AB4 inhibits the activation of NLRP3 inflammasome in vitro
Next, we examined whether AB4 could regulate the activation of NLRP3 inflammasome in macrophages in vitro. BMDMs were pretreated with AB4 (50, 100, and 200μM) for 4h, and then treated with LPS (1μg/ml) for 6h. Meanwhile, CCK-8 results showed that 50-200μM AB4 had no toxic effects on BMDMs (Supporting Information Fig. S3). The mRNA expression of related components in NLRP3 inflammasome was analyzed by qPCR. As shown in Fig. 5A, AB4 (50, 100, and 200μM) significantly inhibited the mRNA expressions of NLRP3, IL-1β, and IL-18 in LPS-primed BMDMs. By contrast, the expression of ASC and Caspase-1 were unaffected by AB4. Moreover, AB4 (50, 100, and 200μM) could significantly inhibit the protein expression of NLRP3, proIL-1β, and IL-18 (Fig. 5B). These data suggested that AB4 might partially regulate NLRP3 inflammasome activation by inhibiting the transcription of NLRP3, pro-IL-1β and IL-18 genes in macrophages. The constituent proteins of the NLRP3 inflammasome are widely considered to be the rate-limiting point regulating inflammasome activation (Song et al., 2021). Western Blot confirmed that AB4 (50, 100, and 200μM) significantly inhibited the protein expressions of NLRP3, Caspase-1 P20, IL-1β p17 and IL-18 in LPS-primed BMDMs (Fig. 5C) and differentiated THP-1 cells (Fig. 5D) in response to ATP (5mM;30min) or nigericin (Nig;10μM;30min), suggesting inactivation of NLRP3 inflammasome by AB4. Consistently, ELISA results also confirmed that IL-1β and IL-18 in the supernatants were suppressed by AB4 in LPS-primed BMDMs and differentiated THP-1 cells in response to nigericin (Fig. 5E). These data suggested that AB4 might also affect the NLRP3 inflammasome assembly stage, namely reducing NLRP3-mediated Caspase-1 activation and inhibiting macrophage IL-1β and IL-18 secretion. Taken together, AB4 inhibited the activation of NLRP3 inflammasome in vitro.