Pulchinenoside B4 alleviates DSS-induced colitis by inhibiting CD1d-dependent NLRP3 inflammasome activation in macrophages.

Ulcerative colitis (UC) represents a significant challenge to global health, underscoring the importance of developing novel alternative anti-colitis agents. Inhibition of the NLRP3 inflammasome in macrophages has emerged as a potential therapeutic strategy for UC. Pulchinenoside B4 (PB4) is a major component of traditional medicinal plants that demonstrated to possess promising anti-inflammatory properties. The aim of the present study was to assess whether PB4 alleviates dextran sodium sulfate (DSS)-induced colitis by inhibiting the NLRP3 inflammasome in macrophages and its potential molecular mechanism. We constructed DSS-induced colitis in C57BL/6 mice, and isolated mouse intestinal macrophages and epithelial cells to investigate the effect of PB4 on NLRP3 inflammasome, and confirmed our findings in DSS-induced NLRP3 mice. In addition, we constructed lipopolysaccharides (LPS)-induced macrophages in vitro and identified the target and molecular mechanism of PB4 through biolayer interference (BLI) and cell thermal migration (CETSA) in conjunction with dss induced macrophage-specific CD1d depletion (CD1d) colitis. This study showed that PB4 had a strong anti-inflammatory effect on WT mice induced by DSS, but the protective effect on NLRP3 mice was no longer enhanced. Interestingly, PB4 inhibited the activation of NLRP3 inflammasome in colon macrophages without affecting intestinal epithelial cells. Mechanistically, PB4 may target CD1d, thereby reducing the AKT-STAT1-PRDX1-NF-κB signaling pathway and ultimately inhibiting the activation of the NLRP3 inflammasome. Macrophage-specific CD1d loss has been shown to reverse the protective effects of PB4. These findings have paved the way for the development of CD1d/NLRP3-based novel anti-colitis agents and will facilitate the future clinical translation of the plant-derived drug PB4.