Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

The protective effect of teprenone in TNBS-induced ulcerative colitis rats by modulating the gut microbiota and reducing inflammatory response.

OBJECTIVE: Ulcerative colitis (UC), a chronic and refractory nonspecific inflammatory bowel disease, affects millions of patients worldwide and increases the risk of colorectal cancer. Teprenone is an acylic polyisoprenoid that exerts anti-inflammatory properties in rat models of peptic ulcer disease. This in vitro and in vivo study was designed to investigate the effects of teprenone on UC and to explore the underlying mechanisms. METHODS: Human intestinal epithelial cells (Caco-2 cells) serve as the in vitro experimental model. Lipopolysaccharide (LPS, 1 µg/mL) was employed to stimulate the production of pro-inflammatory cytokines (interleukin [IL]-6, IL-1ß, and tumor necrosis factor [TNF]-α), Toll-like receptor-4 (TLR4), MyD88 expression, and NF-κB activation. A trinitrobenzene sulfonic acid (TNBS)-induced chronic UC rat model was employed for the in vivo assay. RESULTS: Pro-inflammatory cytokine stimulation by LPS in Caco-2 cells was inhibited by teprenone at 40 µg/mL through the TLR4/NF-κB signaling pathway. Teprenone attenuated TNBS-induced UC, decreased myeloperoxidase and malondialdehyde, induced TLR4 expression and NF-κB activation, and increased glutathione and zonula occludens-1 level in the rat colonic tissue. Moreover, Fusobacterium, Escherichia coli, Porphyromonas gingivalis elevation, and Mogibacterium timidum decline in UC rats were inhibited by teprenone. CONCLUSION: Based on our results, the protective effects of teprenone for UC may be related to its ability to modulate the gut microbiota and reduce the inflammatory response.

Quercetin Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis by Inhibiting the Glucose Regulatory Protein 78 Activation.

Background: The inflammatory bowel diseases (IBD) are significantly influenced by apoptosis and endoplasmic reticulum (ER) stress. Aims: To investigate the effects of quercetin on ER stress-mediated apoptosis in a trinitrobenzene sulfonic acid (TNBS) induced experimental IBD model. Study Design: In vivo animal experimental study. Methods: To demonstrate the effect of quercetin in an experimental colitis model, Control, TNBS, and TNBS+quercetin groups were created with 24 Wistar Albino rats. Colitis was induced by intrarectal administration of 25 mg TNBS. In the TNBS+quercetin group, intragastrically 100 mg/kg quercetin was given for 7 days, immediately after colitis induction. In the TNBS-induced experimental IBD model, we evaluated the effects of quercetin on colonic epithelial cell apoptosis, oxidative stress, ER stress, the mitogen-activated protein kinase c-Jun N-terminal kinase, and the nuclear factor kappa B immunoreactivities, the levels of myeloperoxidase and tumor necrosis factor-α, the disease activity index with colonic histopathologic changes. Results: TNBS administration induced an elevated level of disease activity and oxidative stress indices, inflammation markers, and an increase in the immunoreactivities of nuclear factor kappa B and the mitogen-activated protein kinase c-Jun N-terminal kinase in the colon of the colitis group. Glucose regulatory protein 78, caspase-12 immunoreactivities, and epithelial cell apoptosis also were shown in the colon. However, quercetin improved TNBS-induced histopathological alterations, apoptosis, inflammation, oxidative stress, and ER stress. Conclusion: This study suggests that quercetin has a regulatory effect on ER stress-mediated apoptosis, and thus may be beneficial in treating IBD.

Transient receptor potential melastatin 2 is involved in trinitrobenzene sulfonic acid-induced acute and chronic colitis-associated fibrosis progression in mice.

Crohn's disease, a chronic and recurrent gastrointestinal disease, frequently causes intestinal fibrosis. Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, is activated by reactive oxygen species. This study investigated the role of TRPM2 in acute colitis and chronic colitis-associated fibrosis progression. Acute colitis and chronic colitis-associated fibrosis were induced in TRPM2-deficient (TRPM2KO) and wild-type (WT) mice through single and repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Bone marrow-derived macrophages (BMDMs) from WT and TRPM2KO mice were stimulated using H2O2. In WT mice, a single TNBS injection induced acute colitis with upregulated inflammatory cytokines/chemokines and Th1/Th17-related cytokines, while repeated TNBS injections induced chronic colitis-associated fibrosis with upregulation of fibrogenic factors and Th2-related cytokines. Acute colitis and chronic colitis-associated fibrosis with cytokines/chemokine upregulation and fibrogenic factors were considerably suppressed in TRPM2KO mice. Treating BMDMs with H2O2 increased cytokine/chemokine expression and JNK, ERK, and p38 phosphorylation; however, these responses were significantly less in TRPM2KO than in WT mice. These findings suggest that TRPM2 contributes to acute colitis progression via Th1/Th17-mediated immune responses. Furthermore, TRPM2 may be directly involved in colitis-associated fibrosis induction, likely due to the regulation of Th2/TGF-ß1-mediated fibrogenesis in addition to a consequence of acute colitis progression.

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice. / ç»“è‚ ç‚Žæ¨¡åž‹å°é¼ å† è„ç—›åŠç©´ä½æ•åŒ–çš„å¤–å‘¨ç¥žç»æœºåˆ¶æŽ¢è®¨.

OBJECTIVES: To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. METHODS: Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ï¼»EBï¼½) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. RESULTS: Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. CONCLUSIONS: Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Persistence of 2,4,6-triamino-1,3,5-trinitrobenzene in the environment.

2,4,6-triamino-1,3,5-trinitrobenzene (TATB) is an Insensitive High Explosive (IHE) that is increasingly being used as a safer alternative to traditional energetic materials. However, the high thermal stability of TATB poses challenges for its disposal, particularly through existing open burning methods and its ability to remain in the environment for long period of time. Therefore, this study investigated the persistence of TATB in the environment by conducting small-scale experiments which were designed to examine the resistance of TATB to open burning and to assess unburnt residues. To evaluate the fate and transport of the unburnt materials in soil, laboratory-scale soil column transport studies were conducted to gauge the movement of TATB through soil. The results indicate that TATB exhibits a high resistance to burning, leaving unburnt materials that can persist in soil. The study emphasizes the importance of efficient disposal methods for explosives and highlights the need for further research to understand the environmental impact and toxicity of TATB.

The Structure Properties of Carbon Materials Formed in 2,4,6-Triamino-1,3,5-Trinitrobenzene Detonation: A Theoretical Insight for Nucleation of Diamond-like Carbon.

The structure and properties of nano-carbon materials formed in explosives detonation are always a challenge, not only for the designing and manufacturing of these materials but also for clearly understanding the detonation performance of explosives. Herein, we study the dynamic evolution process of condensed-phase carbon involved in 2,4,6-Triamino-1,3,5-trinitrobenzene (TATB) detonation using the quantum-based molecular dynamics method. Various carbon structures such as, graphene-like, diamond-like, and "diaphite", are obtained under different pressures. The transition from a C sp2- to a sp3-hybrid, driven by the conversion of a hexatomic to a non-hexatomic ring, is detected under high pressure. A tightly bound nucleation mechanism for diamond-like carbon dominated by a graphene-like carbon layer is uncovered. The graphene-like layer is readily constructed at the early stage, which would connect with surrounding carbon atoms or fragments to form the tetrahedral structure, with a high fraction of sp3-hybridized carbon. After that, the deformed carbon layers further coalesce with each other by bonding between carbon atoms within the five-member ring, to form the diamond-like nucleus. The complex "diaphite" configuration is detected during the diamond-like carbon nucleation, which illustrates that the nucleation and growth of detonation nano-diamond would accompany the intergrowth of graphene-like layers.

Catechol-O-Methyltransferase Loss Drives Cell-Specific Nociceptive Signaling via the Enteric Catechol-O-Methyltransferase/microRNA-155/Tumor Necrosis Factor α Axis.

BACKGROUND & AIMS: The etiology of abdominal pain in postinfectious, diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown, and few treatment options exist. Catechol-O-methyltransferase (COMT), an enzyme that inactivates and degrades biologically active catecholamines, plays an important role in numerous physiologic processes, including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS: Colon neurons, epithelial cells, and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT, microRNA-155 (miR-155), and tumor necrosis factor (TNF) α expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-, colon-specific COMT-/-, and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS: Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-α were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-α in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-α) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS: Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-α axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections.

Matrix metalloproteinase 7 contributes to intestinal barrier dysfunction by degrading tight junction protein Claudin-7.

Background: Previous studies implicated matrix metalloproteinases (MMPs), such as MMP-7, in inflammatory bowel diseases (IBD) by showing increased activity during inflammation of the gut. However, the pathophysiological roles of MMP-7 have not been clearly elucidated. Methods: The expression of MMP-7 was assessed in colonic biopsies of patients with ulcerative colitis (UC), in rodents with experimental colitis, and in cell-based assays with cytokines. Wild-type and MMP-7-null mice treated with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid were used for determining the pro-inflammatory function(s) of MMP-7 in vivo. Results: MMP-7 was highly expressed in patients with UC and in rodents with experimental colitis. IL-1ß, IL-4, IL-13, TNFα, or lipopolysaccharide enhanced MMP-7 expression in human colonic epithelial cells, rat colonic smooth muscle cells, and THP-1-derived macrophages. Active MMP-7 degraded tight junction protein Claudin-7 in epithelial cells, cleaved recombinant Claudin-7 in cell-free system, and increased Caco-2 monolayer permeability. Immunostaining of colon biopsies revealed up-regulation of MMP-7 and reduction of Claudin-7 in UC patients. Compared to wild-type mice, Mmp7 -/- mice had significantly less inflammation in the colon upon DSS insult. DSS-induced alterations in junction proteins were mitigated in Mmp7 -/- mice, suggesting that MMP-7 disrupts the intestinal barrier. MMP-7 antibody significantly ameliorated colonic inflammation and Claudin-7 reduction in 2 different rodent models of colitis. Summary: MMP-7 impairs intestinal epithelial barrier by cleavage of Claudin-7, and thus aggravating inflammation. These studies uncovered Claudin-7 as a novel substrate of MMP-7 in the intestinal epithelium and reinforced MMP-7 as a potential therapeutic target for IBD.

Soluble Protein Hydrolysate Ameliorates Gastrointestinal Inflammation and Injury in 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis in Mice.

Inflammatory bowel diseases (IBD) are chronic, recurring gastrointestinal diseases that severely impair health and quality of life. Although therapeutic options have significantly expanded in recent years, there is no effective therapy for a complete and permanent cure for IBD. Well tolerated dietary interventions to improve gastrointestinal health in IBD would be a welcome advance especially with anticipated favorable tolerability and affordability. Soluble protein hydrolysate (SPH) is produced by the enzymatic hydrolysis of commercial food industry salmon offcuts (consisting of the head, backbone and skin) and contains a multitude of bioactive peptides including those with anti-oxidant properties. This study aimed to investigate whether SPH ameliorates gastrointestinal injury in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Mice were randomly assigned to four groups: Control (no colitis), Colitis, Colitis/CP (with control peptide treatment), and Colitis/SPH (with SPH treatment). Colitis was induced by cutaneous sensitization with 1% TNBS on day -8 followed by 2.5% TNBS enema challenge on day 0. Control peptides and SPH were provided to the mice in the Colitis/CP or Colitis/SPH group respectively by drinking water at the final concentration of 2% w/v daily from day -10 to day 4. Then, the colon was harvested on day 4 and examined macro- and microscopically. Relevant measures included disease activity index (DAI), colon histology injury, immune cells infiltration, pro- and anti-inflammatory cytokines and anti-oxidative gene expression. It was found that SPH treatment decreased the DAI score and colon tissue injury when compared to the colitis-only and CP groups. The protective mechanisms of SPH were associated with reduced infiltration of CD4+ T, CD8+ T and B220+ B lymphocytes but not macrophages, downregulated pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-6), and upregulated anti-inflammatory cytokines (transforming growth factor-ß1 and interleukin-10) in the colon tissue. Moreover, the upregulation of anti-oxidative genes, including ferritin heavy chain 1, heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, and superoxide dismutase 1, in the colons of colitis/SPH group was observed compared with the control peptide treatment group. In conclusion, the protective mechanism of SPH is associated with anti-inflammatory and anti-oxidative effects as demonstrated herein in an established mice model of colitis. Clinical studies with SPH as a potential functional food for the prevention or as an adjuvant therapy in IBD may add an effective and targeted diet-based approach to IBD management in the future.

Rapamycin Alleviates 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis through Autophagy Induction and NF-κB Pathway Inhibition in Mice.

Background: Recent genetic studies indicated that variants of autophagy genes were associated with the predisposition of Crohn's disease (CD). The autophagy deficiency may affect the innate and adaptive immunity, which is related to persistent and excessive inflammation of the bowel. However, it remains unclear how autophagy modulates the expression of immune response regulator NF-κB and proinflammatory cytokine TNF-α in CD. Aim: We aimed to investigate the role of rapamycin on the expression of NF-κB p65 and TNF-α in 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis and lipopolysaccharide (LPS)-induced HT-29 cells. Methods: TNBS-induced colitis mice were treated with saline or rapamycin, and the disease activity index (DAI) and histological scores of colonic mucosa were evaluated. The expressions of p65, ATG16L1 and LC3 were detected by western blot and immunohistochemistry staining. The monodansylcadaverine (MDC) staining and transmission electron microscopy were developed to study the autophagy in LPS-induced HT-29 cells. Expression of TNF-α from colon tissue and HT-29 cells were detected by ELISA. The expressions of p65, ATG16L1 and LC3 in active CD patients were also investigated. Results: Significantly more autophagosomes were observed in rapamycin-treated cells than in controls. Rapamycin remarkably upregulated the expression of ATG16L1 and LC3II, inhibited p65 nucleus translocation and secretion of TNF-α both in vivo and in vitro. The expression of both ATG16L1 and LC3II increased in mild to moderate CD specimens, while no significant difference was noted between severe CD and normal controls. The expression of p65 increased notably in severe CD compared to those in mild to moderate patients. Conclusions: In LPS-treated HT-29 cells and TNBS-induced colitis, p65 is overexpressed, which results in exaggerated secretion of TNF-α and induce or worsen the inflammation in the bowel. Rapamycin protects against colitis through induction of autophagy, thus inhibiting the activation of NF-κB pathway and secretion of TNF-α.

Protective Effect of Nanoparticles from Platycladi Cacumen Carbonisata on 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-Induced Colitis in Rats.

Aim: To evaluate the protective effects of Platycladi Cacumen Carbonisata-derived nanoparticles (PCC-NPs) against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis (UC) in rats. Methods: This study developed and characterized novel PCC-NPs synthesized by a green and simple pyrolysis process using Platycladi Cacumen (PC) as the sole precursor. The UC model prepared with rectal instillation of TNBS was used to evaluate the potential efficacy of PCC-NPs, and the underlying mechanisms were preliminarily explored from the perspective of anti-inflammatory and antioxidative stress for the first time. Results: PCC-NPs exhibited low cytotoxicity, good dispersibility and copious surface functional groups. Nanoparticles with diameters ranging from 40-60 nm mainly manifested a therapeutic effect by downregulating tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and upregulating interleukin-10 (IL-10). In addition, PCC-NPs relieved colon injury by inhibiting myeloperoxidase (MPO) activity, limiting the release of malondialdehyde (MDA) and increasing the activity of superoxide dismutase (SOD). Conclusion: Green synthetic PCC-NPs is a potential candidate as a complementary drug for intestinal inflammation of inflammatory bowel disease, and its regulatory mechanisms may be to balance the levels of pro-/anti-inflammatory cytokines and improve resistance to oxidative stress.

A New Rat Model of Pouchitis After Proctocolectomy and Ileal Pouch-Anal Anastomosis Using 2,4,6-Trinitrobenzene Sulfonic Acid.

BACKGROUND: Pouchitis is a common complication after ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis. However, an ideal model remains lacking. Therefore, we aimed to establish an appropriate model resembling human pouchitis. METHODS: Sprague-Dawley rats were randomly assigned to five groups: TNBS group, DSS group, NS group (following IPAA procedure, administrated with TNBS enema, DSS orally, normal saline enema, respectively), NI group (underwent IPAA), and sham group (underwent switch abdominal surgery). General status, weight change, hematochezia, and fecal scores were recorded. Fecal microbiota were counted under a microscope and analyzed by 16S rRNA gene high-throughput sequencing. Specimens of ileal pouch and small intestine (proximal, mid, distal) were collected to evaluate myeloperoxidase and occludin expression by immunohistochemistry and mRNA expression of pro-inflammatory markers by PCR. RESULTS: General status, hematochezia, fecal score, and increased mRNA expression of interleukin-6 and TNF-α in the TNBS group were similar to those in the DSS group, whereas the TNBS-induced model displayed a more stable weight change and more serious dysbacteriosis, not only was fecal bacterial diversity reduced, the dominant microbiota was altered. Histopathology scores of the distal small intestine in the TNBS group were lower compared with those in the DSS group (P < 0.05). A significant difference in myeloperoxidase and occludin expression in the small intestine was also detected between the TNBS and DSS groups. CONCLUSIONS: Our model mimicked the characteristics of human pouchitis and avoided potential side effects in the small intestine, and thus could be employed for further research.

A polymeric diet rich in transforming growth factor beta 2 does not reduce inflammation in chronic 2,4,6-trinitrobenzene sulfonic acid colitis in pre-pubertal rats.

BACKGROUND: Pediatric Crohn's disease is characterized by a higher incidence of complicated phenotypes. Murine models help to better understand the dynamic process of intestinal fibrosis and test therapeutic interventions. Pre-pubertal models are lacking. We aimed to adapt a model of chronic colitis to pre-pubertal rats and test if a polymeric diet rich in TGF-ß2 could reduce TNBS-induced intestinal inflammation and fibrosis. METHODS: Colitis was induced in 20 five-week-old Sprague-Dawley male rats by weekly rectal injections of increasing doses of TNBS (90 mg/kg, 140 mg/kg and 180 mg/kg) for 3 weeks, while 10 controls received phosphate-buffered saline. Rats were anesthetized using ketamine and chlorpromazine. After first administration of TNBS, 10 rats were fed exclusively MODULEN IBD® powder, while remaining rats were fed breeding chow. Colitis was assessed one week after last dose of TNBS by histopathology and magnetic resonance colonography (MRC). RESULTS: Histological inflammation and fibrosis scores were higher in TNBS group than controls (p < 0.05 for both). MRC showed increased colon wall thickness in TNBS group compared to controls (p < 0.01), and increased prevalence of strictures and target sign (p < 0.05). Colon expression of COL1A1, CTGF, α-SMA and COX-2 did not differ between TNBS rats and controls. TNBS colitis was not associated with growth failure. Treatment with MODULEN IBD® was associated with growth failure, increased colon weight/length ratio (p < 0.01), but did not affect histological scores or MRI characteristics. Colon expression of α-SMA was significantly lower in the MODULEN group versus controls (p = 0.005). CONCLUSION: Features of chronic colitis were confirmed in this model, based on MRC and histopathology. Treatment with MODULEN did not reverse inflammation or fibrosis.

Kynurenine plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate-induced colitis in mice.

BACKGROUND: Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is characterized by chronic intestinal inflammation leading to intestinal mucosal damage. Inflammatory bowel disease causes dysregulation of mucosal T cell responses, especially the responses of CD4+ T cells. Previously, we demonstrated that indoleamine-2,3-dioxygenase plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate (TNBS)-induced colitis. Although indoleamine-2,3-dioxygenase exerts immunosuppressive effects by altering the local concentration of tryptophan (Trp) and immunomodulatory Trp metabolites, the specific changes in immune regulation during colitis caused by Trp metabolites and its related enzymes remain unclear. AIM: To investigate role of kynurenine 3-monooxygenase (KMO) in TNBS-induced colitis and involvement of Trp metabolites in maintenance of intestinal homeostasis. METHODS: Colitis was induced in eight-week-old male KMO+/+ or KMO-/- mice of C57BL/6N background using TNBS. Three days later, the colon was used for hematoxylin-eosin staining for histological grading, immunohistochemical or immunofluorescence staining for KMO, cytokines, and immune cells. Inflammatory and anti-inflammatory cytokines were measured using quantitative RT-PCR, and kynurenine (Kyn) pathway metabolites were measured by high-performance liquid chromatography. The cell proportions of colonic lamina propria and mesenteric lymph nodes were analyzed by flow cytometry. RESULTS: KMO expression levels in the colonic mononuclear phagocytes, including dendritic cells and macrophages increased upon TNBS induction. Notably, KMO deficiency reduced TNBS-induced colitis, resulting in an increased frequency of Foxp3+ regulatory T cells and increased mRNA and protein levels of anti-inflammatory cytokines, including transforming growth factor-ß and interleukin-10. CONCLUSION: Absence of KMO reduced TNBS-induced colitis via generation of Foxp3+ regulatory T cells by producing Kyn. Thus, Kyn may play a therapeutic role in colon protection during colitis.

A Mixed-ligand Zn(II)-based Coordination Polymer for Selectively Detect 2,4,6-Trinitrophenol (TNP) and Treatment Effect on Periodontal Diseases via Inhibition Effect on P. gingivalis Growth.

A new difunctional Zn(II) coordination polymer (CP) with the chemical formula of [Zn(TBTA) (L)1.5]n (1) has been synthesized hydrothermally from tetrabromoterephthalic acid (H2TBTA) and 4,4'-bis(imidazole-1-yl)-biphenyl (L) ligands. Furthermore, due to its strong intense emission and open N donor sites, complex 1 could be used as a light-emitting sensor to determine 2,4,6-trinitrophenol (TNP) which has high selectivity and sensitivity. Furthermore, the anti-bacterial effect of the compound against P. gingivalis in vitro was evaluated by measuring the P. gingivalis growth curves after compound treatment. And the RT-PCR assay was performed to detect the relative expression of ragA and ragB, which are important for the P. gingivalis growth. The potential anti-infectious mechanism was further studied by using molecular docking technique.

Anti-Inflammatory Effect of Crude Momordica charantia L. Extract on 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis Model in Rat and the Bioaccessibility of its Carotenoid Content.

Momordica charantia L., known as bitter melon (BM), is a plant that belongs to the family Cucurbitaceae. Aims of this study are to investigate the anti-inflammatory effect of crude BM extract on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model in rat. It was also aimed to determine the content and bioaccessibility of carotenoids of BM. BM was purchased from local markets in Izmir, Turkey. Fruits of BM were lyophilized, powdered, and used in the experiment. Carotenoids were determined by high-performance liquid chromatography. To determine the bioaccessibility of ß-carotene, in vitro digestion was performed. Wistar albino rats were divided into four groups: group A (BM+TNBS), group B (BM), group C (TNBS), and group D (control). BM solution was given 300 mg/(kg·day) for 6 weeks orally. Colitis was induced by 0.25 mL of a solution containing 100 mg/kg 5% (w/v) TNBS in 50% ethanol (w/v) intrarectally after 6 weeks. After sacrification, macroscopic and microscopic evaluations were performed. Myeloperoxidase, cytokines levels (interleukin-17 [IL-17], TNF-alpha, and interleukin-10 [IL-10]) were measured in serum and colonic samples by ELISA test. Institutional Animal Ethics Committee approval was obtained. Total carotenoid content of BM was determined 11.7 mg/g dry weight as ß-carotene equivalents. Bioaccessibility of total carotenoids was determined as 2.1% with in vitro digestion. Pretreatment with crude BM extract significantly reduced weight loss, macroscopic, and microscopic colitis damages in colonic samples (P = .000), (P = .015), and (P = .026), respectively. Serum anti-inflammatory cytokine IL-10 increased significantly in both treatment groups (P = .000). BM is a rich source of carotenoids, but the bioaccessibility of its carotenoids is low. This study displays that BM has protective anti-inflammatory effects on TNBS-induced colitis.

Ras isoforms selectively regulate antigen-specific immune response.

H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.

Elevated expression of the leptin receptor ob­R may contribute to inflammation in patients with ulcerative colitis.

The effect of leptin on ulcerative colitis (UC) has been controversial. The present study aimed to investigate the role of leptin and its receptor ob­R in UC and the underlying mechanism of this role. The level of serum leptin and the protein expression of the leptin receptor ob­R in the colonic mucosa were determined in patients with UC. Experimental colitis was induced through intrarectal administration of 2,4,6­trinitrobenzene sulfonic acid (TNBS) in leptin receptor­deficient Zucker rats (LR­D). The body weight, disease activity index, colon length, and macroscopic and histopathological appearance were evaluated. Furthermore, the myeloperoxidase (MPO) enzyme activity and cytokine levels in colon tissues were also determined. The expression of the signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p­STAT3), nuclear factor (NF)­κB­p65, and Ras homolog gene family member A (RhoA) proteins in colon tissues was assessed. The results revealed that the expression of the leptin receptor ob­R was increased in the colonic mucosa but the serum leptin level was not altered in patients with UC compared with healthy volunteers. The severity of experimental colitis, represented by body weight loss, disease activity index, colon length, and macroscopic and histological changes, was ameliorated in LR­D rats compared with the wild­type (WT) rats. Moreover, the MPO activity; levels of cytokines including interleukin (IL)­1ß, IL­6, and tumor necrosis factor­α; and expression of p­STAT3, NF­κB, and RhoA proteins were reduced in colon tissues of LR­D rats compared with WT rats. In conclusion, activation of the leptin receptor ob­R is an important pathogenic mechanism of UC, and leptin receptor deficiency may provide resistance against TNBS­induced colitis by inhibiting the NF­κB and RhoA signaling pathways.

Nobiletin Enhances Induction of Antigen-Specific Immune Responses in BALB/c Mice Immunized with Ovalbumin.

We examined the effect of nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) on immune response in ovalbumin (OVA)-immunized mice. Treatment with nobiletin increased OVA-specific IL-4 and IL-10 production. In addition, mice that received nobiletin showed higher levels of OVA-specific IgE, IgG and IgG1 production than did control mice. The antibody response to the thymus-independent antigen 2,4,6-trinitrophenyl-Ficoll was not different in the control and nobiletin groups, suggesting that nobiletin does not directly stimulate antibody production. An in vitro study showed that treatment with nobiletin enhanced the ability of antigen presentation of bone marrow-derived dendritic cells. The in vivo and in vitro results indicate that nobiletin regulates immune function.