Indigo naturalis as a potential drug in the treatment of ulcerative colitis: a comprehensive review of current evidence

Intervention of immune cells and inflammatory signaling pathways

Immune cells, inflammasomes, and inflammatory signaling pathways are contributors to intestinal inflammation (Ramos and Papadakis 2019). CD4⁺ T cells can differentiate into various T cells mediated by cytokines, including Th1 cells induced by IFN-γ and IL-2, and Th2 cells induced by IL-4 (Bing et al. 2017). TNF-α and IFN-γ act as effectors of Th1 cells and play pro-inflammatory roles in the pathogenesis of UC (Gao et al. 2016). In contrast, IL-10 is an important anti-inflammatory cytokine in intestinal inflammation. Therefore, the Th1/Th2 imbalance is involved in the development of UC. Indirubin can inhibit CD4⁺ T cell infiltrations in the colonic mucosa, further downregulate Th1-type cytokine levels, and upregulate Th2 cytokine levels to regulate the Th1/Th2 imbalance. Another IL-10-secreting T cell, Fxop3⁺ Treg cells, are also induced to differentiate after indirubin intervention, thus exerting anti-inflammatory effects (Gao et al. 2016).

In addition to the involvement of immune cells, the aberrant activation of inflammatory signaling pathways is conductive to chronic intestinal inflammation. Among them, the NF-κB p65 protein is highly expressed in intestinal mucosal epithelial cells, crypt epithelial cells, and lamina propria monocytes in UC patients (Lu and Zhao 2020). Notably, both the application of IN and indigo or indirubin can inhibit colonic NF-κB signaling pathway activation in vivo by affecting p65 protein and the inhibitor of κB-α(IκBα) activities, which can reduce the secretion of its effector cytokines, TNF-α, IL-1β, and IL-6 (Gao et al. 2016; Yang et al. 2021). Moreover, the activation of the MAPK pathway is associated with chronic inflammation in UC. p38 MAPK, an isoform of MAPK, its phosphorylation has been found to increase significantly in the nucleus of immune effector cells in the mucosal crypt of UC patients (Dahan et al. 2008). The MAPK signaling pathway promotes the M1 macrophage activation, which releases pro-inflammatory cytokines and reactive oxygen species (ROS) to exacerbate inflammatory damage during UC development (Jing, Wang, and Xu 2019). The combination of indirubin and isatin can significantly decrease the phosphorylation of p38 MAPK, c-Jun N-terminal kinase, and extracellular regulated protein kinases, suggesting its extensive inhibitory activity for the MAPK pathway (Gao et al. 2018). Thus, the regulating effects on inflammatory mediators are also a critical mechanism of IN for treating UC.

Inhibition of oxidative stress

Oxidative stress is another necessary risk factor in the pathogenesis of UC (Ma et al. 2023). The production of excess ROS and reactive nitrogen species (RNS) in the intestine, which has exceeded the buffering capacity of the antioxidant defense in the body, leads to lipid peroxidation, intestinal mucosal barrier damage, and bacterial translocation (Wang et al. 2016). Additionally, ROS can act as a second messenger to activate intracellular inflammatory signaling pathways, which participate in chronic intestinal inflammation (Wang, Grivennikov, and Karin 2013)

MPO, nitric oxide synthase (NOS), and cyclooxygenase (COX) can promote endogenous ROS and RNS production by catalyzing chemical reactions (Tian, Wang, and Zhang 2017). MPO is significantly expressed in the inflamed mucosa of UC patients, and COX-2 is also closely associated with colitis (Konturek et al. 2005; Ullman and Itzkowitz 2011). Similarly, inducible nitric oxide synthase (iNOS), an inducible type of NO production, is also widely expressed in mucosal inflammation (Cross and Wilson 2003). After the administration of IN or extracted indigo, indirubin, tryptanthrin, and isatin, the gene expression of COX2 is inhibited, MPO and iNOS levels are reduced, and NO content is decreased, suggesting that IN and its active compounds can reduce oxidative stress by decreasing ROS and RNS production (Gao et al. 2018; Ozawa et al. 2020; Sun et al. 2020; Yang et al. 2021).

Enhancing the body’s antioxidant defense is another crucial strategy for combating oxidative stress. SOD is an intracellular enzymatic antioxidant that can reduce ROS levels by catalyzing the reduction of superoxide to hydrogen peroxide (Kaur and Benov 2020). GSH, on the other hand, is an intracellular, non-enzymatic antioxidant that can form an antioxidant barrier in the intestinal mucosa (Tian, Wang, and Zhang 2017). The application of indirubin and isatin can significantly restore SOD and GSH levels in the DSS-induced UC model (Gao et al. 2018). Further in vitro research reveals that both indigo and indirubin can modulate the nuclear factor erythroid 2-related factor 2-related antioxidant processes to inhibit excess ferroptosis (Yokote et al. 2023). Therefore, the relief of oxidative stress damage by regulating the oxidative/antioxidant balance in the intestine is also one of the important mechanisms of IN.

Regulation of the intestinal microbiota

Differences in intestinal microbiota between UC patients and the healthy population are significant, as manifested by decreased microbial diversity, reduced abundance, and changed abundance of specific groups of organisms. IN can help to restore intestinal microbial homeostasis in experimental colitis (Table 3). The imbalance of the Firmicutes/Bacteroidetes ratio due to decreased Firmicutes abundance and increased Bacteroidetes abundance at the phylum level is a characteristic feature of microbial dysbiosis in IBD patients (Walker et al. 2011). After the intervention of IN, the abundance of Firmicutes is restored, and the abundance of Bacteroidetes is decreased (Adachi et al. 2017; Liang et al. 2019; Sun et al. 2020). At the genus level, some probiotic bacteria, such as Lactobacillus, ruminococcus_1, ruminococcaceae_UCG-005, norank_f_Erysipelotrichaceae, Butyricicoccus, Bifidobacterium, and Turicibacter, are recovered in abundance after IN treatment. On the contrary, the harmful bacteria such as Escherichia-shigella, decreases in abundance after IN application. In conclusion, these research projects demonstrate the potential of IN to regulate host intestinal microbial homeostasis in UC.

Modulation of AhR pathway

The activation of the AhR pathway by indirubin and indigo as ligands to induce downstream immunomodulatory activity is considered the key mechanism of IN in the treatment of UC (Adachi et al. 2001). AhR is a member of the basic helix-loop-helix/Per-Arnt-Sim family proteins that are widely expressed in vertebrate cells (Kewley, Whitelaw, and Chapman-Smith 2004). AhR is located in the cytoplasm. When it is inactivated, it will form a complex multimer with the 90-kDa heat shock protein (HSP90), the AhR-interacting protein (AIP, also known as XAP2 or Ara9), the cochaperone p23, and the c-SRC protein kinase (Gutiérrez-Vázquez and Quintana 2018). When bound to a ligand, AhR in the cytoplasmic matrix translocates to the nucleus, dimerizes with the AhR nuclear translocator, interacts with the xenobiotic response element (XRE) in the promoter region of the target gene, initiates transcription of target genes such as the xenobiotic metabolic enzyme cytochrome P450 1A1 (CYP1A1), and finally induces numerous cascade reactions downstream (Hankinson 1995) (Figure 2).

AhR has been recognized as a key modulator of the host immune and inflammatory systems (Neavin et al. 2018). AhR pathway often plays a protective role in intestinal inflammation (Pernomian et al. 2020). Genome-wide association research has identified AHR as a susceptibility locus for IBD (Liu et al. 2015). It is reported that AHR expression is significantly lower in the intestinal tissues of IBD patients compared to healthy individuals (Monteleone et al. 2011). Also, mice lacking the AHR gene exhibit more severe experimental colitis after DSS induction compared to wild-type mice (Wang et al. 2018b). Moreover, after advanced treatment with the classical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, mice suffer mild colonic inflammation even after administration by DSS (Takamura et al. 2010). These findings demonstrate the important regulatory effect of AhR pathway in UC.

Indeed, the activation of the AhR pathway exerts an ameliorative effect on UC through different mechanisms due to its widespread expression in various immune and non-immune cells in the intestine. Disturbances of the immune system and excessive inflammatory responses have irreplaceable roles in the pathogenesis of UC (Ungaro et al. 2017). AhR is widely involved in the intrinsic and adaptive immunity of the intestine by affecting immune cells. Innate lymphoid cells (ILCs), a cell population including NK cells, ILC1, ILC2, ILC3, and regulatory ILCs, are widely present in the mucosal barrier and influence the innate immunity of host (Li et al. 2018b). All subsets of ILCs can be found in the intestine, and ILC2 participates in the expression of IL-5 and IL-13, two pro-inflammatory cytokines (Li et al. 2018b). The mRNA expression of IL13 shows a significant increase in rectal biopsies from UC patients (Inoue et al. 1999). The activation of the AhR pathway can promote host intestinal immune homeostasis from pathogenic bacteria by suppressing intestinal ILC2 and promoting ILC3 function (Li et al. 2018a). Meanwhile, NKp46 and lymphoid tissue inducer-like subsets are two major IL-22-producing subsets of ILCs. IL-22 is important for suppressing abnormal intestinal immunity by promoting the survival and proliferation of epithelial cells and secreting antimicrobial peptide production (Sonnenberg, Fouser, and Artis 2011). It is shown that the AhR pathway is essential for IL-22 production by ILCs, and its deletion affects the differentiation of IL-22-producing ILC subsets in the mouse intestine (Lee et al. 2011; Zelante et al. 2013). In conclusion, ILCs are possibly central to IBD pathogenesis, and the key regulatory properties of the AhR pathway in immunomodulation are increasing in prominence.

Macrophages and dendritic cells are also important members of intestinal intrinsic immunity. In the development of IBD, microbial exposure stimulated by risk factors, particularly epithelial barrier dysregulation, can stimulate macrophage activation, and the engulfment of microbes by phagocytosis will occur, which further produces higher levels of IL-6, IL-23, IL-12, and TNF-α to promote inflammation (Chang 2020). Dendritic cells, also known as antigen-presenting cells, can provide antigen to primary T cells after antigen capture and then participate in T cell differentiations to control T cell responses and activate adaptive immune pathways (Guermonprez et al. 2002; Chang 2020). The effects of the AhR pathway on macrophages and dendritic cells have been demonstrated, including influence on macrophage polarization and interference with antigen presentation by dendritic cells (Bankoti et al. 2010; Shinde et al. 2018). However, it remains unclear about the specific effects and mechanisms of the AhR pathway on macrophages and dendritic cells in the intestine. Notably, it has shown that mice with AHR deletion in the intestinal lamina propria macrophages and dendritic cells are more susceptible to DSS-induced colitis, which confirms the effects of the AhR pathway on the involvement of macrophages and dendritic cells in intestinal lamina propria immunity (Chng et al. 2016).

AhR pathway can also protect the intestinal barrier and maintain intrinsic immunity by affecting non-immune cells. The intestinal barrier, constructed by intestinal epithelial cells (IECs) and the mucus barrier, is the first defense lines against pathogenic microbial invasion. The deficiency of intestinal barrier function is thought to emerge before UC and is strongly implicated in the pathogenesis of UC (Kobayashi et al. 2020). The rapid regeneration of IECs is driven by the proliferation of intestinal stem cells in the crypt (Barker et al. 2007), but mice with AHR-specific deficiency in IECs cannot maintain the integrity of the epithelial barrier against bacterial infection due to impaired intestinal stem cells differentiation and proliferation (Metidji et al. 2019). Mucin secreted by the goblet cells is the necessary source of mucus. The deficiency of the AHR gene leads to the loss of goblet cells and reduced expression of mucin 2 in mice (Yin et al. 2019). Thus, AhR activity is also essential for the maintenance of the intestinal barrier.

Except for ILCs, T cells, another immune cells are involved in intestinal adaptive immunity, also contribute to the expression of the anti-inflammatory cytokine IL-22 in the intestine (Mizoguchi et al. 2018). Various subtypes of T cells, including CD4⁺ Th17 cells and CD4⁺ Th22 cells, can induce IL-22. In addition, IL-17 is one of the major effector cytokines produced by Th17 cells and can promote inflammation in the intestine (Eyerich, Dimartino, and Cavani 2017). It is reported that Th17 cells are an important source of IL-22 expression in mice. However, only a limited number of Th17 cells can express both IL-22 and IL-17 in humans (Eyerich, Dimartino, and Cavani 2017). In contrast, Th22 cells in humans, which can express IL-22 but not IL-17, play a protective role in intestinal inflammation. The activation of the AhR pathway influences Th17 cells in separate aspects (Qiu et al. 2013). Firstly, the absence of AHR limits the production of IL-22 induced by ILCs, which further leads to an increased abundance of commensal segmented filamentous bacteria that stimulate Th17 cell responses, resulting in increased Th17 cell expression and IL-17 secretion, which reflects an indirect effect of the AhR pathway on Th17 cells. Secondly, the AhR pathway can promote the differentiation of Th17 cells into IL-10-producing Treg type 1 cells that no longer express IL-17 but IL-10 during pathogenic microbial infections (Gagliani et al. 2015).

Another T cell subpopulation, Treg cells, also play an important role in colonic inflammation by expressing anti-inflammatory cytokines, such as IL-10 (Coombes et al. 2005). A dysregulation of Th17/Treg balance, with a decrease of Treg cells and an increase of Th17 cells in the colonic mucosa, is a typical manifestation of colonic inflammation in UC patients (Gong et al. 2016). It is proven that AhR expression is significant in intestinal Treg cells in steady state (Ye et al. 2017). However, the specific absence of the AHR gene in Treg cells leads to an obvious reduction of Treg cells in the intestine, which is linked to the fact that AhR can promote the intestinal homing of Treg cells (Ye et al. 2017). In summary, the activation of the AhR pathway contributes to suppressing the overactive Th17 response, which is necessary to regulate intestinal inflammation in UC.

Another non-negligible effect of AhR on colonic inflammation lies in its close association with the intestinal microbiota. The reduction of microbiota diversity and altered composition structure are the main features of microbiota dysbiosis in UC patients (Sokol et al. 2017). It has been demonstrated that the intestinal microbiota can be affected by AhR pathway in preclinical studies. Wild-type mice receiving normal chow has lower proportions of Firmicutes and higher proportions of Bacteroidetes in the small intestine compared to AHR-deficient mice receiving chow without potential AhR ligands (Korecka et al. 2016). Similarly, the AHR deficiency significantly affects the microbial composition in the cecum of mice, and these alterations are associated with more severe intestinal inflammation (Murray et al. 2016). Additionally, studies based on AhR signaling activation also support these findings. Exogenous AhR ligand uptake can alleviate colonic inflammation, and improve microbiota dysbiosis, or restore mouse intestinal microbes to normal levels through IL-22-induced pathways (Busbee et al. 2020). Taken together, the AhR pathway in the intestine can improve intestinal inflammation and alleviate UC through multiple effects (Figure 3).

The activation of AhR is identified as the key mechanism of IN in the treatment of UC. Indigo and indirubin exhibit potent AhR ligand activity in vitro, even comparable to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Likewise, the activation of AhR ligands in vivo by indigo and indirubin has been demonstrated (Sugihara et al. 2004). Interestingly, Kawai et al. reveal that indigo and indirubin can ameliorate DSS-induced mouse colitis in an AhR pathway-dependent manner (Kawai et al. 2017). The mononuclear cells isolated from mice show a significant increase in Cyp1A1, a downstream factor of AhR. IL-10 and IL-22, the anti-inflammatory cytokines released by immune cells affected by AhR, can be stimulated by IN and indigo. Furthermore, IN and indigo are only involved in AhR pathway in the intestinal mucosa and do not affect the mesenteric lymph nodes or spleen, suggesting that the activation of AhR by IN seems to present intestinal specificity (Kawai et al. 2017). Yokote et al. (2023) have found that the expression of Cyp1A1 in the rectal mucosa of UC patients receiving IN treatment is significantly increased, with indirubin detected in the rectal mucosa of patients. This finding directly provides clinical evidence for IN and its main active ingredient, indirubin, to treat UC by activating the AhR pathway.

Considering the beneficial effects of the AhR pathway on intestinal microbiota, it is reasonable to assume that IN has an indirect influence on intestinal microbiota through the AhR pathway. Sun et al. (2020) find that IN exerts therapeutic effects on experimental colitis in a microbiota-dependent manner when using an antibiotic-induced germ-free mouse model. They further demonstrate that the specific mechanism may be related to the increase of butyrate, a short-chain fatty acid beneficial to intestinal homeostasis, induced by IN. There is a bidirectional interaction between the AhR pathway and the intestinal microbiota. Tryptophan in humans can be metabolized mainly through the kynurenine or serotonin pathway (Gao et al. 2018). The intermediate metabolites produced via the kynurenine pathway, including kynurenic acid, O-aminobenzoic acid, cinnamic acid, and flavoracil, have been shown to be endogenous ligands of AhR (Romani et al. 2014). The kynurenine pathway can be induced by the intestinal microbiota. Moreover, approximately 4-6% of tryptophan can be metabolized by the intestinal microbiota into indole, indican, tryptamine, and skatole, as well as indole acid derivatives (Yokoyama and Carlson 1979). Among them, indole and indolic acid derivatives are also ligands that can activate the intestinal AhR pathway (Gao et al. 2018). Zelante et al. (2013) have reported that lactobacillus can activate AhR pathway by interfering with tryptophan metabolism and induced IL-22 secretion by ILC3, to maintain intestinal mucosal immunity.

Obviously, there is an intricate interplay between the microbiota, AhR pathway, and host intestinal immune system (Rooks and Garrett 2016). The precise mechanism underlying the therapeutic effects of IN in UC is yet to be determined. It is unclear if the efficacy is primarily due to the direct activation of the AhR pathway or if it is mediated through indirect effects on the intestinal microbiota. In any case, AhR may be the core treatment target of IN on UC based on its wide range of regulating effects.