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.