[Mechanism of Chuanshanlong Compound in regulating pyroptosis in gouty arthritis via NLRP3/GSDMD signaling axis]

This study focused on the NOD-like receptor protein 3(NLRP3)/exfoliating agent(GSDMD) signaling axis to investigate the regulatory mechanisms of Chuanshanlong Compound(CCS) on pyroptosis in gouty arthritis(GA). Phorbol 12-myristate 13-acetate(PMA) was used to induce differentiation of human myelomonocytic leukemia cells(THP-1) into macrophages, followed by the establishment of a monosodium urate(MSU) crystal stimulation model. Cells were divided into a blank control group, a model group, a colchicine group, and CCS groups at low, medium, and high doses. ELISA was used to measure the levels of inflammatory cytokines, including interleukin(IL)-1β, IL-18, and tumor necrosis factor(TNF)-α, in the cell supernatant. Flow cytometry was employed to investigate the effects of CCS on macrophage pyroptosis via the NLRP3/GSDMD signaling axis. Western blot analysis was performed to detect the expression of NLRP3, apoptosis-related speck-like protein containing a CARD(ASC), caspase-1, the N-terminal domain of gasdermin D(GSDMD-N), and IL-1β in the supernatant of THP-1 macrophages. Immunofluorescence double staining was used to observe the colocalization of NLRP3 and GSDMD. Co-immunoprecipitation(Co-IP) was performed to validate the protein-protein interaction(PPI) between NLRP3 and GSDMD. Western blot analysis was conducted to assess the effects of the high-dose CCS on the expression levels of NLRP3, caspase-1, and GSDMD-N in NLRP3 and GSDMD overexpression models. ELISA results showed that the high-dose CCS significantly reduced the levels of IL-1β, IL-18, and TNF-α in the cell supernatants. Flow cytometry analysis indicated that the high-dose CCS reduced the proportion of pyroptotic cells induced by MSU, with an effect comparable to that of NLRP3 inhibitor(MCC950) or GSDMD inhibitor(LDC7559). Western blot analysis revealed that the high-dose CCS significantly downregulated the expression levels of NLRP3, ASC, caspase-1, GSDMD-N, and IL-1β. Immunofluorescence double staining and Co-IP further demonstrated that the high-dose CCS attenuated the colocalization and PPI of NLRP3 and GSDMD. In the overexpression models, the high-dose CCS similarly reversed the exacerbation of pyroptosis induced by elevated NLRP3 or GSDMD levels. In summary, CCS reduces the generation of pyroptotic cells by blocking the NLRP3/GSDMD positive feedback loop, thereby inhibiting pyroptosis and alleviating GA.