Discussion
In this study, we demonstrated that AB4 of the three Pulsatilla
saponins AB4, AA3, and 23-HA had the strongest inhibition rate of IL-1β
secretion in macrophages. To assess the anti-inflammatory activity of
AB4, we constructed DSS-induced colitis. We notably found that the AB4
pretreatment group was more effective than the AB4 treatment group and
the 5-ASA positive group. AB4 reduced the severity of colitis in WT mice
by inhibiting the activation of NLRP3 inflammasome and promoting the
balance of inflammatory factors and repair of intestinal epithelial
damage. In contrast, it lost its ability to alleviate DSS-induced
colitis in NLRP3-/- mice. Then, we were surprised to
find that AB4 inhibited NLRP3 inflammasome activation in colonic
macrophages, but not in 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. Together these data indicated that AB4
attenuated DSS-induced colitis by inhibiting
CD1d-dependent
NLRP3 inflammasome activation in macrophages.
Ulcerative
colitis (UC) is a chronic, non-specific, non-infectious, inflammatory
intestinal disease mediated by abnormal immunity caused by multiple
etiological factors (de Lange & Barrett, 2015; Ge et al., 2018; Kaplan,
2015; Torres et al., 2021). UC has become a huge burden to human life
due to its repeated course of the disease, difficult to cure and easy to
cause cancer (de Lange & Barrett, 2015; Ge et al., 2018; Kaplan, 2015;
Torres et al., 2021). During the novel Corona Virus Disease 2019
(COVID-2019) pandemic, the clinical management of UC has always been an
area of high concern for patients and physicians around the world. UC
patients have changed their potential immune response, which may make
them more susceptible to infection (Peng et al., 2020). The standard
treatment for patients with UC is to receive long-term immunosuppressive
and anti-inflammatory therapy, however, this can lead to severe side
effects that limit long-term use (de Lange & Barrett, 2015). Therefore,
there is an urgent need for drugs with high efficacy, low cost and few
side effects.
Pulsatilla decoction (Bai-Tou-Weng-Tang, BTWT) is a famous
Chinese medicine prescription for intestinal diseases caused by
inflammation. The main component of BTWT isPulsatilla
chinensis (Gu, 2018). It contains a large number of triterpenoids
saponins and is considered to be its main active ingredient (Li et al.,
2020; Ma et al., 2020; Zhang et al., 2021). Among them, the contents of
AB4, AA3, and 23-HA are higher (Ip et al., 2017; Wang, Lin, Lin, Yao, &
Zhang, 2019; Yao et al., 2018; Ye & Ji, 2012). In order to further
clarify the main active ingredients of BTWT anti-colitis efficacy. In
this study, we demonstrated that AB4 of the three Pulsatilla
saponins AB4, AA3, and 23-HA had the strongest inhibition rate of IL-1β
secretion in macrophages. Recent studies had shown that AB4 reduced the
levels of inflammatory cytokines IL-1β, IL-6, TNF-α, or NF-κB to
alleviate colitis (Ma et al., 2020; Zhang et al., 2021). To evalute the
anti-inflammatory activity of AB4, we investigated the role of AB4 in
3.0%DSS-induced colitis. The data showed that AB4 (5mg/kg) could
attenuate the severity of colitis in WT mice. Notably, we first found
that the AB4 pretreatment group was more effective than the AB4
treatment group and 5-ASA positive group. Interestingly, we found that
AB4 (5, 10, and 15mg/kg) had a significant dose-dependent effect in
reducing the severity of colitis. Considering drug safety, we found no
significant difference between AB4 (15mg/kg) alone group and the normal
group. In addition, studies had shown that the median lethal dose (LD50)
of AB4 after intravenous injection in mice was 3.36g/kg (Qin et al.,
2019), and the intravenous infusion dose was 2.5g/kg for 14 consecutive
days, with no significant toxic changes such as body weight, liver, and
kidney
function of mice were detected (L. He et al., 2019).
These
data indicated that AB4 had a relatively high safety index and no
adverse reactions had been detected to date. Therefore, as a natural
product with high safety index, AB4 might be a promising drug candidate
for the treatment of colitis. But the exact molecular mechanism remains
unclear.
Activation of the NLRP3 inflammasome plays an important role in
mediating the inflammatory response in UC (Qu et al., 2021; Zmora, Levy,
Pevsner-Fishcer, & Elinav, 2017). Increasing evidence confirms that
blocking NLRP3 inflammasome activation in macrophages is a novel
strategy to block inflammatory and immune responses (Lv et al., 2021;
Zhao et al., 2019). In this paper, we found that the protective effect
of AB4 on DSS-induced colitis in mice was attributable to the inhibition
of NLRP3 inflammasome activation and subsequent stimulation of colon
epithelial cell proliferation, local IL-22 and IL-10 expression. At the
same time, we verified DSS-induced colitis in NLRP3-/-and WT mice, and we found that AB4 lost its protective effect in
NLRP3-/- mice. This confirmed that the protection of
AB4 against DSS-induced colitis depended on the intervention of NLRP3
inflammasome. However, it had been demonstrated that activation of the
NLRP3 inflammasome in intestinal epithelial cells led to the secretion
of IL-18 and contributed to ameliorating intestinal epithelial barrier
dysfunction (Dupaul-Chicoine et al., 2010). Dupaul-Chicoine showed
administration of exogenous recombinant IL-18 could improve the
inflammatory symptoms of DSS-induced colitis, and the colitis was more
severe in NLRP3-/- mice than in WT
mice
(Dupaul-Chicoine et al., 2010). Thus, drugs that selectively inhibit
NLRP3 inflammasome activation in colonic macrophages but not intestinal
epithelial cells have the potential to treat colitis. Our data strongly
suggested that AB4 inhibited the activation of NLRP3 inflammasome in
colonic macrophages, but not in intestinal epithelial cells. Therefore,
we first found that AB4 selectively inhibited the activation of NLRP3
inflammasome in colonic macrophages to attenuate DSS-induced colitis.
Although various stages of signaling involved in NLRP3 inflammasome
activation have been studied, NLRP3 expression is considered to be an
important factor in its associated inflammatory mechanisms (Hirota et
al., 2011; Moreira Lopes et al., 2020; Song et al., 2021; Zaki et al.,
2011). Many regulatory mechanisms had been shown to inhibit NLRP3
inflammasome signaling, the most classic being the activation of the
NF-κB signaling pathway (Guo et al., 2020; Schreiber et al., 1998).
NF-κB plays a key role in the pathogenesis of colon immune cell
infiltration in UC patients and experimental colitis models (Guo et al.,
2020). Our study confirmed that AB4 significantly inhibited the NF-κB
signaling pathway, showing down-regulated expression of p-P65/P65 and
p-IκB/IκB, which is synergistic with NF-κB inhibitor JSH-23. TLR4 can
recognize LPS-activated downstream transcription factor signals and
induce transcription expression of inflammatory genes (Sheng et al.,
2021). Notably, we found that AB4 was not associated with the activation
of the NLRP3 inflammasome signaling pathway involved in TLR4. However,
this contradicts previous research (Zhang et al., 2021). The difference
in conclusions might be caused by the difference in administration
concentration, action time, mouse background, and intestinal microbe.
PRDX1
is a peroxidase reductase that plays an important regulatory role in
reactive oxygen species scavenging, cell proliferation, differentiation,
apoptosis, and inflammation (Y. He et al.,
2019).
A
recent study showed that PRDX1 expression was increased in DSS-induced
colitis, and silencing PRDX1 expression inhibited
DSS-induced
inflammation and apoptosis, thereby ameliorating colonic injury in rats
(N. Wu et al., 2021). Hansen demonstrated that extracellular PRDX1
promoted the activation of NF-κB by inducing the phosphorylation of IκBα
(Hansen, Moriarty-Craige, & Jones, 2007). In this study, we found that
AB4 inhibited PRDX1 protein expression in LPS-challenged macrophages.
AKT signaling pathway is involved in the regulation and release of
pro-inflammatory cytokines, plays an important role in the occurrence
and development of UC, and can mediate the expression of LPS-induced
PRDX1 (Bast, Fischer, Erttmann, & Walther, 2010; Q. Wu et al., 2021).
Considering the inhibitory effect of AB4 on PRDX1, we further
investigated its effect on AKT signaling. Western Blot confirmed that
AB4 inhibited AKT/STAT1 signaling pathway synergically with AKT
inhibitor MK2206. Moreover, AKT agonist SC79 reversed the inhibitory
effect of AB4 on the AKT-STAT1-PRDX1-NF-κB-NLRP3 signaling pathway.
Similarly, in vivo studies also confirmed that AB4 inhibited NLRP3
inflammasome activation through the AKT-STAT1-PRDX1-NF-κB signaling
pathway.
The molecule CD1d has the antigen-presenting effect and is a member of
the glycoprotein CD1 family. The homology between human CD1d and mouse
CD1d1 is more than 95% (Ishii et al., 1993; Olszak et al., 2014; Sheng
et al., 2021). Multiple studies have confirmed that CD1d-related immune
pathways have an important effect on UC (Fuss et al., 2004; Huang et
al., 2016; Lee et al., 2019; Mizoguchi et al., 2002; Olszak et al.,
2014). It had been reported that CD1d-/- mice were
more sensitive to DSS-induced colitis, CD1d expressed in colonic
intestinal epithelial cells of mice binds to the exogenous glycolipid
ligand α-galactothenamide (α-GalCer) and induced the activation of NKT
cells in a CD1d restrictive manner, thus alleviating DSS-induced colitis
(Saubermann et al., 2000). Moreover, CD1d could transmit interactive
signals that trigger CD1d expressing intestinal epithelial cells to
produce anti-inflammatory cytokine IL-10 and heat shock protein 110
(HSP110) to relieve DSS-induced colitis in mice (Olszak et al., 2014).
Targeting this mechanism may help improve the treatment of UC and
prevent colitis-related colorectal cancer. A recent work had
demonstrated that CD1d1 negatively regulated the expression of NLRP3
inflammasome (Cui et al., 2020). Therefore, we hypothesized that AB4
might play a protective role in colitis by regulating the NLRP3
inflammasome through the CD1d signaling pathway. First, we performed
molecular dynamics simulations to assess the stability of AB4 and CD1d.
The results showed that AB4 was successfully fitted to the catalytic
domain and formed multiple interactions with the internal residues of
CD1d (data not shown). Next, we constructed 3.0% DSS-induced colitis in
WT mice and found that AB4 significantly enhanced the expression of CD1d
protein in colon tissue and colon macrophages. Surprisingly,
macrophage-specific CD1d depletion had been shown to reverse the
protective effects of AB4 on the NLRP3 inflammasome and
DSS-induced
colitis,
which
validated that the CD1d-dependent NLRP3 axis was a preferential
signaling pathway. These results demonstrated for the first time that
AB4 might trigger the endogenous negative signaling of CD1d, inhibiting
the expression of NLRP3 inflammasomes through the AKT-STAT1-PRDX1-NF-κB
signaling cascade, and alleviating DSS-induced colitis in mice. As CD1d
is an MHC-like transmembrane protein, it contains 336 amino acids and 10
amino acids in the cytoplasmic tail. Therefore, whether AB4 directly
targets CD1d or indirectly targets CD1d, as well as the specific amino
acid sites are worth further investigation.
In summary, our study confirmed that AB4 alleviated inflammatory damage
in DSS-induced colitis by reducing the expression of inflammatory
factors and improving intestinal barrier damage. Notably, we found for
the first time that CD1d might be a therapeutic target for AB4. AB4
targets macrophages CD1d thus to reduce AKT-STAT1-PRDX1-NF-κB signaling
cascade, eventually inhibiting the activation of NLRP3 inflammasome and
ameliorating DSS-induced colitis (Fig. 8).
As
the main active component of BTWT, AB4 lays the groundwork for a better
understanding of BTWT’s clinical efficacy from an active ingredient
perspective, and also lays a theoretical foundation for the future
systematic study of the corresponding mechanism of action of BTWT. More
importantly, new resources have been provided for the treatment of UC.
However, the comprehensive safety evaluation and treatment optimization
of AB4 in the clinical application is worthy of further study.