Abstract
Background and
Purpose:Abnormal activation of the NLRP3 inflammasome in macrophages is closely
associated with Ulcerative colitis (UC), and targeting the NLRP3
inflammasome has been proposed as a potential therapeutic approach, but
the underlying mechanism by which it regulates intestinal inflammation
remains unclear. Anemoside B4 (AB4) has anti-inflammatory activity, but
whether it alleviates UC by inhibiting the activation of NLRP3
inflammasome remains unclear. More importantly, the molecular targets of
AB4 remain unknown.
Experimental Approach: We explored the role of AB4 in the
development of dextran sodium sulfate (DSS)-induced colitis in wild-type
(WT) mice and its effect on NLRP3 inflammasome. Next, we isolated
intestinal macrophages and epithelial cells, and validated them in
DSS-induced NLRP3-deficient (NLRP3-/-) mice. The
target and molecular mechanism of AB4 were identified in
lipopolysaccharides (LPS)-induced macrophages in vitro and DSS-induced
macrophage-specific CD1d depletion (CD1d-/-) mice in
vivo.
Key Results: This study showed that AB4 had a strong
anti-inflammatory effect DSS-induced colitis in WT mice, whereas the
protective effects were lost in
NLRP3-/-mice. Interestingly, AB4 inhibited the activation of NLRP3 inflammasome
in colonic macrophages without affecting intestinal epithelial cells.
Mechanistically,
AB4
might target CD1d thus reducing the AKT-STAT1-PRDX1-NF-κB signaling
pathway, eventually inhibiting the activation of NLRP3 inflammasome.
Macrophage-specific CD1d depletion had been shown to reverse the
protective effect of AB4.
Conclusions and
Implications: Our data showed that AB4 attenuated DSS-induced colitis
by inhibiting CD1d-dependent NLRP3 inflammasome activation in
macrophages. Therefore, as a natural product with
high
safety index, AB4 might be considered a promising candidate drug for the
treatment of colitis.
Introduction
Ulcerative colitis (UC) is a type of nonspecific inflammatory bowel
disease (IBD), which starts from the rectum and extends continuously to
proximal segments of the colon (Ge, Li, Gong, & Zhu, 2018). With the
rapid development of industrialization and modernization, the global
incidence of UC has also been rising continuously. The main pathological
lesions are mucosal ulcers, and the typical clinical symptoms are
diarrhea, rectal bleeding, and weight loss. Repeated episodes of UC
increase the cumulative risk of colorectal cancer (CRC) by 18-20%,
which undoubtedly brings serious mental burden and psychological
pressure to UC patients and seriously affects their normal life (Ge et
al., 2018; Torres et al., 2021). Although the exact cause of UC is
uncertain at present, the activation of the mucosal immune system and
the subsequent pathological cytokines play roles in the generation of UC
(Ge et al., 2018; Torres et al., 2021). At present, UC patients can only
receive long-term immunosuppressive and anti-inflammatory treatment,
such as glucocorticoids, immune-suppressants, biological agents, and
5-aminosalicylic acid (5-ASA), as well as even require surgery, which is
limited due to more side effects or high recurrence rate (de Lange &
Barrett, 2015; Kaplan, 2015). In this case, it is urgently needed to
develop highly effective drugs with fewer side effects, long-term
control ability of inflammation development, and stabilization of
intestinal microenvironment.
Macrophages are abundant in colon samples from UC patients and animal
models, which play an essential function in the occurrence, development
and resolution of inflammation (Moreira Lopes, Mosser, & Goncalves,
2020). Macrophages can respond to the damage-associated molecular
patterns (DAMPs) and the pathogen-associated molecular patterns (PAMPs),
enhance the recruitment, and activate other innate and adaptive immune
cells to amplify intestinal inflammation (de Lange & Barrett, 2015;
Moreira Lopes et al., 2020). The NLR family pyrin domain containing 3
(NLRP3) inflammasome is a multiprotein complex consisting of NLRP3, the
apoptosis-associated peck-like protein with CARD domain (ASC), and
Caspase-1. NLRP3 is a well-studied inflammasome, and numerous types of
research have revealed that the NLRP3 activation of macrophages plays an
important role in mediating UC inflammatory response (Hirota et al.,
2011; Moreira Lopes et al., 2020; Song et al., 2021; Zaki, Lamkanfi, &
Kanneganti, 2011). Activation of the NLRP3 inflammasome is mediated by
two key steps: Priming and assembling. (Hirota et al., 2011; Moreira
Lopes et al., 2020; Song et al., 2021; Zaki et al., 2011). The priming
step is mediated by activation of nuclear factor kappa-B (NF-κB)
signaling to up-regulate the transcription of inflammasome-related
proteins (NLRP3, pro-interleukin (IL)-1β and IL-18). The assembling
signal is induced by various triggers, such as adenosine 5′-triphosphate
(ATP), potassium (K+) efflux, mitochondrial reactive
oxygen species (mtROS), or lysosomal destabilization/rupture, induces
the assembly of NLRP3 inflammasome (Hirota et al., 2011; Song et al.,
2021; Zaki et al., 2011). Activation of NLRP3 inflammasome promotes the
cleavage of Caspase-1 as well as the maturation and secretion of
pro-inflammatory cytokines IL-1β and IL-18 (Hirota et al., 2011; Moreira
Lopes et al., 2020; Song et al., 2021; Zaki et al., 2011). Many studies
have confirmed that the inhibition of NLRP3-mediated IL-1β and IL-18
production in macrophages improves dextran sulfate sodium (DSS)-induced
inflammation (Bauer et al., 2010; Hirota et al., 2011; Sun et al.,
2015). Toward this end, searching for drug candidates targeting NLRP3
inflammasome activation is an effective anti-inflammatory strategy for
the potential treatment of UC.
Natural
products provide a new source of compounds for the treatment of UC due
to their abundant resources, definite efficacy, few side effects, and
low price (Gu, 2018; Newman & Cragg,
2020).Pulsatilladecoction (Bai-Tou-Weng-Tang, BTWT) is a classic Chinese herbal formula
for the treatment of intestinal bacterial diseases in humans (Gu, 2018).
In recent years, many basic studies have verified the anti-colitis
efficacy of BTWT (Canxing, Xinlong, Jian, Yue, & Xiaobo, 2017; Gu,
2018; Xiaomei et al., 2018). However, the active ingredient of BTWT’s
anti-colitis activity remains uncertain, which limits the discovery of
its biological mechanisms, and hinders the further translation of BTWT
into standard clinical application. Anemoside B4 (AB4) is a main natural
saponin component isolated from the root of Pulsatilla Chinensis,which can be
used
as a quality control index (Li, Zou, Han, Deng, & Weinshilboum, 2020).
Recently, AB4 has been shown to possess antibacterial, anti-diarrhea,
anti-inflammatory, anti-endotoxin, anti-tumor, and immunomodulatory (Han
et al., 2022; L. He et al., 2019; Li et al., 2020; Ma et al., 2020; Yuan
et al., 2020; Zhang et al., 2021). Therefore, we hypothesized that AB4
might be a main component of BTWT anti-colitis.
It
has been previously shown that AB4 protects against colitis by
regulating inflammatory responses such as NF-κB, IL-6, and tumor
necrosis factor (TNF)-α or gut microbiota (Han et al., 2022; Ma et al.,
2020; Zhang et al., 2021). However,
whether
AB4 alleviates UC by inhibiting the activation of NLRP3 inflammasome
remains unclear. What’s more, the molecular target of AB4 remains
unknown. In this study, we verified AB4’s protective effect on
DSS-induced colitis. The mechanistic study highlighted that AB4
inhibited NLRP3 inflammasome activation by targeting macrophage CD1d to
regulate AKT-STAT1-PRDX1-NF-κB signaling, thereby attenuating
DSS-induced colitis.