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.