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.