AB4 inactivates AKT-STAT1-PRDX1-NF-κB signaling in macrophages
Subsequently, we explored how AB4 regulated NLRP3 inflammasome
activation. NF-κB is a key activator of inflammation, which primes the
activation of NLRP3 inflammasome by promoting the transcription of
NLRP3, IL-1β, IL-18 (Schreiber, Nikolaus, & Hampe, 1998). First, we
assessed the effects of AB4 on NF-κB signaling in vitro. the results
showed AB4 (50, 100, and 200μM) significantly decreased the protein
expression of
NF-κB
p65 phosphorylation and nuclear factor κB (IκBα) phosphorylation in
LPS-challenged BMDMs (Fig. 6A) and differentiated THP-1 cells (Fig. 6B),
indicating an inhibitory action of AB4 on NF-κB signaling. Meanwhile,
inhibition of
NF-κB
P65 and IκBα phosphorylation with NF-κB inhibitor JSH-23 (25μM;10h) also
attenuated LPS-challenged up-regulation of NLRP3, proIL-1β, and IL-18,
both at protein (Fig. 6C) and mRNA (Fig. 6D) levels in BMDMs, which is
synergistic with AB4. These data indicated that the classical NF-κB
signaling pathway mediated AB4-dependent inhibition of NLRP3, IL-1β, and
IL-18. TLR4 can recognize the downstream transcription factor signals
initiated by LPS and cause the transcriptional expression of
inflammatory genes (Sheng et al., 2021). However, our result showed that
AB4 was not associated with TLR4-involved activation of NLRP3
inflammasome signaling (Supporting Information Fig. S4).
Next, we explored the direct signaling events of NF-κB inactivation by
AB4. Peroxiredoxin 1 (PRDX1), a protein capable of promoting NF-κB
activation by inducing IκBα phosphorylation, is considered a competitive
molecule for the transcriptional control of inflammatory genes (Cui et
al., 2020; Ishii et al., 1993).Western Blot confirmed that
AB4
(50, 100, and 200μM) significantly reduced LPS-challenged PRDX1 protein
expression in BMDMs (Fig. 6A) and differentiated THP-1 cells (Fig. 6B).
Studies had shown that LPS-dependent PRDX1 expression was mediated by
Protein kinase B (AKT)/signal transducer and activator of transcription
1(STAT1) signaling (Cui et al., 2020). In agreement, AB4 (50, 100, and
200μM) significantly reduced phosphorylation of AKT and STAT1 in
LPS-challenged BMDMs (Fig. 6A) and differentiated THP-1 cells (Fig. 6B).
Western Blot confirmed that AKT inhibitor MK2206 (20μM; 10h) effectively
inhibited LPS-challenged AKT and STAT1 phosphorylation, resulting in the
reduction of PRDX1 expression, phosphorylation of IκBα and P65, and the
down-regulation of NLRP3, proIL-1β, and IL-18 (Fig. 6E). By contrast,
AKT agonist SC79 (20μM; 12h) could reverse the inhibitory effect of AB4
on LPS-challenged p-AKT, p-STAT1, PRDX1, p-P65, p-IκBα, NLRP3, proIL-1β
and IL-18 proteins in BMDMs (Fig. 6F). In keeping with this, we also
demonstrated that AB4 (5, 10, and 15mg/kg) could reduce the expression
of p-AKT/AKT, p-STAT1/STAT1, PRDX1, p-P65/P65, p-IκBα/IκBα proteins in
colonic homogenates of 3.0% DSS-induced WT mice
(Fig. 6G). These data suggested that
AB4 might inhibit NLRP3 inflammasome activation by inactivating NF-κB by
inhibiting AKT/STAT1-mediated PRDX1 expression.