Administration Of Trinitrobenzene Sulfonic Acid Research Articles

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 relevanceChinese 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 studyThis 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 methods61 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-1β, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iβ, 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. ResultsGQD 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-1β, 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-1β. 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. ConclusionsGQD 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.

Taurohyodeoxycholic acid alleviates trinitrobenzene sulfonic acid induced ulcerative colitis via regulating Th1/Th2 and Th17/Treg cells balance

AimsTaurohyodeoxycholic acid (THDCA), a natural 6α-hydroxylated bile acid, exhibits intestinal anti-inflammatory effects. This study aimed to explore the efficacy of THDCA on ulcerative colitis and to reveal its mechanisms of action. Main methodsColitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to mice. Mice in the treatment group were gavage THDCA (20, 40, and 80 mg/kg/day) or sulfasalazine (500 mg/kg/day) or azathioprine (10 mg/kg/day). The pathologic markers of colitis were comprehensively assessed. The levels of Th1-/Th2-/Th17-/Treg-related inflammatory cytokines and transcription factors were detected by ELISA, RT-PCR, and Western blotting. The balance of Th1/Th2 and Th17/Treg cells was analyzed by Flow cytometry. Key findingsTHDCA significantly alleviated colitis by improving the body weight, colon length, spleen weight, histological characteristics, and MPO activity of colitis mice. THDCA reduced the secretion of Th1-/Th17-related cytokines (IFN-γ, IL-12p70, IL-6, IL-17A, IL-21, IL-22, and TNF-α) and the expressions of transcription factors (T-bet, STAT4, RORγt, and STAT3), but increase the production of Th2-/Treg-related cytokines (IL-4, IL-10, and TGF-β1) and the expressions of transcription factors (GATA3, STAT6, Foxp3, and Smad3) in the colon. Meanwhile, THDCA inhibited the expressions of IFN-γ, IL-17A, T-bet, and RORγt, but improved the expression of IL-4, IL-10, GATA3, and Foxp3 in the spleen. Furthermore, THDCA restored the proportion of Th1, Th2, Th17, and Treg cells, and balanced the Th1/Th2 and Th17/Treg immune response of colitis mice. SignificanceTHDCA can alleviate TNBS-induced colitis via regulating Th1/Th2 and Th17/Treg balance, which may represent a promising treatment for patients with colitis.

Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation.

Background & AimsNADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process.MethodsThe NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed.ResultsWe identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid–induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage.ConclusionsThe ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.

Effect of Arrabideae chica (crajiru) extract in a model of induced experimental colitis in rats

Objective: This study aimed to investigate the effect of A. chica extract on the evolution of experimental rectocolitis in rats, and the expression of the pro-inflammatory cytokines TNF-a, IL-1β and IL-6 in colonic tissue. Methods: Wistar rats weighing 275±23g were distributed into 4 groups of 6 animals each. Rectocolitis was induced in rats by rectal administration of trinitrobenzene sulfonic acid (TNBS). Seventy-two hours after TNBS injection, animals were treated daily for 6 days. Groups: 1. Normal control group without induction of rectocolitis. Received 0.9% saline injection v.o. by gavage during treatment. 2. TNBS rectocolitis group, treated with normal saline (SN) by gavage (TNBS+SN); 3. TNBS rectocolitis group treated with A. chica extract (ACE), receiving a daily dose of 300 mg of A. chica extract by gavage (TNBS+ACE);4. TNBS rectocolitis group treated with mesalazine, receiving a daily dose of 100 mg/kg of mesalazine orally (TNBS+MEZ). Macroscopic examination of the colon and dosing of TNF-α, IL-1β and IL-6 in colon tissue were performed. Results: There was a reduction in weight in animals treated only with TNBS+NS. No difference in weight was observed comparing the animals treated with ACE and MEZ. In the control group no mucosal ulcers or edema of the colon wall were observed. Several mucosal ulcers, edema and hyperemia occurred in the colon of rats in the TNBS+SN group. In two of the animals in this group there was colon perforation, tamponated by omentum. A reduction of mucosal ulcers number in the TNBS+ACE (crajiru) group was seen, compared to the TNBS+SN and TNBS+MEZ group. There was a significant reduction of TNF-α, IL-1β and IL-6 in the colon tissue of animals treated with crajiru extract, TCBS+ACE group, when compared to the control group (p<0.001), TNBS+SN group, and TNBS+MEZ groups (p<0.001). Conclusion: This is the first study to show that A. chica extract positively influences the treatment of TNBS/induced rectocolitis through its antiinflamatory activity. More comprehensive studies are needed to understand the underlying mechanisms.

Anterior Cingulate Cortex Mediates Hyperalgesia and Anxiety Induced by Chronic Pancreatitis in Rats

Central sensitization is essential in maintaining chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. Here, we examined the role of the anterior cingulate cortex (ACC) in the pathogenesis of abdominal hyperalgesia in a rat model of CP induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). TNBS treatment resulted in long-term abdominal hyperalgesia and anxiety in rats. Morphological data indicated that painful CP induced a significant increase in FOS-expressing neurons in the nucleus tractus solitarii (NTS) and ACC, and some FOS-expressing neurons in the NTS projected to the ACC. In addition, a larger portion of ascending fibers from the NTS innervated pyramidal neurons, the neural subpopulation primarily expressing FOS under the condition of painful CP, rather than GABAergic neurons within the ACC. CP rats showed increased expression of vesicular glutamate transporter 1, and increased membrane trafficking and phosphorylation of the N-methyl-D-aspartate receptor (NMDAR) subunit NR2B and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluR1 within the ACC. Microinjection of NMDAR and AMPAR antagonists into the ACC to block excitatory synaptic transmission significantly attenuated abdominal hyperalgesia in CP rats, which was similar to the analgesic effect of endomorphins injected into the ACC. Specifically inhibiting the excitability of ACC pyramidal cells via chemogenetics reduced both hyperalgesia and comorbid anxiety, whereas activating these neurons via optogenetics failed to aggravate hyperalgesia and anxiety in CP rats. Taken together, these findings provide neurocircuit, biochemical, and behavioral evidence for involvement of the ACC in hyperalgesia and anxiety in CP rats, as well as novel insights into the cortical modulation of painful CP, and highlights the ACC as a potential target for neuromodulatory interventions in the treatment of painful CP.

Dilodendron bipinnatum Radlk. extract alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration, TNF-α and IL-1β concentrations, IL-17 and COX-2 expressions, supporting mucus production and promotes an antioxidant effect

Ethnopharmacological relevanceDilodendron bipinnatum (Sapindaceae) stem bark decoction and macerate were used to treat uterine inflammation, pain in general, dermatitis and bone fractures. These homemade preparations also have diuretic, stimulant, expectorants and sedative effects and are effective in treating worm infections in the Brazilian Pantanal population. Our previous research confirmed the anti-inflammatory activity of the hydroethanolic extract of inner stem bark of D. bipinnatum (HEDb). AimThis work aimed to investigate the efficacy of HEDb in ameliorating experimental colitis in rats and to elucidate the possible mechanisms involved in the anti-ulcerative colitis properties of HEDb in rats and Caco-2 cell line. Materials and methodsThe effects on cell viability, IL-8 and TNF-α in human colon adenocarcinoma (Caco-2) were determined by flow cytometer and ELISA. Wistar rats (n = 6–7) were orally gavaged with, vehicle (0.9% saline), HEDb at doses of 20, 100 or 500 mg/kg, or mesalazine at a dose of 500 mg/kg, at 48, 24 and 1 h prior to the administration of trinitrobenzene sulfonic acid via rectal administration to induce colitis. The anti-inflammatory effects of HEDb were assessed macroscopically, by myeloperoxidase (MPO) activity and for glutathione (GSH) concentration in the colon. Additionally, colonic histopathological analyses of UC severity were conducted by different staining methods (H&E, PAS and toluidine blue). Pro-inflammatory cytokines TNF-α and IL-1β were quantified in colonic tissue by ELISA and colonic expressions of COX-2 and IL-17 were analyzed by western blotting. ResultsHEDb was shown to be non-cytotoxic with mean viability of 80% in Caco-2 cells. HEDb pre-treatments of 1, 5 or 20 μg/mL significantly reduced TNF-α production in Caco-2 cells by 21.8% (p < 0.05), 60.5 and 82.1% (p < 0.001) respectively following LPS treatment compared to LPS alone. However, no change in IL-8 production was observed. HEDb pre-treatment of rats subjected to TNBS significantly (p < 0.001) reduced colonic lesion score. Higher doses (100 and 500 mg/kg) caused a sharp downregulation of haemorrhagic damage, leukocyte infiltration, edema and restoration of mucus production. Moreover, mast cell degranulation was inhibited. Colonic MPO activity was reduced following all doses of HEDb, reaching 51.1% ± 1.51 (p < 0.05) with the highest dose. GSH concentration was restored by 58% and 70% following 100 and 500 mg/kg of HEDb, respectively. The oral treatment of HEDb at doses 20, 100 and 500 mg/kg decreased the concentrations of TNF-α and IL-1β at all doses in comparison to vehicle treated control. In addition, HEDb inhibited the COX-2 and IL-17 expressions with maximal effect at 500 mg/kg (60.3% and 65% respectively; p < 0.001). In all trials, the effect of HEDb at all doses being 20, 100 and 500 mg/kg was statistically comparable to mesalazine (500 mg/kg). ConclusionsHEDb reduces colonic damage in the TNBS colitis model and relieves oxidative and inflammatory events, at least in part, by increasing mucus production, reducing leukocyte migration and reducing TNF-α (in vivo and in vitro), IL-1β, IL-17 and COX-2 expression. Therefore, HEDb requires further investigation as a candidate for treating IBD.

Increased Mucosal Thrombin is Associated with Crohn's Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation.

Background and AimsThrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn’s disease.MethodsThrombin activity was studied in tissues from Crohn’s disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid.ResultsActive forms of thrombin were increased in Crohn’s disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models.ConclusionsOur data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Impact of extracum of curcuma longa in a composition of rectal suppositoria on of lipid peroxidal oxidation indications in the thick intestiness in experimental Crohn’s disease

The aim. To conduct a comparative analysis of the effect of turmeric extract and 5-aminosalicylic acid (5-ASA) in the composition of rectal suppositories in clinical symptoms and the content of lipid peroxidation products in the mucous membrane of the large intestine in experimental Crohn’s disease (CD).Materials and methods. The experiment was performed on 70 non-linear white Wistar rats. CD was modeled by rectal administration of trinitrobenzenesulfonic acid, rectal suppositories were used each 12 hours: with 5-ASA in a dose of 50 mg, with 10% turmeric extract of original composition, weighing 0.3 g each. On the 3rd, 5th and 7th day of CD, the clinical status was determined by the modified Disease activity index scale and the content of lipid peroxidation products in the large intestine mucosa.Results. In the dynamics of CD in the colon, the content of primary, secondary and final lipid peroxidative products in the heptane and isopropanol phases increases; the severity of clinical signs (weight loss, diarrhea, blood in the fecal masses) increases with increasing content of POL. The use of rectal suppositories with turmeric extract in case of CD reduces the severity of clinical symptoms, reduces the content of lipid peroxidation products mainly in the isopropanol phase of the colon lipid extract; an association was established between the index of clinical activity of CD and the content of lipid peroxidation products in the large intestine. The effectiveness of local application of turmeric extract in the composition of rectal suppositories is comparison with the effectiveness of the use of rectal suppositories with 5-ASA in terms of the clinical activity index and the content of lipid peroxidation products in the large intestine.

The effect of Hericium erinaceum on the prevention of chemically induced experimental colitis in rats.

Background/AimsThe aim of this study is to investigate the effects of the Hericium erinaceum on an experimental colitis model.MethodsTwenty-four Wistar albino were included in this study. Rats were divided into three groups. Group 1 (n = 8) was sham group. Group 2 is the group of chemically induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) resulting in colitis. Group 3 (n = 8) is the group that was treated 7 days before and 7 days after with H. erinaceum resulting in colitis. The activity of colitis was evaluated macroscopically and microscopically in rats. In other words, nitric oxide (NO) levels, malondialdehyde (MDA), interleukin 6 (IL-6), nuclear factor-kappa B (NF-κB) and, tumor necrosis factor-α (TNF-α) in addition to the myeloperoxidasem (MPO) activities was determined.ResultsThe rate of TNBS-induced colitis caused to increase the level of MDA activities meaningfully in the colitis group than the control group. The results indicated that MDA (p = 0.001), NO (p = 0.001), IL-6 (p = 0.001), MPO (p = 0.878), TNF-α (p = 0.001), and NF-κB levels of treatment group decreased in the blood and colon tissues because of the H. erinaceum treatment when compared to the colitis group. H. erinaceum treatment was related to the declining of MDA, NF-κB, NO, IL-6, and TNF-α levels.ConclusionsH. erinaceum had a positive effect on the colitis by reducing oxidative damage in blood and tissue.

Effect of a bioactive product SEL001 from Lactobacillus sakei probio65 on gut microbiota and its anti-colitis effects in a TNBS-induced colitis mouse model.

This study underpins the therapeutic potential of SEL001, a bioactive product isolated from Lactobacillus sakei probio65, in terms of its anti-inflammatory properties and its effect on gut-microbiota in a TNBS-induced ulcerative colitis mouse model. Ulcerative colitis was developed in mice by intra rectal administration of trinitrobenzene sulfonic acid. Bioactive product SEL001 (50 mg/kg b.w.) was administered orally. Myeloperoxidase activity was measured using 3,3′, 5,5′-tetramethylbenzidine. The entire colon was sampled for post-mortem clinical assessment. Colonic injury was assessed through histological and histomorphometric examinations. The 454 pyrosequencing and QIIME pipeline were used for gut microbiota analysis and statistical analysis were conducted using R. mRNA extraction from colon tissue and RT-PCR approaches were employed to determine the changes in the level of specific biomarker genes associated with UC. The results depict that SEL001 significantly lowered pro-inflammatory cytokines, including CD4, TNF-α, and interleukin-6. Examination of clinical and histopathological traits revealed that SEL001 was effective and potent in reducing the inflammatory signatures of UC to a similar extent as did by the standard drug mesalamine (5-ASA). Pyro-sequencing 16S data revealed that the reduction in the major member of phylum Firmicutes, which has been previously associated with a higher risk of UC. The SEL001, an anti-inflammatory bioactive product originated from a probiotic strain L. sakei probio65 could be an alternative therapeutic agent for treatment of UC.

Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats

Central sensitization plays a pivotal role in the maintenance of chronic pain induced by chronic pancreatitis (CP), but cortical modulation of painful CP remains elusive. This study was designed to examine the role of anterior insular cortex (aIC) in the pathogenesis of hyperalgesia in a rat model of CP. CP was induced by intraductal administration of trinitrobenzene sulfonic acid (TNBS). Abdomen hyperalgesia and anxiety were assessed by von Frey filament and open field tests, respectively. Two weeks after surgery, the activation of aIC was indicated by FOS immunohistochemical staining and electrophysiological recordings. Expressions of VGluT1, NMDAR subunit NR2B and AMPAR subunit GluR1 were analyzed by immunoblottings. The regulatory roles of aIC in hyperalgesia and pain-related anxiety were detected via pharmacological approach and chemogenetics in CP rats. Our results showed that TNBS treatment resulted in long-term hyperalgesia and anxiety-like behavior in rats. CP rats exhibited increased FOS expression and potentiated excitatory synaptic transmission within aIC. CP rats also showed up-regulated expression of VGluT1, and increased membrane trafficking and phosphorylation of NR2B and GluR1 within aIC. Blocking excitatory synaptic transmission significantly attenuated abdomen mechanical hyperalgesia. Specifically inhibiting the excitability of insular pyramidal cells reduced both abdomen hyperalgesia and pain-related anxiety. In conclusion, our findings emphasize a key role for aIC in hyperalgesia and anxiety of painful CP, providing a novel insight into cortical modulation of painful CP and shedding light on aIC as a potential target for neuromodulation interventions in the treatment of CP.

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis

Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Tripartite motif-containing (TRIM) 21 negatively regulates intestinal mucosal inflammation through inhibiting TH1/TH17 cell differentiation in patients with inflammatory bowel diseases

Tripartite motif-containing (TRIM) 21 has been implicated in the pathogenesis of several types of autoimmune diseases. We sought to elucidate TRIM21 expression in patients with inflammatory bowel diseases (IBDs) and its role in regulating intestinal mucosal inflammation. TRIM21 expression was analyzed in the inflamed mucosa of patients with IBDs by means of quantitative RT-PCR and immunohistochemistry. Peripheral blood CD4+ T cells were transfected with lentivirus-expressing TRIM21 (LV-TRIM21) or LV-sh-TRIM21, and cytokine expression was determined by using quantitative RT-PCR and ELISA. TRIM21-/- mice were generated, and trinitrobenzene sulfonic acid- and CD45RBhighCD4+ T cell-induced colitis models were established to determine its role in induction of intestinal inflammation. TRIM21 was expressed predominantly in CD4+ T cells and decreased markedly in the inflamed mucosa of patients with IBDs compared with healthy control subjects. Ectopic expression of TRIM21 inhibited IBD CD4+ T cells to differentiate into TH1 and TH17cells, whereas downregulation of TRIM21 had the opposite effects. TRIM21-/- mice had more severe colitis after administration of trinitrobenzene sulfonic acid compared with wild-type mice, which was characterized by increased expression of IFN-γ, TNF-α, and IL-17A in the colon. TRIM21-/-CD45RBhighCD4+ T cells reconstituted into recombination-activating gene (Rag1)-/- mice induced more severe colitis than in wild-type control mice. Mechanistically, interferon regulatory factor 3 was identified as a functional downstream target of TRIM21 in that silencing of interferon regulatory factor 3 suppressed TRIM21-/-CD4+ T-cell differentiation into TH1 and TH17cells. TRIM21 plays a protective role in mucosal inflammation through inhibiting TH1 and TH17cell differentiation. Thus TRIM21 might serve as a potential therapeutic target for the treatment of IBDs.

The effect of sphingosine-1-phosphate on colonic smooth muscle contractility: Modulation by TNBS-induced colitis.

AimIncreased levels of circulating sphingosine-1-phosphate (S1P) have been reported in ulcerative colitis. The objective of this study was to examine the effect of S1P on colonic smooth muscle contractility and how is it affected by colitis.MethodsColonic inflammation was induced by intrarectal administration of trinitrobenzene sulfonic acid. Five days later colon segments were isolated and used for contractility experiments and immunoblotting.ResultsS1P contracted control and inflamed colon segments and the contraction was significantly greater in inflamed colon segments. S1P-induced contraction was mediated by S1PR1 and S1PR2 in control and S1PR2 in inflamed colon segments. S1PR3 did not play a significant role in S1P-induced contractions in control or inflamed colon. S1PR1, S1PR2 and S1PR3 proteins were expressed in colon segments from both groups. The expression of S1PR1 and S1PR2 was significantly enhanced in control and inflamed colon segments, respectively. S1PR3 levels however were not significantly different between the two groups. Nifedipine significantly reduced S1P-induced contraction in control but not inflamed colon segments. Thapsigargin significantly reduced S1P-induced contraction of the inflamed colon. GF 109203X and Y-27632, alone abolished S1P-induced contraction of the control but not inflamed colon segments. Combination of GF 109203X, Y-27632 and thapsigargin abolished S1P-induced contraction of inflamed colon segments.ConclusionS1P contracted control colon via S1PR1 and S1PR2 and inflamed colon exclusively via S1PR2. Calcium influx (control) or release (inflamed) and calcium sensitization are involved in S1P-induced contraction. Exacerbated response to S1P in colitic colon segments may explain altered colonic motility reported in patients and experimental models of inflammatory bowel disease.

Neonatal immune challenge followed by adult immune challenge induces epigenetic‐susceptibility to aggravated visceral hypersensitivity

Abdominal pain is one of the major symptoms of inflammatory Bowel Disease (IBD). The inflammatory mediators released by colon inflammation are known to sensitize the afferent neurons, which is one of the contributors to abdominal pain. However, not all IBD patients have abdominal pain, and some patients report abdominal pain during remission, suggesting contributions of other pathological factors to abdominal pain in IBD. Epidemiological studies found early-life gastrointestinal infections a risk factor for IBD symptoms and adult-life gastrointestinal infections may trigger the onset of IBD. We investigated the hypothesis that neonatal colon immune challenge followed by an adult colon immune challenge upregulates spinal cord BDNF that aggravates visceral sensitivity over and above that induced by adult colon immune challenge alone. We induced neonatal and adult colon immune challenges by intraluminal administration of trinitrobenzene sulfonic acid to the rat colon. We found that neonatal immune challenge triggers epigenetic programming that upregulates tyrosine hydroxylase in the locus ceruleus when these rats are subjected to an adult colon immune challenge. The upregulation of locus ceruleus tyrosine hydroxylase, upregulates norepinephrine in the cerebrospinal fluid that acts on adrenergic receptors to enhance pCREB binding to the cAMP response element, which recruits histone acetylene transferase (HAT) to the BDNF gene to enhance its transcription resulting in aggravated visceromotor response to colorectal distension. HAT and adrenergic receptor antagonists block the aggravation of visceral sensitivity. HAT and adrenergic receptor inhibitors may serve as alternates to opioids and NSAIDS in suppressing abdominal pain in IBD.

BDNF modulates intestinal barrier integrity through regulating the expression of tight junction proteins.

Brain-derived neurotrophic factor (BDNF) may play a vital role in the homeostatic regulation of intestinal barrier integrity. We aimed to investigate the physiological role of BDNF in maintaining the intestinal epithelial barrier using postinflammatory irritable bowel syndrome (PI-IBS) mice and explore the underlying molecular mechanisms using intestinal epithelial cells in vitro. Postinflammatory-IBS mice were induced by intrarectal administration of trinitrobenzene sulfonic acid and allowed to recover for 28days. Frequency of defecation, fecal water content, colonic epithelial integrity and expressions of BDNF and tight junction (TJ) proteins (occludin, ZO-1, claudin-1, claudin-2) of the PI-IBS mice were investigated. Based on the results of animal studies, we further performed RT-PCR and Western blots to assess how BDNF stimulation and BDNF knockdown impacted TJ proteins in the ht-29 intestinal epithelial cells. Water content of stools was significantly increased in the PI-IBS mice compared with controls. Colonic mucosa from the PI-IBS mice displayed epithelial barrier defects and exhibited increased protein expressions of BDNF and claudin-2 and decreased protein expressions of occludin, ZO-1 and claudin-1. Furthermore, a siRNA against BDNF in the ht-29 cells could effectively suppress BDNF gene and protein expressions, and subsequently reduce TJ gene and protein levels. When the ht-29 cells were incubated with different doses of exogenous BDNF, significant increases of occludin, ZO-1 and claudin-1 and decreases of claudin-2 protein were observed. BDNF may play a role in regulating intestinal epithelial barrier via affecting the expression of TJ proteins.

Abnormal cannabidiol attenuates experimental colitis in mice, promotes wound healing and inhibits neutrophil recruitment.

BackgroundNon-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice.MethodsColitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 μl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration.ResultsTNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC.ConclusionsAbn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-016-0129-0) contains supplementary material, which is available to authorized users.

Core Fucosylation on T Cells, Required for Activation of T-Cell Receptor Signaling and Induction of Colitis in Mice, Is Increased in Patients With Inflammatory Bowel Disease

Attachment of a fucose molecule to the innermost N-glycan in a glycoprotein (core fucosylation) regulates the activity of many growth factor receptors and adhesion molecules. The process is catalyzed by α1-6 fucosyltransferase (FUT8) and required for immune regulation, but it is not clear whether this process is dysregulated during disease pathogenesis. We investigated whether core fucosylation regulates T-cell activation and induction of colitis in mice, and is altered in patients with inflammatory bowel disease (IBD). Biopsy samples were collected from inflamed and noninflamed regions of intestine from patients (8 with Crohn's disease, 4 with ulcerative colitis, and 4 without IBD [controls]) at Osaka University Hospital. Colitis was induced in FUT8-deficient (Fut8(-/-)) mice and Fut8(+/+) littermates by administration of trinitrobenzene sulfonic acid. Intestinal tissues were collected and analyzed histologically. Immune cells were collected and analyzed by lectin flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction, as well as for production of cytokines and levels of T-cell receptor (TCR) in lipid raft fractions. T-cell function was analyzed by intraperitoneal injection of CD4(+)CD62L(+) naïve T cells into RAG2-deficient mice. Levels of core fucosylation were increased on T cells from mice with colitis, compared with mice without colitis, as well as on inflamed mucosa from patients with IBD, compared with their noninflamed tissues or tissues from control patients. Fut8(-/-) mice developed less-severe colitis than Fut8(+/+) mice, and T cells from Fut8(-/-) miceproduced lower levels of T-helper 1 and 2 cytokines. Adoptive transfer of Fut8(-/-) T cells to RAG2-deficient mice reduced the severity of colitis. Compared with CD4(+) T cells from Fut8(+/+) mice, those from Fut8(-/-) mice expressed similar levels of TCR and CD28, but these proteins did not contain core fucosylation. TCR complexes formed on CD4(+) T cells from Fut8(-/-) mice did not signal properly after activation and were not transported to lipid rafts. Core fucosylation of the TCR is required for T-cell signaling and production of inflammatory cytokines and induction of colitis in mice. Levels of TCR core fucosylation areincreased on T cells from intestinal tissues of patients with IBD; this process might be blocked as a therapeutic strategy.

Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling.

Periostin is a matricellular protein that interacts with various integrin molecules on the cell surface. Although periostin is expressed in inflamed colonic mucosa, its role in the regulation of intestinal inflammation remains unclear. We investigated the role of periostin in intestinal inflammation using Postn-deficient (Postn-/-) mice. Intestinal epithelial cells (IECs) were transfected by Postn small interfering RNAs. Periostin expression was determined in colon tissue samples from ulcerative colitis (UC) patients. Oral administration of dextran sulfate sodium (DSS) or rectal administration of trinitrobenzene sulfonic acid, induced severe colitis in wild-type mice, but not in Postn-/- mice. Administration of recombinant periostin induced colitis in Postn-/- mice. The periostin neutralizing-antibody ameliorated the severity of colitis in DSS-treated wild-type mice. Silencing of Postn inhibited inteleukin (IL)-8 mRNA expression and NF-κB DNA-binding activity in IECs. Tumor necrosis factor (TNF)-α upregulated mRNA expression of Postn in IECs, and recombinant periostin strongly enhanced IL-8 expression in combination with TNF-α, which was suppressed by an antibody against integrin αv (CD51). Periostin and CD51 were expressed at significantly higher levels in UC patients than in controls. Periostin mediates intestinal inflammation through the activation of NF-κB signaling, which suggests that periostin is a potential therapeutic target for inflammatory bowel disease.

Imiquimod-induced CCR9 Ameliorates murine TNBS Colitis.

To investigate whether Imiquimod (IMQ) as TLR7 ligand protects mice from colonic inflammation and the mechanisms underlying in such immunoregulatory conditions. METHODS: Murine colitis was induced to Balb/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ. Colitis was evaluated by body weight decreases and by histological score. Also colonic mRNA expression was measured by RT-PCR. To confirm the induction of Regulatory T cells (Tregs) by type-1 IFN from pDCs, we generated mouse bone marrow-derived pDCs and co-cultured these with CD4+ T cells isolated from mouse spleen with or without IMQ stimulation. Cytokine production in the culture supernatant was measured by ELISA and the number of Tregs were analyzed by flow cytometry. Spleen and mesenteric lymph nodes (MLN) from IMQ-treated mice were collected, and mRNA expressions of cytokine were measured by RT-PCR and cytokine productions were measured by ELISA. Tregs and chemokine expressions were analyzed in colon of TNBS-induced colitis mouse by immunohistochemistry. RESULTS: Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis. In the colons of IMQ-treated mice, mRNA expression of TNF-α was decreased, and strong expressions of IL-6, IFN-β and TGF-β were detected. IL-10 and TGF-β productions were increased in the supernatant of co-cultured cells stimulated with IMQ, although we were unable to detect Treg differentiaton in IMQ-stimulated co-cultured cells. In MLN of IMQ-treated mice, strong expressions of TLR7, IFN-β, TGF-β and Foxp3 mRNA were detected. IL-10 production from MLN cells was also increased in the IMQ-treated group. Finally, Tregs in the inflamed colon and CCR9 in MLN of IMQ-treated mice were detected. CONCLUSION: These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9, which regulates accumulation of Tregs in the inflamed colon.