Background
Crohn’s disease (CD) and ulcerative colitis (UC), the main clinical phenotypes of inflammatory bowel diseases (IBD), are chronic relapsing inflammatory disorders that affect the gastrointestinal tract. IBD are thought to result from an inappropriate and continuing inflammatory response to commensal microbes in a genetically susceptible host. Environmental triggers such as increased hygiene, drug use, stress, smoking, and diet influence the onset of the disease [
1]. Over the past decades, naturally occurring alkaloids from plant or medicinal herb sources have sparked considerable interest because of their significant anti-inflammatory and antioxidant properties [
2‐
4].
Alkaloids—a class of natural bioactive compounds derived from amino acids that contain one or more heterocyclic nitrogen atoms—are produced by a wide range of living organisms, such as bacteria, fungi, plants, and animals [
5]. In plants, alkaloids are produced as secondary metabolites in response to environmental biotic or abiotic interactions, and they confer protection through a range of insecticidal, antimicrobial and pharmacological attributes. The anti-inflammatory activities of plant-derived alkaloids have been documented in several animal models of disease, including respiratory distress [
6], spinal cord injury [
7], hepatic fibrosis [
8], cancer [
9], and IBD [
10,
11]. The protective effects of alkaloids have been attributed to amelioration of inflammatory responses and colonic oxidative stress [
12,
13], promotion of epithelial barrier function [
14], and positive regulation of gut microbiota [
15].
Pyridine alkaloids present in tobacco (
Nicotiana tabacum) have been the subject of intensive research. Nicotine, the major alkaloid in tobacco, accounts for approximately 95% of the total alkaloid content of tobacco, while the structurally related nornicotine and anatabine are the most abundant minor pyridine alkaloids, accounting for 4 to 5% of total alkaloids [
16]. Other pyridine alkaloids in tobacco, such as anabasine, anabaseine, and cotinine, are present in smaller amounts [
17]. Nicotine and all minor tobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholine receptors (nAChRs) [
18]. Tobacco nAChR agonists such as nicotine, anatabine, anabasine, anabaseine, and cotinine display protective effects in animal models of several inflammatory conditions, including sepsis [
19], Parkinson’s disease [
20], Alzheimer’s disease [
21], and IBD [
22].
Several in vitro and in vivo studies have shown a clear nicotine-dependent positive effect on inflammatory processes [
23,
24]. In a previous study, oral nicotine administration reduced the severity of DSS-induced colitis and reduced colonic TNFα synthesis, while subcutaneous injection or minipump infusion had no effect, highlighting the crucial role of administration route for the protective effects of nicotine in DSS colitis [
25]. Nicotine has also been shown to attenuate the severity of DSS colitis and expression of IL-6 in CD4T cells [
23]. A recent study suggested that nicotine ameliorates DSS-induced inflammation through inhibition of signal transducer and activator of transcription (STAT)3 in gut-infiltrated lymphocytes and intestinal epithelial cells [
26]. Recently, nicotine was shown to cause a decrease in leukocyte recruitment, disease activity index (DAI), and histological score in DSS colitis and block TNF-mediated expression of mucosal vascular addresin cell adhesion molecule-1 in endothelial cells. These authors concluded that nicotine suppresses inflammation through downregulation of adhesion molecules in the gut [
27]. Nonetheless, clinical studies on the efficacy and tolerability of nicotine have shown that therapeutic application of nicotine for treatment of UC is limited because of frequent adverse effects and nicotine inconsistent efficacy in maintaining remission in UC patients [
28,
29].
Anatabine is found in plants of the Solanacea family, which includes tobacco, peppers, tomato, and eggplant [
30]. Little is known about the biological properties of anatabine, although several studies have suggested that this alkaloid is a potential candidate compound for anti-inflammatory drug development [
21,
31]. Anatabine was marketed in the US as a dietary supplement under the name Anatabloc. In an internet-based survey, approximately 90% of all users rated the effect of anatabine supplementation as good or excellent for joint pain, stiffness, and functionality [
32]. Anatabine has been shown to inhibit lipopolysaccharide (LPS)-induced pro-inflammatory gene expression as well as NF-κB and STAT3 phosphorylation in human neuroblastoma SH-SY5Y, HEK293, human microglia, and human blood mononuclear cells [
33] as well as in the brain and spleen of mouse models of autoimmune encephalomyelitis [
31] and Alzheimer’s disease [
33]. In SH-SY5Y cells, anatabine also reduced the expression of beta-secretase 1—the rate limiting enzyme for β-amyloid peptide production, which is a major hallmark of Alzheimer’s disease—through inhibition of NF-κB activation [
21].
In this study, we aimed to assess the anti-inflammatory effects of the tobacco alkaloids nicotine and anatabine in the established DSS mouse model of UC [
34]. Our results show that oral administration of anatabine, but not nicotine, ameliorates the clinical manifestations of DSS treatment in mice. The results of gene expression analysis indicated that anatabine had a partial restorative effect on global DSS-induced gene expression profiles, while nicotine only had minimal effects. Moreover, multi-analyte profiling (MAP) showed that anatabine, but not nicotine, suppresses the production of IL-6, IL-1α, TNFα, granulocyte-colony stimulating factor (G-CSF), and keratinocyte chemoattractant (KC) while increasing IL-10 expression in the colon of DSS-treated mice. For an overview of the study concept and analytical procedures, see “Online Resource
1”.
Discussion
Our study shows that oral administration of anatabine, but not nicotine, reduces the clinical manifestations of DSS-induced colitis in a mouse model. In line with these findings, anatabine demonstrated a global downregulatory effect on DSS-induced gene expression changes in the colon, whereas the effects of nicotine were more limited. In particular, the results of gene expression profiling further supported the reduction of inflammatory signaling processes upon anatabine treatment, including suppression of IL-6 signaling (as shown by GSA findings) and TLR signaling (as shown by the results of network perturbation analysis and GSA). MAP also showed a significant decrease in the abundance of IL-6, KC, TNFα, IL-1α, and G-CSF and an increase in the expression of IL-21 and the anti-inflammatory cytokine IL-10.
Several studies have reported on the anti-inflammatory effects of nicotine on the DSS mouse model of UC [
27]. Subcutaneous administration of nicotine was shown to ameliorate tissue injury in DSS colitis and IL-6 expression in CD4T cells via α7-nAChRs [
23]. Nicotine was also shown to decrease the activation of STAT3 through induction of miR-124 in gut-infiltrated lymphocytes and intestinal epithelial cells [
26]. Strikingly, AlSharari et al. observed that oral, but not subcutaneous injection of nicotine, ameliorated intestinal inflammation and colonic TNFα expression in DSS-treated mice [
25]. In spite of the reported beneficial effects, our results do not support a protective effect of orally administered nicotine on DSS colitis. Of note, we found that nicotine significantly reduced DSS-associated intestinal bleeding, which was the only clinical parameter affected by this tobacco alkaloid. The vasoconstrictor effects of nicotine are well stablished [
38]. In the gut mucosa, nicotine decreases blood flow [
39], and cigarette smoking decreases rectal blood flow to normal levels in patients with UC [
40]. However, how changes in blood flow affect the pathophysiology of UC is still unclear. The possible therapeutic use of nicotine to induce remission in UC patients has been evaluated in five clinical studies [
41‐
45] and two meta-analyses [
28,
46]. These studies have demonstrated a variable efficacy of nicotine therapy in induction of remission [
28], with several studies showing no effect [
41,
44]. Moreover, a high frequency of adverse events increased the withdrawal rate in the nicotine group in some studies, thus limiting the therapeutic benefit of nicotine [
29].
To the best of our knowledge, the present study is the first to assess the impact of anatabine on experimental colitis. Gene expression analysis of distal colon biopsy specimens highlighted the anti-inflammatory properties of anatabine in multiple functional categories, including “Immune Responses”, “Signal Transduction”, and “Extracellular Matrix Organization”, which in turn encompassed several TLR and cytokine signaling pathways. Genes contributing to the downregulation of TLR cascades included TLR2, TLR4, and TLR6 and a number of downstream signaling factors shared by several TLRs, including MYD88, RIPK2, IRAK3, IRAK4, and NOD1 (nucleotide-binding oligomerization domain-containing protein 1), as well as the nuclear factors ELK1, Fos, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)1, activating transcription factor (ATF)1, and ATF2 (See
Online Resource of the respective gene lists). For the cytokine signaling pathways, the contributing genes included IL-6 and IL-6 receptor α, IFNγ, IL-4, CXCL10, IL-22 receptor α2, IL-2 receptor α, TNF receptor superfamily member 1A, IL-17α, and IL-17 receptor α, JAK1, JAK2, STAT3, and STAT4. Members of the NF-κB signaling pathway also contributed to the overall reduction in inflammatory cascades, including NF-κB p65, p105, and p100 subunits, IκBα, and the NF-κB activating protein TAB3. In agreement with the results of transcriptional analysis, MAP findings showed a significant decrease in DSS-associated IL-6, KC, TNFα, IL-1α, and G-CSF protein expression and an increase in IL-10 expression in the presence of anatabine. Strikingly, while TLR downstream factors modulated by anatabine are shared by most TLRs, the majority of cytokine-associated signaling molecules are specific for each pathway. The seemingly pleiotropic regulatory effects of anatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different pro-inflammatory signaling cascades.
Previous studies using in vitro and in vivo disease models have demonstrated the anti-inflammatory effects of anatabine [
21,
31]. Paris et al. showed that this pyridine alkaloid reduced the plasma levels of IL-1β, IL-6, and IL-17A as well as the expression of IL-1β, INFγ, and TNFα in the spleen of experimental autoimmune encephalomyelitis mice [
31]. These authors also showed that anatabine suppressed STAT3 and NF-κB phosphorylation in the spleen and brain of these mice [
31]. Anatabine also prevented LPS- and TNFα-induced NF-κB and STAT3 phosphorylation in SH-SY5Y and HEK cells, human microglia, and human blood mononuclear cells [
33]. Additionally, anatabine prevented LPS-induced IL-1β expression in human whole blood as well as IL-1β, IL-6, and TNFα production in the plasma, kidney, and spleen in the LPS mouse model [
33]. Phosphorylated STAT3, TNFα, and IL-6 were also downregulated in the presence of anatabine in a transgenic mouse model of Alzheimer’s disease [
33].
Our results on the effects of anatabine in the DSS mouse model of UC are also in line with the findings of a substantial number of studies demonstrating the protective effects of natural alkaloids in several animal models of colitis [
13,
47]. Intraperitoneal administration of the minor tobacco alkaloid and nicotinic receptor agonist anabaseine was shown to reduce tissue damage, myeloperoxidase activity, and colonic TNFα expression in a TNBS mouse model of colitis [
22]. These mice also showed reduced NF-κB activation in lamina propria mononuclear cells, while mice administered a nicotinic receptor antagonist presented worse colitis symptoms than those treated with TNBS alone [
22]. The algae alkaloid caulerpin reduces DSS colitis by suppressing NF-κB activation and subsequently inhibiting the colonic production of TNFα, IFNγ, IL-6, IFNγ, and IL-17 [
48]. Oral administration of berberine also ameliorates DSS-induced colitis and downregulates the expression of TNFα, IFNγ, KC, and IL-17 in colonic macrophages [
49]. The plant-derived alkaloid
N-methylcytisine and the tea alkaloid theophylline mitigate colitis by downregulating TNFα, IL-1β, and IL-6 expression in DSS and acetic acid models of colitis, respectively [
50,
51]. Induction of the anti-inflammatory cytokine IL-10 in the presence of natural alkaloids has also been reported. Thus, indirubin ameliorates DSS-induced colitis by suppressing the expression of colonic TNFα, IFNγ, and IL-2 and upregulating IL-10 [
52].
Additionally, indole alkaloids caulerpin and isatin have been shown to increase the expression of IL-
10 in DSS and TNBS models of IBD, respectively [
48,
53]
. Of note, our results show an increase in the abundance of IL-21 and a tendency towards increase in colonic IL-1β levels in the presence of anatabine. Although IL-21 expression is increased in many chronic inflammatory disorders, genetic deficiency of IL-21 is associated with IBD, and inhibition of IL-21 in the early phases of some inflammatory disorders exacerbates disease development, suggesting the dual role of IL-21 in the control of immune responses [
54]. IL-21 also promotes IL-22 expression in mucosal T-cells through a mechanism involving STAT3, retinoid-related orphan receptor γt, and aryl hydrocarbon receptor, thereby helping protect immunodeficient mice from DSS colitis [
55]. Interestingly, IL-21 has been recently shown to induce IL-1β production in dendritic cells through a STAT3-dependent but NF-κB-independent mechanism, thereby suggesting a link between IL-21 and IL-1β [
56].
Mounting evidence suggests that alkaloids—in particular isoquinoline alkaloids present in traditional medicine herbs—exert their anti-inflammatory effects through regulation of NF-κB and STAT3 signaling pathways. For example, sanguinarine and cavidine suppress the expression of NF-κB p65 subunit, thereby reducing colonic TNFα and IL-6 in acetic acid-induced colitis [
2,
11]. In the TNBS mouse model of colitis, berberine reduces IFNγ, IL-1α, IL-6, IL-17, and TNFα expression in colonic tissues and sera and suppresses Th1 and Th17 cells through reduction of STAT1, STAT3, and NF-κB phosphorylation [
57]. Oral administration of boldine attenuates DSS-induced colitis and reduces colonic phosphorylation of STAT3 and NF-κB p65 subunit as well as the expression of TNFα, IL-6, and IL-17 [
58]. Demethyleneberberine, tetrandrine, and norisoboldine show protective effects in the DSS colitis model and reduce the levels of colonic IL-1β, TNFα, and phosphorylated NF-κB p65 subunit [
10,
59,
60].
According to the present GSA findings, genes affected by anatabine in the Reactome category “signal transduction” encompassed numerous GPCR-associated factors, including several G protein subunits, adenylate cyclases, Rho guanine nucleotide exchange factors, factors involved in Ca
2+-mediated events, and phosphoinositide 3-kinase (PI3K)-associated signaling factors. The latter included the ubiquitously expressed class I PI3K isoforms p110α and p110γ (also known as PIK3CA and PIK3CG, respectively), PI3K regulatory subunit (PIK3R)5, PIK3R6, and AKT3. Similarly, several other alkaloids have been shown to reduce PI3K signaling cascades in the DSS mouse model of colitis. For example, quinolizidine alkaloids oxymatrine and aloperine ameliorate DSS colitis by inhibiting the PI3K/Akt pathway and T-cell responses [
61,
62]. Plant-derived alkaloids evodiamine and rutaecarpine inhibit corticosterone production by decreasing the activity of cAMP-related pathways in rat zona fasciculata–reticularis cells [
63]. Sinomenine, an isoquinoline alkaloid which ameliorates colitis and inflammatory gene expression in several mouse models of IBD [
64,
65], also reduces the levels of cAMP, intracellular Ca
2+, and phosphorylated CREB in morphine-treated SH-SY5Y cells [
66]. Strikingly, our findings suggested that factors contributing to anatabine-associated reduction of GPCR cascades included a wide range of cyclic nucleotide phosphodiesterases (PDEs) —including several subtypes of PDE1, PDE3, PDE4, PDE7, PDE8, and PDE10—which modulate the intracellular concentrations of cAMP and cyclic guanosine monophosphate (cGMP) by regulating their rates of degradation [
67]. In this regard, several naturally occurring alkaloids, such as caffeine and tolafentrine, also inhibit PDE activities [
68,
69]. The opium alkaloid papaverine has been identified as a potent and specific inhibitor of PDE10A, with the ability to trigger cAMP and cGMP accumulation as well as CREB phosphorylation in several animal behavioral models [
70,
71]. Taken together, our results suggest that anatabine exerts a dual effect on GPCR-associated pathways hindering the expression of GPCR-associated factors, while enhancing the accumulation of intracellular cAMP through reduction of PDE expression levels. Further research is needed to
understand the overall impact of anatabine on GPCR-mediated responses.
Interestingly, in the present study, anatabine reduced the expression of factors in the Reactome category “Extracellular Matrix Organization”. Genes contributing to this reduction encompassed a wide range of factors involved in ECM deposition, including fibronectin 1, fibulin (FBLN)1, FBLN2, elastin, laminin, lysyl oxidases
(collagen cross-linking enzymes), and fibrillar, network-forming, transmembrane, and fibril-associated collagens. Likewise, we observed a tendency towards reduced expression of a number of genes involved in ECM protein degradation, including several disintegrins and metalloproteinases with thrombospondin motifs, MMPs (including MMP8; MMP14; the gelatinases MMP2 and MMP9; and the stromelysins MMP3, MMP7, MMP10, and MMP12), and TIMP1 and TIMP2. Gene expression analysis identified factors involved in fibroblast proliferation and activation, including vimentin, various members of the TGFβ family, fibroblast growth factors, and fibroblast growth factor receptor (FGFR)1, FGFR2, and FGFR3. Overall, our data point to the possibility that the anti-inflammatory effects of anatabine span the regulation of IBD-associated fibrogenic responses, including fibroblast activation, ECM protein deposition, and breakdown of ECM proteins. A previous study highlighted the protective effects of intraperitoneally administered pentoxifylline—a xanthine alkaloid derived from the seeds of the cacao tree (
Theobroma cacao)—on TNBS colitis in rats through limiting TGFβ1 accumulation and MMP3 and MMP9 activation [
12]. Because wound-healing responses follow tissue injury and inflammation, it is plausible that the downregulation of extracellular matrix organization factors observed in the presence of anatabine is a consequence of reduced DSS inflammation rather than a direct effect of anatabine [
72].
DSS induces an UC-like pathology through the breakdown of the gut epithelium [
34]. Loss of epithelial barrier function in IBD is caused by enhanced apoptosis of epithelial cells and alterations in the architecture of epithelial tight junctions. Our results show that anatabine regulates the production of several pro-inflammatory cytokines involved in barrier disruption, including TNFα. TNFα influences the expression and the localization of tight junction proteins, thereby contributing to a leaky gut barrier [
73] and anti-TNFα therapies have been shown to reduce intestinal epithelial cell apoptosis in IBD patients [
74]. Some of the primary factors affected by anatabine are associated with GPCR signaling, which has been shown to regulate tight junction sealing. Thus, Gα
12/13 triggers the dissociation of occludin and claudin-1 from the tight junction complex through the phosphorylation of zonula occludens (ZO)-1 and ZO-2, thereby increasing the paracellular permeability of the epithelial barrier [
75]. Taken together, our results point to that part of the protective effects on DSS-colitis observed in the presence of anatabine could be ascribed to the downregulation of factors promoting the breakdown of the intestinal epithelial barrier.
An important limitation of our study is that it is not possible to discriminate between the individual contributions of the different cell types in intestinal mucosa to the overall anti-inflammatory response triggered by anatabine. Further research is needed to elucidate whether anatabine-mediated changes affect primarily one cell population, which in turn influences other cell types, or whether anatabine has synergistic or antagonistic effects in different cell types.
Our computational transcriptomic analysis allowed us to identify potential mechanism of anatabine in a highly un-targeted manner. This approach gave us the opportunity to discover multiple signaling pathways concurrently targeted by anatabine that are important for the development of IBD instead of focusing on one single signaling cascade. The pleiotropic effects observed in many natural bioactive compounds in general and anatabine in particular allows them to avoid the development of resistance due to the activation of alternative pathways, an issue observed with most single-target drugs [
76]. Our study paves the way for further translational studies focusing on IBD-relevant molecular mechanisms modulated by anatabine.
In summary, our results show that oral administration of anatabine, but not nicotine, ameliorates the clinical symptoms of DSS colitis and regulates the expression of several factors involved in inflammatory and immune responses in the DSS mouse model of UC. Our data suggest that anatabine is a promising therapeutic agent for IBD treatment.