoneal injection of thioacetamide (TAA), and the success of modeling was confirmed through behavioral, histological, and enzymatic assessments. The expression levels of ANXA1, along with those of cytokines and proteins associated with NLRP3 inflammasome activation, were analyzed via Western blotting and enzyme-linked immunosorbent assay (ELISA). The biologically active domain of ANXA1, AC2-26, was employed to investigate the effects of ANXA1 in HE in vitro and in vivo. Results We detected increased ANXA1 expression in microglia from HE model mice and brain tissues from cirrhosis patients. Microglia cultured under hyperammonemic conditions in vitro exhibited increased aNLRP3 inflammasome activation and increased secretion of inflammatory mediators, including ANXA1, TNF-α, IL-1β, and IL-6. Importantly, AC2-26 treatment effectively inhibited NLRP3 inflammasome activation and cytokine production by microglia, and this effect was diminished by the ANXA1 receptor antagonist Boc2, suggesting that ANXA1 plays a role in limiting microglia-mediated neuroinflammation by modulating NLRP3 inflammasome activation. In addition, microglia-derived cytokines reduced tight junction protein expression by endothelial cells under hyperammonemic conditions, indicating impaired blood‒brain barrier (BBB) function, which was ameliorated by AC2-26 treatment, suggesting that ANXA1 can protect BBB integrity in HE. Furthermore, we demonstrated that AC2-26 administration alleviated HE symptoms in a mouse model. Conclusion In conclusion, our findings indicate that ANXA1 counteracts NLRP3 inflammasome activation and mitigates neuroinflammation in HE, suggesting a potential therapeutic strategy for managing HE in the clinic.