Marine Hydroquinone Zonarol Prevents Inflammation and Apoptosis in Dextran Sulfate Sodium-Induced Mice Ulcerative Colitis

Background and Aim

We previously identified an anti-inflammatory compound, zonarol, a hydroquinone isolated from the brown algae Dictyopteris undulata as a marine natural product. To ascertain the in vivo functions of zonarol, we examined the pharmacological effects of zonarol administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of ulcerative colitis (UC). Our goal is to establish a safe and effective cure for inflammatory bowel disease (IBD) using zonarol.

Methods and Results

We subjected Slc:ICR mice to the administration of 2% DSS in drinking water for 14 days. At the same time, 5-aminosalicylic acid (5-ASA) at a dose of 50 mg/kg (positive control) and zonarol at doses of 10 and 20 mg/kg, were given orally once a day. DSS-treated animals developed symptoms similar to those of human UC, such as severe bloody diarrhea, which were evaluated by the disease activity index (DAI). Treatment with 20 mg/kg of zonarol, as well as 5-ASA, significantly suppressed the DAI score, and also led to a reduced colonic ulcer length and/or mucosal inflammatory infiltration by various immune cells, especially macrophages. Zonarol treatment significantly reduced the expression of pro-inflammatory signaling molecules, and prevented the apoptosis of intestinal epithelial cells. Finally, zonarol protected against in vitro lipopolysaccharide (LPS)-induced activation in the RAW264.7 mouse macrophage cell line.

Conclusions

This is the first report that a marine bioproduct protects against experimental UC via the inhibition of both inflammation and apoptosis, very similar to the standard-of-care sulfasalazine, a well-known prodrug that releases 5-ASA. We believe that the oral administration of zonarol might offer a better treatment for human IBDs than 5-ASA, or may be useful as an alternative/additive therapeutic strategy against UC, without any evidence of side effects.     

Genetic Analysis of Susceptibility to Dextran Sulfate Sodium-Induced Colitis in Mice

The genetic basis for differential sensitivity of inbred mice to inflammatory bowel disease induced by dextran sulfate sodium (DSS) is unknown. Susceptible C3H/HeJ were outcrossed to partially resistant C57BL/6J mice. F2 and N2 progeny were phenotyped by evaluating histopathologic lesions in large intestine detected 16 days after a 5-day period of feeding 3.5% DSS. Screening for DSS colitis (Dssc) loci revealed quantitative trait loci (QTL) on Chr 5 (Dssc1) and Chr 2 (Dssc2). These traits contributed additively, explaining 17.5% of the variation in total colonic lesions. Additional QTL on Chr 18 and 1 that collectively explained 11% of the variation in total colon lesions were indicated. In the cecum, only a putative QTL on Chr 11 was associated with pathology (lesion severity) in the cecum. Reduced DSS susceptibility was observed in congenic stocks in which the highly susceptible NOD/Lt strain carried putative resistance alleles from either B6 on Chr 2 or from the highly resistant NON/Lt strain on Chr 9. We conclude that multiple genes control susceptibility to DSS colitis in mice. PossibleDssccandidate genes are discussed in terms of current knowledge of inflammatory bowel disease susceptibility loci in humans.     

Lacticaseibacillus rhamnosus LS8 Ameliorates Azoxymethane/Dextran Sulfate Sodium-Induced Colitis-Associated Tumorigenesis in Mice via Regulating Gut Microbiota and Inhibiting Inflammation

Probiotics and Antimicrobial Proteins - Gut microbiota dysbiosis may promote the process of colorectal cancer (CRC). Lacticaseibacillus rhamnosus LS8 (LRL) is a potential gut microbiota regulating...     

Effects of Orally Administered Bovine Lactoperoxidase on Dextran Sulfate Sodium-Induced Colitis in Mice

The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.     

Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis

Doublecortin-like kinase 1 (Dclk1), a microtubule-associated kinase, marks the fifth lineage of intestinal epithelial cells called tuft cells that function as tumor stem cells in Apc mutant models of colon cancer. In order to determine the role of Dclk1 in dextran sulfate sodium (DSS) induced colonic inflammation both intestinal epithelial specific Dclk1 deficient (Villin^Cre;Dclk1^f/f) and control (Dclk1^f/f) mice were fed 3% DSS in drinking water for 9 days, allowed to recover for 2 days, and killed. The clinical and histological features of DSS-induced colitis were scored and immunohistochemical, gene expression, pro-inflammatory cytokines/chemokines, and immunoblotting analyses were used to examine epithelial barrier integrity, inflammation, and stem and tuft cell features. In DSS-induced colitis, Villin^Cre;Dclk1^f/f mice demonstrated exacerbated injury including higher clinical colitis scores, increased epithelial barrier permeability, higher levels of pro-inflammatory cytokines and chemokines, decreased levels of Lgr5, and dysregulated Wnt/b-Catenin pathway genes. These results suggest that Dclk1 plays an important role in regulating colonic inflammatory response and colonic epithelial integrity.     

Opposite Role of Tumor Necrosis Factor Receptors in Dextran Sulfate Sodium-Induced Colitis in Mice

Tumor necrosis factor-α (TNF-α) is a key factor for the pathogenesis of inflammatory bowel diseases (IBD), whose function is known to be mediated by TNF receptor 1 (TNFR1) or 2. However, the precise role of the two receptors in IBD remains poorly understood. Herein, acute colitis was induced by dextran sulfate sodium (DSS) instillation in TNFR1 or 2−/− mice. TNFR1 ablation led to exacerbation of signs of colitis, including more weight loss, increased mortality, colon shortening and oedema, severe intestinal damage, and higher levels of myeloperoxidase compared to wild-type counterparts. While, TNFR2 deficiency had opposite effects. This discrepancy was reflected by alteration of proinflammatory cytokine and chemokine production in the colons. Importantly, TNFR1 ablation rendered enhanced apoptosis of colonic epithelial cells and TNFR2 deficiency conferred pro-apoptotic effects of lamina propria (LP)-immune cells, as shown by the decreased ratio of Bcl-2/Bax and enhanced nuclear factor (NF)-κB activity.     

Auraptene Decreases the Activity of Matrix Metalloproteinases in Dextran Sulfate Sodium-Induced Ulcerative Colitis in ICR Mice

Previously we reported that auraptene was a potent suppressant for matrix metalloproteinase (MMP)-7 expression in HT-29 human colon cancer cells. In the present study, we examined the effects of auraptene on MMP-2, -7, and -9 expression in colonic mucosa from dextran sulfate sodium (DSS)-induced ulcerative colitis mice. Auraptene remarkably suppressed the DSS-induced gelatinolytic activity of MMP-7 as well as the expression of MMP-2 and -9, suggesting that it might be useful in anti-metastatic therapies via the targeting of MMPs.     

Heme Oxygenase-1 Ameliorates Dextran Sulfate Sodium-induced Acute Murine Colitis by Regulating Th17/Treg Cell Balance

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn''s disease, is a group of autoimmune diseases characterized by nonspecific inflammation in the gastrointestinal tract. Recent investigations suggest that activation of Th17 cells and/or deficiency of regulatory T cells (Treg) is involved in the pathogenesis of IBD. Heme oxygenase (HO)-1 is a protein with a wide range of anti-inflammatory and immune regulatory function, which exerts significantly protective roles in various T cell-mediated diseases. In this study, we aim to explore the immunological regulation of HO-1 in the dextran sulfate sodium-induced model of experimental murine colitis. BALB/c mice were administered 4% dextran sulfate sodium orally; some mice were intraperitoneally pretreated with HO-1 inducer hemin or HO-1 inhibitor stannum protoporphyrin IX. The results show that hemin enhances the colonic expression of HO-1 and significantly ameliorates the symptoms of colitis with improved histological changes, accompanied by a decreased proportion of Th17 cells and increased number of Tregs in mesenteric lymph node and spleen. Moreover, induction of HO-1 down-regulates retinoic acid-related orphan receptor γt expression and IL-17A levels, while promoting Treg-related forkhead box p3 (Foxp3) expression and IL-10 levels in colon. Further study in vitro revealed that up-regulated HO-1 switched the naive T cells to Tregs when cultured under a Th17-inducing environment, which involved in IL-6R blockade. Therefore, HO-1 may exhibit anti-inflammatory activity in the murine model of acute experimental colitis via regulating the balance between Th17 and Treg cells, thus providing a possible novel therapeutic target in IBD.     

Different Effects of Three Selected Lactobacillus Strains in Dextran Sulfate Sodium-Induced Colitis in BALB/c Mice

Aim

To analyze the changes of different Lactobacillus species in ulcerative colitis patients and to further assess the therapeutic effects of selected Lactobacillus strains on dextran sulfate sodium (DSS)-induced experimental colitis in BALB/c mice.

Methods

Forty-five active ulcerative colitis (UC) patients and 45 population-based healthy controls were enrolled. Polymerase chain reaction (PCR) amplification and real-time PCR were performed for qualitative and quantitative analyses, respectively, of the Lactobacillus species in UC patients. Three Lactobacillus strains from three species were selected to assess the therapeutic effects on experimental colitis. Sixty 8-week-old BALB/c mice were divided into six groups. The five groups that had received DSS were administered normal saline, mesalazine, L. fermentum CCTCC M206110 strain, L. crispatus CCTCC M206119 strain, or L. plantarum NCIMB8826 strain. We assessed the severity of colitis based on disease activity index (DAI), body weight loss, colon length, and histologic damage.

Results

The detection rate of four of the 11 Lactobacillus species decreased significantly (P < 0.05), and the detection rate of two of the 11 Lactobacillus species increased significantly (P < 0.05) in UC patients. Relative quantitative analysis revealed that eight Lactobacillus species declined significantly in UC patients (P < 0.05), while three Lactobacillus species increased significantly (P < 0.05). The CCTCC M206110 treatment group had less weight loss and colon length shortening, lower DAI scores, and lower histologic scores (P < 0.05), while the CCTCC M206119 treatment group had greater weight loss and colon length shortening, higher histologic scores, and more severe inflammatory infiltration (P < 0.05). NCIMB8826 improved weight loss and colon length shortening (P < 0.05) with no significant influence on DAI and histologic damage in the colitis model.

Conclusions

Administration of an L. crispatus CCTCC M206119 supplement aggravated DSS-induced colitis. L. fermentum CCTCC M206110 proved to be effective at attenuating DSS-induced colitis. The potential probiotic effect of L. plantarum NCIMB8826 on UC has yet to be assessed.     

Bacteria Penetrate the Inner Mucus Layer before Inflammation in the Dextran Sulfate Colitis Model

Background

Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described.

Principal Findings

When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.

Conclusion

DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.     

Treatment with Extracellular Vesicles from Giardia lamblia Alleviates Dextran Sulfate Sodium-Induced Colitis in C57BL/6 Mice

Inflammatory bowel disease (IBD) is a chronic and recurrent illness of the gastrointestinal tract. Treatment of IBD traditionally involves the use of aminosalicylic acid and steroids, while these drugs has been associated with untoward effects and refractoriness. The absence of effective treatment regimen against IBD has led to the exploration of new targets. Parasites are promising as an alternative therapy for IBD. Recent studies have highlighted the use of parasite-derived substances, such as excretory secretory products, extracellular vesicles (EVs), and exosomes, for the treatment of IBD. In this report, we examined whether EVs secreted by Giardia lamblia could prevent colitis in a mouse model. G. lamblia EVs (GlEVs) were prepared from in vitro cultures of Giardia trophozoites. Clinical signs, microscopic colon tissue inflammation, and cytokine expression levels were detected to assess the effect of GlEV treatment on dextran sulfate sodium (DSS)-induced experimental murine colitis. The administration of GlEVs prior to DSS challenge reduced the expression levels of pro-inflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma. Our results indicate that GlEV can exert preventive effects and possess therapeutic properties against DSS-induced colitis.     

Extracellular Vesicles Derived from Gut Microbiota,Especially Akkermansia muciniphila,Protect the Progression of Dextran Sulfate Sodium-Induced Colitis

Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammatory bowel disease (IBD). However, owing to the complexity of the gut microbiota, our understanding of the roles of commensal and pathogenic bacteria in the maintenance of immune homeostasis in the gut is evolving only slowly. Here, we evaluated the role of gut microbiota and their secreting extracellular vesicles (EV) in the development of mucosal inflammation in the gut. Experimental IBD model was established by oral application of dextran sulfate sodium (DSS) to C57BL/6 mice. The composition of gut microbiota and bacteria-derived EV in stools was evaluated by metagenome sequencing using bacterial common primer of 16S rDNA. Metagenomics in the IBD mouse model showed that the change in stool EV composition was more drastic, compared to the change of bacterial composition. Oral DSS application decreased the composition of EV from Akkermansia muciniphila and Bacteroides acidifaciens in stools, whereas increased EV from TM7 phylum, especially from species DQ777900_s and AJ400239_s. In vitro pretreatment of A. muciniphila-derived EV ameliorated the production of a pro-inflammatory cytokine IL-6 from colon epithelial cells induced by Escherichia coli EV. Additionally, oral application of A. muciniphila EV also protected DSS-induced IBD phenotypes, such as body weight loss, colon length, and inflammatory cell infiltration of colon wall. Our data provides insight into the role of gut microbiota-derived EV in regulation of intestinal immunity and homeostasis, and A. muciniphila-derived EV have protective effects in the development of DSS-induced colitis.     

New Perspective on Dextran Sodium Sulfate Colitis: Antigen-Specific T Cell Development during Intestinal Inflammation

CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens.     

Crucial Role for CD69 in the Pathogenesis of Dextran Sulphate Sodium-Induced Colitis

CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In this study, we used CD69-deficient (CD69 KO) mice to assess the role of CD69 in the pathogenesis of dextran sulphate sodium (DSS)-induced acute and chronic colitis. The severity of colitis was assessed by the survival rate, clinical signs, colon length, histological examination and the expression of cytokines and chemokines in the large intestines. Both acute and chronic colitis were attenuated in the CD69 KO mice, as reflected by the lower lethality, weight loss, clinical signs, and improved histological findings. CD69⁺ cells infiltrated extensively into the inflamed mucosa of the colon in WT mice after DSS treatment. Experiments with the transfer of WT CD4 T cells into CD69 KO mice restored the induction of colitis. The administration of an anti-CD69 antibody also inhibited the induction of the DSS-induced colitis. These results indicate that CD69 expressed on CD4 T cells plays an important role in the pathogenesis of DSS-induced acute and chronic colitis, and that CD69 could be a possible therapeutic target for colitis.     

Addition of Berberine to 5-Aminosalicylic Acid for Treatment of Dextran Sulfate Sodium-Induced Chronic Colitis in C57BL/6 Mice

Ulcerative colitis (UC) is a common chronic remitting disease but without satisfactory treatment. Alternative medicine berberine has received massive attention for its potential in UC treatment. Conventional therapies with the addition of berberine are becoming attractive as novel therapies in UC. In the present study, we investigated the preclinical activity of a conventional oral 5-aminosalicylic acid (5-ASA) therapy plus berberine in experimental colitis. A subclinical dose of 5-ASA (200 mg/kg/day) alone or 5-ASA plus berberine (20 mg/kg/day) was orally administered for 30 days to C57BL/6 mice with colitis induced by three cycles of 2% dextran sulfate sodium (DSS). The disease severity, inflammatory responses, drug accumulation and potential toxicity of colitis mice were examined. The results showed that comparing to 5-ASA alone, 5-ASA plus berberine more potently ameliorated DSS-induced disease severity, colon shortening, and colon histological injury. Further, the up-regulation in mRNA level of colonic TNF-α as well as NFκB and JAK2 phosphorylation caused by DSS were more pronouncedly reversed in animals treated with the combination therapy than those treated with 5-ASA alone. Moreover, the addition of berberine to 5-ASA more significantly inhibited lymphocyte TNF-α secretion of DSS mice than 5-ASA alone. In the meanwhile, no extra drug accumulation or potential toxicity to major organs of colitis mice was observed with this combination treatment. In summary, our studies provide preclinical rationale for the addition of berberine to 5-ASA as a promising therapeutic strategy in clinic by reducing dose of standard therapy.     

mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

It has been established that mammalian target of Rapamycin (mTOR) inhibitors have anti-inflammatory effects in models of experimental colitis. However, the underlying mechanism is largely unknown. In this research, we investigate the anti-inflammatory effects of AZD8055, a potent mTOR inhibitor, on T cell response in dextran sulfate sodium (DSS)-induced colitis in mice, a commonly used animal model of inflammatory bowel diseases (IBD). Severity of colitis is evaluated by changing of body weight, bloody stool, fecal consistency, histology evaluation and cytokine expression. We find that AZD8055 treatment attenuates DSS-induced body weight loss, colon length shortening and pathological damage of the colon. And AZD8055 treatment decreases colonic expression of genes encoding the pro-inflammatory cytokines interferon-γ, interleukin (IL)-17A, IL-1β,IL-6 and tumor necrosis factor(TNF)-a and increases colonic expression of anti-inflammatory cytokines IL-10. We show that AZD8055 treatment decreases the percentages of CD4+ T cells and CD8+ T cells in spleen, lymph nodes and peripheral blood of mice. We also find that AZD8055 treatment significantly reduces the number of T helper 1(TH1) cells and TH17 cells and increases regulatory T (Treg) cells in the lamina propria and mesenteric lymph nodes. Furthermore, we demonstrates that AZD8055 suppresses the proliferation of CD4+ and CD8+ T cells and the differentiation of TH1/TH17 cells and expands Treg cells in vitro. The results suggest that, in experimental colitis, AZD8055 exerts anti-inflammatory effect by regulating T helper cell polarization and proliferation.     

Overexpression of GATA-3 in T Cells Accelerates Dextran Sulfate Sodium-Induced Colitis

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis includes genetic, environmental, and immunological factors, such as T helper cells and their secreted cytokines. T helper cells are classified as Th1, Th2, and Th17 cells. However, it is unclear which T helper cells are important in UC. Dextran sulfate sodium (DSS)-induced colitis is a commonly used model of UC. In this study, we induced DSS colitis in Th1 dominant (T-bet transgenic (Tg)) mice, Th2 dominant (GATA-3 Tg) mice, and Th17 dominant (RORγt Tg) mice to elucidate the roles of T helper cell in DSS colitis. The results showed that GATA-3 Tg mice developed the most severe DSS colitis compared with the other groups. GATA-3 Tg mice showed a significant decreased in weight from day 1 to day 7, and an increased high score for the disease activity index compared with the other groups. Furthermore, GATA-3 Tg mice developed many ulcers in the colon, and many neutrophils and macrophages were detected on day 4 after DSS treatment. Measurement of GATA-3-induced cytokines demonstrated that IL-13 was highly expressed in the colon from DSS-induced GATA-3 Tg mice. In conclusion, GATA-3 overexpression in T-cells and IL-13 might play important roles in the development of DSS colitis.