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.