Investigation of the mechanism of NLRP3/GSDMD signaling axis regulating GA cell pyroptosis based on in vivo and in vitro experiments

Abstract Objective:This study investigated the regulatory mechanism of the NLRP3/GSDMD pathway in GA pyroptosis using network pharmacology and in vitro and in vivo experiments. Methods:In this study, network pharmacology was used to predict the mechanism of pyroptosis in Gouty arthritis, and HE, ELISA, Real-time PCR, Western blot, immunoprecipitation, and flow cytometry were used to further investigate the mechanism of NLRP3/GSDMD signal axis in GA rat model induced by MSU and THP-1 cells induced by MSU. Results:When gout arthritis pyroptosis occurred, the NOD-like receptor pathway was activated, as determined by network pharmacology. In the rat model of GA induced by MSU, the swelling degree, pathological damage, serum biochemical indices, and inflammatory factors of the joints were significantly increased, and the expressions of key pyroptosis proteins such as NLRP3, GSDMD, ASC, Caspase-1, and IL-1β were significantly increased in ankle joints, indicating the existence of a NOD-like receptor pathway mediating pyroptosis during the acute attack of GA. The results of NLRP3 and GSDMD inhibitor intervention showed that inhibitor intervention significantly inhibited the expression of pyroptosis protein. At the same time, it was found that the activation of NLRP3 inflammatory bodies was inhibited, and the expressions of GSDMD, ASC, Caspase-1 and IL-1β protein were down-regulated, but the expression of NLRP3 protein was not affected by inhibiting the activation of GSDMD and down-regulating the expression of IL-1β. The results of the cell experiment revealed that the inhibitor group prevented MSU-induced Nod-like receptor-mediated pyroptosis in THP-1. Co-precipitation results also revealed a close interaction between NLRP3 and GSDMD. Conclusion:Inhibiting the NLRP3/GSDMD signaling axis can further reduce the occurrence of pyroptosis and thus alleviate the inflammatory response in acute Gouty arthritis attacks. Through a Caspase-1-dependent mechanism, NLRP3 regulates the process of pyroptosis in the occurrence of Gouty arthritis.