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.