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.