4 Discussion
In the present study, it was firstly
demonstrated that TBⅡ can repress NLRP3 expression via suppressing NF-κB
signaling, an upstream event of the
key transcriptional regulator of NLRP3, and thereby mitigating
DSS-induced intestinal permeability and inflammatory response.
Consequently, the DSS-induced colonic injury and colitis were alleviated
partially through the inhibition of NLRP3-mediated the mutual crosstalk
between epithelial cells and macrophages (Figure 9). Our study
discovered that TBⅡ is a novel inhibitor of NLRP3, thus making it as a
promising candidate compound for treating UC.
Ulcerative colitis is an inflammatory bowel disease, and its incidence
is increasing year by year. Colitis occurs in all age groups and is most
common in young people, leading to lifelong morbidity and even mortality
(Mai, Wu, Wang, Su, Cheng & Zhang, 2019). TBⅡ has been reported to have
a variety of pharmacological activities, and in this study, we confirmed
the protective effect of TBⅡ against DSS induced UC in mice. The main
clinical manifestations of UC include weight loss, shortened colon,
diarrhea, and hematochezia (Krausova et al., 2021). These parameters
could be used to assess the severity of UC. The DSS-induced colitis
model mimics, to some extent, simulated the key immune and
histopathological features of human colitis (Li et al., 2019).
Interestingly, we found that TBⅡ significantly improved the weight loss,
diarrhea, intestinal bleeding, and colon shortening in mice induced by
DSS. Oxidative stress played an
important role in the occurrence or development of colitis (Liu et al.,
2019). In vivo studies showed that TBⅡ significantly increased SOD and
GSH content and decreased MDA content in colon tissue. According to H&E
staining images, DSS caused colonic tissue ulcers involving in the
submucosa, and inflammatory cells infiltrated into the submucosa. TBⅡ
alleviated this inflammation. These data confirmed that TBⅡ could
relieve colitis induced by DSS.
In the pathogenesis of UC, one of the key features of DSS-induced
colitis in mice resembling human UC is associated with the disruption of
intestinal epithelial barrier that enables for electrolyte and nutrient
absorption, but constraints intestinal pathogens entering the body
(Gasaly, de Vos & Hermoso, 2021). The damaged intestinal epithelial
barrier is subsequently caused the enhanced intestinal permeability and
inflammatory infiltration, and deteriorated the intestinal tumor
microenvironment (Xu et al., 2021), suggesting that intestinal mucosal
barriers play an important role in protecting homeostasis against the
chaotic invasion of many antigens from the external environment (Gasaly,
de Vos & Hermoso, 2021). Therefore, recovering of the damaged
intestinal mucosal barrier represents a crucial step for UC
therapeutics. The intestinal mucosal barrier is strictly regulated by
the intact integrity of the intestinal epithelium which is arranged by a
monolayer of intestinal epithelial cells (Turner, 2009). To maintain the
intestinal barrier, the intestinal epithelial cells can be connected
with each other through tight junctions, which are composed of several
intestinal barrier proteins, such as occludins, cadherins, claudins, and
are fastened to the cytoskeleton via zonula occludens (ZO)-1(Spalinger
et al., 2020; Turner, 2009). Deficiency of these key proteins was
connection with the promoted intestinal permeability and damaged
function of intestinal epithelial barrier in IBD(Grill, Neumann,
Hiltwein, Kolligs, Schneider & sciences, 2015; Krishnan & McCole,
2017; Luissint et al., 2019; Mir et al., 2016). Consistently, we found
that the DSS-induced the expression of tight junctions protein
(E-CADHERIN and OCCLUDIN) was
decreased, while the serum FITC-dextran levels was increased,
demonstrating a damaged intestinal epithelial barrier was generated in
DSS-induced colitis mice. The damaged intestinal epithelial barrier was
accompanied with the elevated intestinal permeability, and subsequently
led to increased activity of MPO in plasma and colon tissues, and
infiltration of F4/80+ macrophages in colon tissues, exacerbating the
progression of colitis. Similar to CNCM I-3690 report (Laval et al.,
2015), as expected, TBⅡ treatment can reverse the mentioned-above
effects via enhancing the colonic expression of E-CADHERIN and OCCLUDIN,
thereby reducing intestinal permeability and inflammatory infiltration.
Next, to address the underlying mechanism how TBⅡ alleviates DSS-induced
the injured intestinal barrier, RNA-seq was performed and the results
revealed that several inflammatory pathway signaling including Leukocyte
transendothelial migration and NOD-like receptor pathways were
profoundly enriched in TBⅡ-treated colitis mice by KEGG pathway
analysis. Among these pathway signaling, NOD-like receptor pathways was
particularly attracted our interesting, because its activation was
strongly involved in the damaged intestinal barrier and development of
IBD (Li, Liu, Wang, Liu, Xu & chemistry, 2023). As for the most
extensive study of NOD-like molecule, NLRP3 plays a pivotal role in the
inflammation development and colitis progression. Enhanced expression
was observed in the inflamed colon tissue of IBD patients and
DSS-induced the colitis (Liu et al., 2017). Apparently, its activation
contributed to deterioration of DSS-induced IBD, while its deficiency or
pharmacological inhibition exerted a prominent amelioration(Liu et al.,
2023). Indeed, we also observed an increased expression of NLRP3 in
colitis mice and LPS+ATP-treated HCoEpiC epithelial cells (Figure.3 and
Figure 4E), an effect that was considerably reversed by TBⅡ treatment,
and thereby reducing the generation of NLRP3-mediated pro-inflammatory
cytokines (eg. IL-1β,TNF-α). Our results revealed an essential role of
NLRP3 in TBⅡ-mediated intervention, because we observed that both
MCC950-mediated NLRP3 pharmacological inhibition/adenovirus-mediated
NLRP3 overexpression in cell models and NLRP3 deletion in DSS-induced
colitis mice model partly impaired TBⅡ-mediated the protective effects
against LPS+ATP/DSS-induced inflammation, respectively, which is agreed
with Simiao Qiao results (Qiao et al., 2020). Accordingly, TBⅡ-mediated
NLRP3 inhibition contributed to the decrease of the release of
pro-inflammatory cytokines, and consequently resulting in mitigating
colitis/inflammation-associated damaged function of intestinal barrier
via reinforcing the tight junctions (eg. enhancing E-CADHERIN and
OCCLUDIN expression) (Figure. 4 A and B ).
Damaged intestinal barrier can accelerate the input of intestinal
pathogens, penetrate into mucous lamina propria, induce immune cell
recruitment, and consequently cause intestinal inflammation (Ordás,
Eckmann, Talamini, Baumgart & Sandborn, 2012). Moreover, amplified
inflammatory infiltration in the
colon resulted in enhanced colon permeability, and further speeded up
the disruption of intestinal
epithelial barrier (Gadaleta et al., 2011). Indeed, except for the
intestinal epithelial cells themselves, a number of increasing evidences
demonstrated that the communication
between epithelial cells and macrophages also contributed to promote the
disrupted function of intestinal barrier and inflammation(Spalinger et
al., 2020). Since TBⅡ could suppress NLRP3-triggered inflammation and
reverse the elevated colon permeability and injured intestinal barrier,
indicating a possibility that the protective effect of TBⅡ is likely
associated with the regulation of mutual crosstalk between colonic
epithelial cells and macrophages via probably the inhibition of
NLRP3-dependent mechanism. To examine this hypothesis, CM-associated
culture was performed. We found that restoring the intestinal barrier
via enhancing the tight junctions in colonic epithelial cells after TBⅡ
treatment can significantly repress CM-associated the inflammation in
THP-1 cells, and vice versa (Figure 5). However, co-treatment TBⅡ with
NLRP3 inhibitor MCC950 can, in part, impair the anti-inflammatory effect
and the protective effect of intestinal barrier (Figure 5). Our study
indicates that the ameliorative effect of TBⅡ in colitis mice may be
associated with the blockage of the interaction between colonic
epithelial cells and macrophages, probably in a the pattern of NLRP3
inhibition mechanism.
Under normal physiological conditions, the expression of NLRP3
inflammasome proteins, a multi-protein complexes, which is comprised
NLRP3 itself, the adaptor ASC and pro-caspase-1, is constitutively low
in the colonic epithelial cells and macrophages of WT mice (Sun, Ouyang,
Zeng & Wu, 2022). However, abnormal elevated activation of NLRP3
inflammasome was clearly observed in the colon of IBD patients,
DSS-induced colitis mice and IL-10
knockout mice (Liu et al., 2017). The pathogenic role of NLRP3
inflammasome activation in inflammatory bowel diseases of both mice and
humans, even if the underlying mechanism of its activation remains
largely confusing thus far. Usually, the activation of NLRP3
inflammasome requires two different steps, namely priming (signal 1) and
activation (signal 2). In the priming step, this signal starts the
recognition of molecules of pattern recognition receptors, such as LPS,
TNF-α and IL-1β to activate NF-κB, which induce the expression of NLRP3,
pro-IL-1β and pro-IL-18 (Peng et al., 2020). In the activation step, the
signal 2 can be any pathogen/danger-associated molecular patterns
(PAMP and DAMP) recruiting
inflammasome assembly and subsequent downstream secretion of IL-1β and
IL-18 (Urwanisch, Luciano & Horejs-Hoeck, 2021). Indeed, activation of
both NF-κB and NLRP3 inflammasome have been involved in inflammatory
infiltration and the progression of IBD. Crucially, we found that TBⅡ
treatment mitigated the elevated expression of both NF-κB and NLRP3 in
the DSS-induced colon tissue of mice and LPS+ATP-induced cells
(Figure.7). Importantly, co-treatment TBⅡ with NF-κB inhibitor
BAY11-7082 impaired TBⅡ-mediated NLRP3 inhibition, and thereby reducing
the IL-1β generation (Figure 7). Taken together, our results suggested
that TBⅡ-mediated NLRP3 inhibition may probably be related with the
suppression of NF-κB.
In summary, based on these studies, we provides the first evidences that
TBⅡ, a natural saponin, alleviated DSS-induced colitis via alleviating
the damaged intestinal barrier and inflammatory response. And the
mechanism may be possibly through the inactivation of NLRP3
inflammasome-mediated the suppression of communication between colonic
epithelial cells and macrophages, thereby not only reducing the
inflammatory interaction between macrophages and the gut, but also
repairing intestinal mucosal damage. Overall, our new findings suggest
that TBⅡ could be a promising adjunctive therapeutic strategy for the
treatment of intestinal inflammation.