Figure 2. AB4 attenuates DSS-induced colon injury. Mice were pretreated with AB4 (5, 10, and 15mg/kg) for 7 days, followed by DSS-induced colitis, the serum, and the colons were collected on day 7. (A ) Measurement of serum permeability tracer FITC-dextran. (B ) The protein expression of Occludin, Claudin-1 and ZO-1 was detected by Western Blot. (C ) H&E staining analysis of histopathological changes (left) and semi-quantitative scoring of histopathology (right) and the images was taken at 200x magnification (scale bar: 50μm). (D ) The infiltration of F4/80+ macrophages in colonic tissues were detected by immunofluorescence, and the images were taken at 200x magnification (scale bar: 50μm). Data are presented as mean ± SD. **P < 0.01 vs. Normal group; #P < 0.05, ##P < 0.01 vs. DSS group.
AB4 specifically inhibits NLRP3 inflammasome activation in colonic macrophages
As previously reported, increased production of inflammatory cytokines in serum and colon is an important hallmark of DSS-induced colitis (de Lange & Barrett, 2015; Moreira Lopes et al., 2020). We examined the regulation of AB4 on the secretion of inflammatory cytokines in DSS-induced colitis. Indeed, AB4 (5, 10, and 15mg/kg) inhibited the secretion of pro-inflammatory cytokines secretion in the serum after the DSS challenge, such as IL-1β, IL-18, IL-6, inducible NOS (iNOS) and TNF-α (Fig. 3A). As an important component of innate immunity, NLRP3 inflammasome plays an important role in the development of UC, and is the main and key source of inflammatory cytokines IL-1β and IL-18. Targeting NLRP3 inflammasome has been shown to have a definite therapeutic effect (Hirota et al., 2011; Moreira Lopes et al., 2020; Song et al., 2021; Zaki et al., 2011). To investigate the regulatory role of AB4 on the NLRP3 inflammasome in DSS-induced colitis, we evaluated both mRNA and protein levels of related cytokines in collected colons. AB4 (5, 10, and 15mg/kg) exhibited significant inhibition on protein expression of NLRP3, ASC, Caspase-1 p20, IL-1β p17, and IL-18 in the colons of colitis mice (Fig. 3B). In parallel, AB4 significantly decreased the mRNA levels of NLRP3, Caspase-1, IL-1β, IL-18, IL-6 and TNF-α (Fig. 3C). IL-10 is a typical anti-inflammatory cytokine, and both IL-22 and IL-10 seem to maintain the integrity of the colonic epithelium (Huber et al., 2012; Q. Wu et al., 2021). We observed that AB4 enhanced the expression of IL-10 and IL-22 proteins in colonic homogenates of DSS-induced colitis, and enhanced the mRNA level of IL-10 (Fig. 3B and C). In agreement, we found that the colonic tissues from AB4 administration mice expressed high levels of proliferative cell nuclear antigen (PCNA) (Fig. 3B). Therefore, we hypothesized that AB4 might inhibit the expression of NLRP3 inflammasome and the release of inflammatory cytokines, thereby ameliorating impaired intestinal barrier function and alleviating DSS-induced colitis. To determine whether AB4-inactivated NLRP3 inflammasome was derived from macrophages or intestinal epithelial cells, we isolated these two types of cell lines from different groups of mice. Interestingly, Western Blot and ELISA results showed that AB4 significantly inhibited the protein expression of NLRP3, Caspase-1 p20, IL-1β and IL-18 in colonic macrophages (Fig. 3D), but did not affect the expression in intestinal epithelial cells (Fig. 3E).
To further confirmed that the relief of AB4 from DSS-induced colitis depended on the intervention of NLRP3 inflammasome, we verified it in DSS-induced NLRP3-knockout (NLRP3-/-) mice and WT mice. DSS-induced NLRP3-/-mice exhibited considerably less weight loss, lower DAI score (Fig. 4A and B), and longer colons presentation (Fig. 4C) as compared to WT mice, supporting a critical role of NLRP3 in the development of colitis. However, it was worth noting that the protective effect of AB4 on the DSS challenge was lost in NLRP3-/- mice (Fig. 4A-C). H&E staining showed that the epithelial damage of WT mice colon tissue was more severe and crypt loss than NLRP3-/-mice and AB4 improved the damage and crypt loss of colon tissue in WT mice but had no significant effect on NLRP3-/- mice (Fig. 4D). These data suggested that inhibition of NLRP3 inflammasome activation might be one of the main mechanisms by which AB4 attenuated DSS-induced inflammatory injury in colitis.