hUC-MSCs therapy for Crohn's disease: efficacy in TNBS-induced colitis in rats and pilot clinical study.

BACKGROUND: The use of mesenchymal stem cells (MSCs) has recently emerged as a promising new therapeutic strategy for many diseases including perianal fistulizing Crohn's disease (CD). Whether hUC-MSCs can promote the healing of luminal ulcer in CD has not been studied so far. METHODS: The model of TNBS-induced colitis in rats was used to confirm the efficacy of hUC-MSCs in the treatment of CD. Then, seventeen CD patients refractory to or unsuitable for currently available therapies were enrolled and received once submucosal local injection through colonoscopy combined with once intravenous drip on the next day. All patients received a 24-week follow-up. Clinical and laboratory assessments were monitored at baseline, week 4, 8, 12, and 24. Endoscopic evaluations were conducted at baseline and week 12. Mucosal specimens were obtained at the margin of lesions by endoscopy biopsies and used for RNA sequencing. Two hUC-MSCs co-culture systems were established in vitro, one with the mucosa specimens and the other with M1 macrophages induced from THP1. The expressions of genes representing inflammation (TNFα, IL-6, and IL-1ß) and intestinal barrier function (ZO1, CLAUDIN1, and CDH1) were tested by RT-PCR. FINDINGS: hUC-MSCs treatment increased body weight and decreased disease activity index (DAI), colon macroscopic damage index (CMDI), and histopathological score (HPS) of rats with TNBS-induced colitis. The results of the clinical study also showed that this mode of hUC-MSCs application was associated with regression of intestinal ulceration. Eight patients (47%) got endoscopic responses (SES-CD improvement of ≥50% from baseline) and three patients (17.65%) got mucosal healing (SES-CD is zero), with a parallel improvement of clinical and laboratory parameters without serious adverse events. RNA sequencing showed hUC-MSCs therapy was associated with an upregulation of transcripts linked to intestinal epithelial barrier integrity and a downregulation of inflammatory signaling pathways in the intestinal mucosa, especially the TNF signaling pathway, IL-17 signaling pathway, and TLR signaling pathway. RNA expression of intestinal epithelial tight junction protein (ZO1, CLAUDIN1, and CDH1), and the RNA expression of major intestinal inflammatory factors in CD (IL-1ß, IL-6, and TNFα, p < 0.001 for all) were improved significantly. Moreover, hUC-MSCs could attenuate the polarization of M1 macrophage induced from THP1, thereby decreasing the mRNA expression of IL-1ß, IL-6, and TNFα significantly (p < 0.05 for all). TSG-6 expression was evaluated in hUC-MSCs culture supernatant after treatment with TNFα, IFNγ, and LPS for 48 h. And hUC-MSCs could inhibit the phosphorylation of JAK/STAT1 in the intestinal mucosa of CD patients. INTERPRETATION: hUC-MSCs transplantation alleviated TNBS-induced colitis in rats. In this pilot clinical study, preliminary data suggested that this approach to administering hUC-MSCs might have potential for clinical efficacy and manageable safety in treating refractory CD, potentially providing hope for better outcomes. No serious adverse events were observed. FUNDING: This work was funded by General Program of National Natural Science Foundation of China (Grant No. 82270639), the Scientific research project of Shanghai Municipal Health Committee (Grant No. 202240001), Specialty Feature Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzb2022-05), Shanghai East Hospital Youth Research and Cultivation Foundation program (Grant No. DFPY2022015), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, Technology Development Project of Pudong Science, Technology and Economic Commission of Shanghai (Grant No. PKJ2021-Y08), Key Disciplines Group Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZxq2022-06), Medical discipline Construction Project of Pudong Health Committee of Shanghai (Grant No. PWYgf2021-02) and National Natural Science Foundation of China (Grant No. 82300604).

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.

Shaoyao decoction alleviates TNBS-induced ulcerative colitis by decreasing inflammation and balancing the homeostasis of Th17/Treg cells.

BACKGROUND: Ulcerative colitis (UC) is a persistent and non-specific inflammatory condition that mainly affects the bowels and has challenging treatment. UC has a growing incidence and significantly affects the well-being of patients. Many medications used to treat UC can disrupt the metabolism and immune system homeostasis, frequently leading to significant adverse effects. Hence, exploring alternative therapies, such as traditional Chinese medicine and probiotics, has recently emerged as a primary research hotspot owing to their safety. Although the therapeutic mechanism of Shaoyao decoction has not been clarified, it has demonstrated a beneficial clinical effect on UC. AIM: This study aimed to assess the effect of Shaoyao decoction on a rat model of UC and investigate its underlying mechanisms. METHODS: The rat model of UC was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The extent of damage to the intestines was assessed using the disease activity index (DAI), colonic mucosa damage index (CMDI), and histological scores. Immunohistochemistry was employed to detect the tissue levels of interleukin (IL)-17, transforming growth factor (TGF)-ß1, and IL-10. Additionally, the proportion of Th17 and Treg cells was detected using flow cytometry. In colon tissue, the levels of forkhead box (Fox)p3, RAR-related orphan receptor (ROR)γt, IL-6, p-STAT3, and STAT3 proteins were quantified by Western blotting. RESULTS: Treatment with Shaoyao decoction enhanced the overall health of rats and reduced colonic damage. Additionally, Shaoyao decoction significantly alleviated the severity of DAI, CMDI, and HS. The proportion of Th17 cells was reduced, and the proportion of Treg cells was increased by Shaoyao decoction. The expression of IL-17 and RORγt was suppressed by Shaoyao decoction, while the expression of IL-10, TGF-ß1, and Foxp3 was increased. The expression of IL-6, p-STAT3, and STAT3 was decreased by Shaoyao decoction. CONCLUSION: The Shaoyao decoction alleviates the symptoms of TNBS-induced UC by decreasing inflammation and mitigating intestinal damage while preserving the balance between Th17 and Treg. Shaoyao decoction modulates the IL-6/STAT3 axis, thereby regulating the balance between Th17 and Treg cells.

[Diosmetin regulates intestinal immune balance by inhibiting PI3K/AKT signaling to relieve 2, 4, 6-trinitrobenzene sulfonic acid-induced Crohn's disease-like colitis in mice].

OBJECTIVE: To investigate the therapeutic mechanism of diosmetin on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease (CD)-like colitis in mice. METHODS: Wild-type C57BL/6 mice were randomized into control group, TNBS-induced CD-like colitis group (TNBS group) and 50 mg·kg-1·d-1 diosmetin-treated group (n=8). Disease activity (DAI) scores, body weight changes, histological scores, colon lengths and colon mucosal levels of TNF-α, IFN-γ, and IL-17A were measured to evaluate the severity of colitis. The changes of T lymphocyte subsets (Th1/Th2 and Th17/Treg) in the mesenteric lymph nodes were analyzed by flow cytometry. Network pharmacology and molecular docking were used to analyze the effect of diosmetin on PI3K/AKT pathway. RESULTS: Compared with TNBS group, diosmetin treatment significantly lowered DAI scores, histological scores, body weight loss and colon mucosal levels of TNF-α, IFN-γ, and IL-17A (P < 0.05) and increased the colon length of the rat models, but these improvements did not reach the control levels (P < 0.05). Diosmetin significantly lowered the percentages of Th1/Th17 cells in the mesenteric lymph nodes in TNBS-treated mice, which remained higher than the control levels (P < 0.05); The percentages of Th2/Treg cells were significantly higher in diosmetin group than in TNBS group (P < 0.05) and the control group (P < 0.05). Network pharmacologic analysis identified 46 intersection targets of diosmetin and CD, and among them AKT1, EGFR, SRC, ESR1, MMP9 and PTGS2 were the top 6 core targets. GO and KEGG analyses showed that the PI3K/AKT signaling pathway was closely related with the therapeutic effect of diosmetin on CD-like colitis. Molecular docking suggested strong binding of diosmetin to the key core targets. Diosmetin significantly reduced the levels of p-PI3K and p-AKT in the colon mucosa in TNBS-treated mice (P < 0.05), but their levels remained higher than those in the control group (P < 0.05). CONCLUSION: Diosmetin ameliorates TNBS-induced CDPlike colitis in mice possibly by regulating Th1/Th2 and Th17/Treg balance to improve intestinal immune disorder through inhibition of PI3K/AKT signaling.

Cornus mas L. Extract Targets the Specific Molecules of the Th17/Treg Developmental Pathway in TNBS-Induced Experimental Colitis in Rats.

Given that one of the crucial events in the pathogenesis of inflammatory bowel disease is the loss of homeostasis between Th17 and Treg cells, targeting the specific molecules of the Th17/Treg axis developmental pathway is a promising strategy for inflammatory bowel disease prevention and treatment. The current study aimed to assess the impact of cornelian cherry (Cornus mas L.) extract, rich in iridoids and polyphenols known for their potential anti-inflammatory activity, at two doses (20 or 100 mg/kg) on the crucial factors for Th17/Treg cell differentiation in the course of experimental colitis and compare this action with that of sulfasalazine. This study was conducted on the biobank colon tissue samples collected during the previous original experiment, in which colitis in rats was induced by trinitrobenzenesulfonic acid (TNBS). The levels of IL-6, RORγt, total STAT3, p-STAT3, and Foxp3 were determined by ELISA. The expression of PIAS3 mRNA was quantified by qPCR. Cornelian cherry extract at a dose of 100 mg/kg counteracted the TNBS-induced elevation of IL-6, RORγt, and p-STAT3 levels and a decrease in Foxp3 level and PIAS3 mRNA expression, while given concomitantly with sulfasalazine was more effective than sulfasalazine alone in reversing the TNBS-induced changes in IL-6, RORγt, total STAT3, p-STAT3, Foxp3 levels, and PIAS3 mRNA expression. The beneficial effect of cornelian cherry extract on experimental colitis may be due to its immunomodulatory activity reflected by the influence on factors regulating the Th17/Treg axis.

Possible Curative Effects of Boric Acid and Bacillus clausii Treatments on TNBS-Induced Ulcerative Colitis in Rats.

Crohn's disease (CD) and ulcerative colitis (UC) are two chronic relapsing inflammatory bowel diseases (IBD). Although there are several treatment options available to improve the symptoms of IBD patients, there is no effective treatment that provides a definitive solution. In the present study, we aim to investigate the antioxidative/anti-inflammatory effects of oral administration of boric acid and Bacillus clausii in a rat trinitrobenzenesulfonic acid (TNBS)-induced colitis model. The effects of boric acid and B. clausii were examined in serum and colon tissues with the help of some biochemical and histological analyses. Elevated inflammation and oxidative damage were found in the blood and colon tissue samples in the TNBS-induced group according to the complete blood count (CBC), tumor necrosis factor (TNF) alpha, interleukin-35 (IL-35), malondialdehyde (MDA), glutathione peroxidase (GPx), myeloperoxidase (MPO), nitric oxide (NO), and histological findings. Particularly, the highest IL-35 level (70.09 ± 12.62 ng/mL) in the combined treatment group, highest catalase activity (5322 ± 668.1 U/mg protein) in the TNBS-induced group, and lower relative expression of inducible nitric oxide synthase in the TNBS-induced group than the control group were striking findings. According to our results, it can be concluded that boric acid showed more curative effects, even if B. clausii probiotics was partially ameliorative.

Roseburia intestinalis stimulates TLR5-dependent intestinal immunity against Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disorder characterized by intestinal immune dysfunction. Multiple factors, including gut dysbiosis, are involved in the pathogenesis of CD. However, the effect of commensal bacteria on controlling the inflammatory response in individuals with CD remains unclear. METHODS: We detected Toll-like receptor 2 (TLR2), TLR4, and TLR5 expression in Roseburia intestinalis (R. intestinalis)-treated mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Then, we quantified the signs of colonic inflammation, the proportion of regulatory T cells (Tregs) and the expression of thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-ß in TLR-5-deficient (Tlr5-/-) mice, bone marrow chimera mice (generated using wild-type (WT) and Tlr5-/- mice), and anti-TSLP/anti-TGFß-treated C57BL/6 mice with colitis induced by TNBS. In vitro, we used the lipopolysaccharide (LPS)-stimulated human intestinal epithelial cell line Caco-2 as an inflammatory colon cell model treated with or without the TLR5-siRNA intervention in the presence of R. intestinalis and incubated human monocyte-derived dendritic cells (DCs) with the supernatant of Caco-2 cells. Then, we cocultured human CD4+ T cells with the aforementioned DCs to determine the differentiation of Tregs. Additionally, samples from patients with CD were collected to analyse the correlation between TLR5/TSLP/TGFß expression and the percentage of R. intestinalis. FINDINGS: Here, we show that R. intestinalis inhibits the development of CD by increasing the differentiation of anti-inflammatory Tregs. Mechanistically, R. intestinalis stimulates TSLP production in intestinal epithelial cells (IECs) through TLR5 but not TLR2 or TLR4. TSLP produced by IECs specifically induces the secretion of interleukin-10 (IL-10) and TGFß from DCs, which is necessary for subsequent Treg differentiation. Consequently, the depletion of TLR5 (using Tlr5-/- mice) or inhibition of TSLP (using anti-TSLP neutralizing antibodies) attenuates the protective effect of R. intestinalis on experimental colitis in mice. Importantly, the expression of TSLP in patients with CD is positively correlated with the level of R. intestinalis. INTERPRETATION: These findings reveal the previously unknown mechanism of R. intestinalis-mediated intestinal immune regulation, which may provide the basis for new therapeutic strategies for CD. FUNDING: This work was funded by the National Natural Science Foundation of China (81670504 and 81970494), the Key Project of Research and Development Plan of Hunan Province (2019SK2041) and the Changsha Municipal Natural Science Foundation (kq2014258).

Targeting Lineage-Specific Transcription Factors and Cytokines of the Th17/Treg Axis by Novel 1,3,4-Oxadiazole Derivatives of Pyrrolo[3,4-d]pyridazinone Attenuates TNBS-Induced Experimental Colitis.

The pharmacotherapy of inflammatory bowel disease (IBD) is still not fully effective and safe. Attempts to search for new IBD drugs remain an incessant research aim. One of the novel approaches is targeting the developmental pathway molecules and effector cytokines of Th17/Treg axis. This study aimed to elucidate the impact of new pyrrolo[3,4-d]pyridazinone derivatives, compounds 7b, 10b, or 13b, on the course of experimental colitis in rats and to assess whether these new compounds may influence Th17/Treg axis. Rats were pretreated with studied compounds intragastrically before intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid used for colitis induction. Body weight loss, disease activity index, colon index, and colon tissue damage were analyzed to evaluate the severity of colitis. The colonic levels of RORγt, STAT3, CCR6, Foxp3, IL-6, IL-10, IL-17, TNF-α, IL-23, and PGE2 were assessed. Pretreatment with compounds 7b and 13b alleviated the severity of colitis and concomitantly counteracted the increased levels of RORγt, STAT3, CCR6, IL-6, IL-17, IL-23, TNF-α, and PGE2. The beneficial effect of compounds 7b and 13b may be due to the decrease in the levels of Th17-specific transcription factors and cytokines. The studied compounds might therefore constitute a promising therapeutic strategy in Th17/Treg imbalance-driven inflammatory conditions such as 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.

Anti-Inflammatory Effect of Topiramate in a Chronic Model of TNBS-Induced Colitis.

Inflammatory bowel disease (IBD) is characterized by a chronic and relapsing inflammatory response in the gastrointestinal tract, resulting in severe symptoms such as abdominal pain, vomiting, diarrhea, bloody stools, and weight loss. Currently, there is no cure, and the pharmacological treatment includes drugs that induce and keep the patient in remission, not reversing the underlying pathogenic mechanism. These therapies, in the long term, may cause various side effects and complications, which has increased the need to investigate new, more effective, and safer pharmacological approaches. In preclinical studies, topiramate has demonstrated a potential anti-inflammatory effect by inhibiting the production of several pro-inflammatory cytokines. This study aimed to investigate the effect of topiramate in a chronic TNBS-induced colitis model in rodents. Experimental colitis was induced by four intrarectal administrations of 1% TNBS in female CD-1 mice. Topiramate 10 and 20 mg were administered intraperitoneally for 14 days. Several parameters were evaluated, such as bodyweight, alkaline phosphatase (ALP), fecal hemoglobin, fecal calprotectin, tumor necrosis factor (TNF)-α, and interleukin (IL)-10. Topiramate reduces TNBS-induced colonic damage in a model of chronic experimental colitis and normalizes the stool consistency and anus appearance. Additionally, topiramate significantly reduced the concentration of ALP, fecal hemoglobin, fecal calprotectin, TNF-α, and IL-10, demonstrating it to be a promising pharmacological approach for the treatment of IBD in the future.

Buspirone Ameliorates Colon Inflammation in TNBS-Induced Rat Acute Colitis: The Involvement of TLR4/NF-kB Pathway.

Inflammatory bowel disease (IBD) is an inflammatory situation involving the whole digestive system. This illness includes ulcerative colitis and Crohn's disease. According to scientific research, the immune system plays an essential part in developing this disease. Recently, buspirone has been discovered to have anti-inflammatory properties. As a result, this research aims to see if buspirone provides anti-inflammatory effects in a rat model of TNBS-induced colitis. Control, TNBS, dexamethasone (2 mg/kg), and buspirone (5, 10, and 20 mg/kg) were randomly given to six groups of 36 male Wistar rats. Colitis was induced by intrarectal instillation of TNBS in all research groups except the control group, and rats were meliorated with dexamethasone and buspirone. Macroscopic and microscopic lesions appeared after colitis induction, while therapy with dexamethasone and buspirone significantly improved the lesions. TLR4 and pNF-κB expression were also enhanced during colitis induction. On the other hand, the administration of dexamethasone or buspirone resulted in a considerable reduction in their expression. Tissue TNF-α and MPO activity were enhanced after induction of colitis in terms of biochemical variables; however, administration of dexamethasone or buspirone reduced TNF-α and MPO activity. Eventually, in an animal model of severe colitis, buspirone displayed anti-inflammatory characteristics via lowering the TLR4/NF-ĸB signaling pathway's activity in an animal model of acute colitis.

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.

Mesenchymal Stem Cells Alleviate Inflammatory Bowel Disease Via Tr1 Cells.

Mesenchymal stem cells (MSCs) have been used to achieve exciting therapeutic outcomes in many animal studies and clinical trials for various autoimmune diseases, including inflammatory bowel disease (IBD). Type 1 regulatory T (Tr1) cells are the main source of interleukin (IL) 10 in the intestine. Whether Tr1 cells are involved during MSC-mediated IBD treatment is unclear. We treated a murine model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis with human umbilical cord-derived MSCs (hUCMSCs) and found that the disease severity was alleviated significantly in a dose-dependent manner. hUCMSCs increased the proportion of Tr1 cells and decreased that of T helper (Th)-1 and Th17 cells in the spleen and mesenteric lymph nodes in different stages of colitis. We found that the upregulation of Tr1 cells by hUCMSCs was abrogated after blocking indoleamine-2,3-dioxygenase (IDO), and IDO knockdown in hUCMSCs reversed the increase in Tr1 cell proportions caused by hUCMSCs in colitis. Moreover, hUCMSCs inhibited apoptosis and promoted the proliferation of Tr1 cells. Our results suggest that Tr1 cells play an important role in the amelioration of IBD by MSCs, and they are the target population for the alleviation of IBD by MSCs, providing meaningful references for the study of therapeutic mechanisms of MSCs in other inflammatory diseases.

Rectal roflumilast improves trinitrobenzenesulfonic acid-induced chronic colitis in rats.

Roflumilast, a highly selective oral phosphodiesterase IV inhibitor, exerts anti-inflammatory and anti-fibrotic effects. Oral roflumilast causes gastrointestinal side effects, especially vomiting, which could be reduced by administering roflumilast via off-label routes. Inhaled roflumilast reportedly improved inflammatory and histopathological changes in asthmatic mice. The current study investigated the effects of oral and rectal roflumilast on trinitrobenzenesulfonic acid (TNBS)-induced chronic colitis in rats, an experimental model resembling human Crohn's disease. Five groups of rats (n=8) were used: normal control, TNBS-induced colitis, and three TNBS-treated colitic groups, which received oral sulfasalazine (500 mg·kg-1·day-1), oral roflumilast (5 mg·kg-1·day-1), or rectal roflumilast (5 mg·kg-1·day-1) for 15 days after colitis induction. Then, the following were assessed: the colitis activity score, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-6 serum levels, colonic length, and myeloperoxidase, malonaldehyde, and glutathione levels. Histological examinations employed H&E, Masson trichrome, and PAS stains in addition to immunostaining for KI-67 and TNF-α. The TNBS-induced colitis rats showed significant increases in disease activity scores, serum TNF-α, IL-2, and IL-6 levels, and colonic myeloperoxidase and malonaldehyde content. They also showed significant decreases in colonic length and glutathione levels in addition to histopathological and immunohistochemical changes. All the treatments significantly improved all these changes. Sulfasalazine provided the greatest improvement, followed by oral roflumilast, and then rectal roflumilast. In conclusion, both oral and rectal roflumilast partially improved TNBS-induced chronic colitis, suggesting the potential of roflumilast as an additional treatment for Crohn's disease.

Efficacy of a functional food ingredient from Ensete superbum Roxb. Cheesman peel in reducing the severity of ulcerative colitis in a murine model.

Ulcerative colitis is a recurrent inflammatory illness of the colon with an elevated risk of developing colon cancer. The drugs presently used to treat UC cause adverse effects and are limited to symptomatic treatments. To overcome these constraints, naturally derived novel alternative therapies are being tested. Ensete superbum Roxb. Cheesman (wild banana) is used as a folk medicinal plant to treat stomach aches, diarrhea, and dysentery. Previous research has shown that a peel dioxane (PD) fraction obtained from a ripe peel aqueous extract of E. superbum Roxb. Cheesman possesses in vitro antioxidant, anti-inflammatory and anti-colon cancer effects. Furthermore, it has been shown to alleviate 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced ulcerative colitis (UC) in rats. The current study intended to evaluate its efficacy as a functional dietary component added to cold pressed orange juice in colitic rats, and deduce the molecular processes involved in UC amelioration. The PD fraction in orange juice ameliorated colitis by modulating the oxidative stress and inflammatory parameters in the damaged tissue with improved healing activity as indicated by a lower disease activity index (DAI) score. The ameliorative effect was related to the inhibition of the nuclear factor-κB (NF-κB) signaling pathway by downregulating the expression levels of NFκBp65, TNF-α, IL-6 and IL-1ß, followed by the recovery of epithelial barrier integrity. The ameliorating effects were statistically similar (p > 0.05) to those of the standard sulfasalazine treated population. The results suggest that PD fractions can be used as a new functional food and as an adjuvant to prevent IBDs due to their anti-colitic effect; however, it needs to be confirmed in clinical trials.

Bone marrow mesenchymal stem cells combined with Atractylodes macrocephala polysaccharide attenuate ulcerative colitis.

The aim of the present study was to explore the effects of bone marrow mesenchymal stem cells (BMSCs), combined with Atractylodes macrocephala polysaccharide (AMP), in an experimental model of ulcerative colitis. BMSCs were first isolated, cultured, and identified by flow cytometry. A rat model of colitis was established by trinitrobenzene sulfonic acid (TNBS) injection. Rats were treated with BMSCs with or without AMP for 1 or 2 weeks. H&E staining was performed to assess the extent of histological injury. IEC-6 and BMSCs were co-cultured and treated with AMP. Cell migration was measured using the Transwell assay, whilst the levels of cytokines in the rat blood samples were detected using ELISA. In addition, cytokine levels in the cell supernatant were measured by microarray. The results showed that BMSCs were successfully isolated. BMSCs treatment could markedly alleviate injury according to histological analysis and regulate inflammatory cytokine production in this rat model of TNBS-induced colitis, where a higher number of BMSCs was found in the intestinal tract, compared to the model. AMP not only potentiated the effects of BMSCs on preventing TNBS-induced colitis but also promoted BMSC homing to the injured tissue and regulated cytokines. Furthermore, BMSCs and AMP promoted the migration of IEC in vitro and influenced multiple genes. In conclusion, AMP treatment improved the therapeutic effects of BMSCs on ulcerative colitis, potentially providing a novel clinical treatment strategy for colitis.

Magnetic resonance colonography assessment of acute trinitrobenzene sulfonic acid colitis in pre-pubertal rats.

Pre-pubertal murine models of acute colitis are lacking. Magnetic resonance colonography (MRC) is a promising minimally invasive tool to assess colitis. We aimed to: 1/ Adapt a model of acute experimental colitis to pre-pubertal rats and determine whether MRC characteristics correlate with histological inflammation. 2/ Test this model by administering a diet supplemented in transforming growth factor ß2 to reverse inflammation. Twenty-four rats were randomized at weaning to one of 3 groups: Trinitrobenzene Sulfonic Acid (TNBS) group (n = 8) fed a standard diet, that received an intra-rectal 60 mg/kg dose of TNBS-ethanol; Control group (n = 8) fed standard diet, that received a dose of intra-rectal PBS; TNBS+MODULEN group (n = 8) that received a dose of TNBS and were exclusively fed MODULEN-IBD® after induction of colitis. One week after induction of colitis, rats were assessed by MRC, colon histopathology and inflammation markers (Interleukin 1ß, Tumor necrosis factor α, Nitric Oxide Synthase 2 and Cyclooxygenase 2). TNBS induced typical features of acute colitis on histopathology and MRC (increased colon wall thickness, increased colon intensity on T2-weighted images, target sign, ulcers). Treatment with MODULEN-IBD® did not reduce signs of colitis on MRC. Inflammatory marker expression did not differ among study groups.

HIF-Overexpression and Pro-Inflammatory Priming in Human Mesenchymal Stromal Cells Improves the Healing Properties of Extracellular Vesicles in Experimental Crohn's Disease.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-ß-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn's disease and likely other immune-mediated inflammatory diseases.

Evaluation of the effect of Dahuang-Mudan decoction on TNBS-induced colitis using UPLC-QTOF/MS-based metabolomic analysis.

Dahuang-Mudan decoction (DMD) is a formula that has been widely used as a complementary treatment for inflammatory bowel disease (IBD). However, the mechanism of action of DMD in IBD has not been clearly elucidated. Therefore, we developed a metabolomics-based method to evaluate the effects and potential mechanisms of DMD in a 2,4,6-trinitobenzene sulfonic acid (TNBS)-induced colitis model. The ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) method combined with multiple analysis approaches including principal component analysis, partial least square discriminant analysis and orthogonal partial least square discriminant analysis were used to investigate the different urinary metabolites. We identified 29 potential biomarkers of TNBS-induced colitis that returned to normal conditions after DMD administration. Pathway analysis indicated that changes in these metabolites were associated with cysteine and methionine metabolism, citric acid cycle, glycolysis and glycolic regeneration, pyruvate metabolism, biosynthesis of valine, leucine and isoleucine, biosynthesis of primary bile acids, glycine, serine and threonine metabolism, caffeine metabolism, arginine and proline metabolism and phenylalanine metabolism. It is worth noting that DMD has potential therapeutic effects on TNBS-induced colitis, which functions by restoring the balance of multiple disturbed pathways to a normal condition. This study suggests the reliability of metabolomics-based approaches to identifying biomarkers and pathways, which facilitate further investigation of the potential mechanisms of DMD.

The efficacy of curcumin on PDGF expression and NF-kappa B pathway: TNBS-induced colitis.

BACKGROUND: Curcumin is an antioxidant and anti-inflammatory molecule known to be a potent inhibitor of nuclear factor kappa B (NF-kappa B). In this study, we aimed to investigate the therapeutic effects of curcumin on colitis induced by a 2,4,6-trinitrobenzene sulfonic acid (TNBS). METHODS: After the induction of colitis under anesthesia, 42 rats were divided into six groups as follows; the curcumin oral group, curcumin (20 mg/kg); the corn oil oral group, corn oil (20 mg/kg) using gastric gavage, the curcumin rectal group, curcumin; the corn oil rectal group, corn oil; the control group, 1 mL saline solution (0.9% NaCl) were administered using the rectal route. In the sham group, only rectal catheterization was performed. At the end of the seven day, the blood and intestinal tissue samples were obtained for histopathological examination and for MPO, MDA, NO, PDGF, IL-6, TNF-alpha, NF-kappaB. RESULTS: The macroscopic damage score was significantly higher in curcumin oral, corn oil oral and saline groups when compared to the sham group (p<0.05). The significant differences between groups were evaluated using the biochemical analysis of intestinal tissue for IL-6, NO, NF-κB, PDGF, TNF-α, MDA, MPO (p<0.05). NF-κB levels of blood in curcumin oral, curcumin rectal, sham, corn oil oral, corn oil rectal groups were significantly increased when compared to saline rectal group (p≤0.001). NF-κB serum levels of corn oil rectal and control group (p≤0.001) were lower than the sham group (p=0.012). CONCLUSION: The effects of curcumin improved possibly by modulating the NF-κB signaling pathway should be considered against colitis alone or in combination with the conventional anti-colitic therapies in future studies.