Gut bacteria exacerbates TNBS-induced colitis and kidney injury through oxidative stress.

Gut microbiota has been implicated in the initiation and progression of various diseases; however, the underlying mechanisms remain elusive and effective therapeutic strategies are scarce. In this study, we investigated the role and mechanisms of gut microbiota in TNBS-induced colitis and its associated kidney injury while evaluating the potential of dietary protein as a therapeutic intervention. The intrarectal administration of TNBS induced colitis in mice, concurrently with kidney damage. Interestingly, this effect was absent when TNBS was administered intraperitoneally, indicating a potential role of gut microbiota. Depletion of gut bacteria with antibiotics significantly attenuated the severity of TNBS-induced inflammation, oxidative damage, and tissue injury in the colon and kidneys. Mechanistic investigations using cultured colon epithelial cells and bone-marrow macrophages unveiled that TNBS induced cell oxidation, inflammation and injury, which was amplified by the bacterial component LPS and mitigated by thiol antioxidants. Importantly, in vivo administration of thiol-rich whey protein entirely prevented TNBS-induced colonic and kidney injury. Our findings suggest that gut bacteria significantly contribute to the initiation and progression of colitis and associated kidney injury, potentially through mechanisms involving LPS-induced exaggeration of oxidative cellular damage. Furthermore, our research highlights the potential of dietary thiol antioxidants as preventive and therapeutic interventions.

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.

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.

Phyllanthus emblica L. polysaccharides ameliorate colitis via microbiota modulation and dual inhibition of the RAGE/NF-κB and MAPKs signaling pathways in rats.

Pharmacological treatments for colitis have limited efficacy and side effects. Plant polysaccharides improve colitis by modulating the gut microbiota. However, the specific benefits of Phyllanthus emblica L. polysaccharides (PEPs) in colitis remain unclear. Therefore, this study aimed to assess the physical characteristics and health advantages of PEP in rats subjected to 2,4,6-trinitrobenzene sulfonic acid (TNBS) treatment. The results showed that PEP (1.226 × 103 kDa) was an α-acidic pyran heteropolysaccharide rich in galactose and galacturonic acid. Prefeeding rats with PEP significantly decreased the levels of NO, MDA, proinflammatory cytokines (IL-6, IL-1ß, TNF-α), apoptosis, and the activities of mucinase and ß-glucuronidase. These changes were accompanied by increases in the levels of anti-inflammatory cytokines (IL-4, IL-10) and antioxidant enzymes (SOD, catalase, GPx) in colitis rats. Mechanistically, PEP suppressed the abundance of inflammatory-related bacteria (Bacteroides, Intestinimonas, and Parabacteroides) while promoting the growth of short-chain fatty acid (SCFA)-producing bacteria (Romboutsia, Clostridium_sensu_stricto_1, and Lactobacillus), along with an increase in SCFA secretion. SCFAs may engage with the GPR43 receptor and inhibit downstream HDAC3, consequently downregulating the activation of the RAGE/NF-κB and MAPK pathways. In conclusion, PEP demonstrated preventive effects through its antioxidant, anti-inflammatory, and microbiota modulation properties, thereby ameliorating TNBS-induced colitis in rats.

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.

Oxyberberine ameliorates TNBS-induced colitis in rats through suppressing inflammation and oxidative stress via Keap1/Nrf2/NF-κB signaling pathways.

BACKGROUND: Ulcerative colitis (UC) is a chronic, unspecific inflammatory bowel disorder lacking effective therapeutic targets and radical drugs. Oxyberberine (OBB), a novel intestinal flora-elicited oxidative metabolite of berberine (BBR), has been revealed to exhibit diverse pharmacological properties. PURPOSE: In this follow-up study, we attempted to shed light on the possible therapeutic effect and latent mechanism of OBB on 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-evoked UC in rats. METHODS: UC rats were established via a gentle enema of TNBS. Rats were sacrificed after intragastric administration of drugs for seven days. The weight reduction, disease activity index, macroscopic and histological colonic alterations were assessed. Further investigation on molecular mechanisms was conducted by ELISA, qRT-PCR, immunohistochemistry, or Western blot. RESULTS: OBB treatment remarkably decreased the weight loss, macroscopic scores, and colonal weight/length ratio, as well as mitigated the colonic pathological deterioration and MPO vitality in colitis rats, achieving a superior protective effect to BBR. Additionally, OBB modulated the disequilibrium between pro- and anti-inflammatory factors by promoting the production of IL-13 and IL-4, and lowering the contents of TNF-α, IL-2, IL-8, and IL-22. Furthermore, OBB pretreatment dramatically ameliorated oxidative stress via enhancing antioxidant defense genes expressions (including HO-1, GCLM, GCLC, and NQO-1), thereby increasing SOD and GSH, and decreasing MDA and ROS activities. Furthermore, OBB strikingly restrained the translocation of NF-κB p65 and phosphorylation of IκBα, promoted HO-1 expression, Keap1 degradation and Nrf2 nuclear translocation. CONCLUSION: The study firstly indicated that OBB had a superior therapeutic effect than BBR against TNBS-elicited colitis in rats. The protective effect of OBB might be closely related to the modulation of Keap1/Nrf2/NF-κB-mediated inflammatory response and oxidant stress. The evidences highlight the potentiality of OBB as a prospective candidate for the amelioration of colitis.

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.

[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.

Response Variability to Drug Testing in Two Models of Chemically Induced Colitis.

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively-bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening.

Radix Panacis quinquefolii Extract Ameliorates Inflammatory Bowel Disease through Inhibiting Inflammation.

OBJECTIVE: To investigate the anti-inflammatory activity of Radix Panacis quinguefolii root extract (RPQE) and its therapeutic effects on inflammatory bowel disease (IBD). METHODS: The 72-hour post-fertilization zebrafish was used to generate the local and systematic inflammation models through tail-amputation and lipopolysaccharide (LPS)-induction (100 µ g/mL), respectively. The Tg(zlyz:EGFP) zebrafish was induced with 75 µ g/mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) for establishing the IBD model. The tail-amputated, LPS-, and TNBS-induced models were subjected to RPQE (ethanol fraction, 10-20 µ g/mL) administration for 12 and 24 h, respectively. Anti-inflammatory activity of RPQE was evaluated by detecting migration and aggregation of leukocytes and expression of inflammation-related genes. Meanwhile, TNBS-induced fish were immersed in 0.2% (W/V) calcein for 1.5 h and RPQE for 12 h before photographing to analyze the intestinal efflux efficiency (IEE). Moreover, the expression of inflammation-related genes in these fish was detected by quantitative polymerase chain reaction. RESULTS: Subject to RPQE administration, the migration and aggregation of leukocytes were significantly alleviated in 3 zebrafish models (P<0.01). Herein, RPQE ameliorated TNBS-induced IBD with respect to a significantly reduced number of leukocytes, improved IEE, and inhibited gene expression of pro-inflammatory factors (P<0.05 or P<0.01). CONCLUSION: RPQE exhibited therapeutic effects on IBD by inhibiting inflammation.

Immunohistochemical characterization of transient receptor potential vanilloid types 2 and 1 in a trinitrobenzene sulfonic acid-induced rat colitis model with visceral hypersensitivity.

Transient receptor potential vanilloid type 2 (TRPV2) and type 1 (TRPV1) are originally identified as heat-sensitive TRP channels. We compared the expression patterns of TRPV2 and TRPV1 in the rat distal colon and extrinsic primary afferent neurons, and investigated their roles in visceral hypersensitivity in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rats. Both TRPV2 and TRPV1 expressions in the colon, dorsal root ganglion (DRG), and nodose ganglion (NG) were significantly upregulated in the TNBS-induced colitis model. TRPV2 cell bodies co-localized with the intrinsic primary afferent marker NeuN and the inhibitory motor neuronal marker nNOS in the myenteric plexus. TRPV2 expressions were further detected in the resident macrophage marker ED2 in the mucosa. In contrast, no TRPV1-expressing cell bodies were detected in the myenteric plexus. Both TRPV2- and TRPV1-positive cell bodies in the DRG and NG were double-labeled with the neuronal retrograde tracer fluorescent fluorogold. Large- and medium-sized TRPV2-positive neurons were labeled with the A-fiber marker NF200, calcitonin gene-related peptide (CGRP), and substance P (SP) in the DRG while small-sized TRPV1-positive neurons were labeled with the C-fiber markers IB4, CGRP, and SP. TRPV2- and TRPV1-positive NG neurons were labeled with NF200 and IB4. TNBS treatment increased p-ERK1/2-positive cells in TRPV2 and TRPV1 neurons but did not affect the TRPV2 and TRPV1 subpopulations in the DRG and NG. Both TRPV2 and TRPV1 antagonists significantly alleviated visceral hypersensitivity in TNBS-induced colitis model rats. These findings suggest that intrinsic/extrinsic TRPV2- and extrinsic TRPV1-neurons contribute to visceral hypersensitivity in an experimental colitis model.

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.

Radix Panacis quinquefolii Extract Ameliorates Inflammatory Bowel Disease through Inhibiting Inflammation / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-inflammatory activity of Radix Panacis quinguefolii root extract (RPQE) and its therapeutic effects on inflammatory bowel disease (IBD).@*METHODS@#The 72-hour post-fertilization zebrafish was used to generate the local and systematic inflammation models through tail-amputation and lipopolysaccharide (LPS)-induction (100 µ g/mL), respectively. The Tg(zlyz:EGFP) zebrafish was induced with 75 µ g/mL 2,4,6-trinitrobenzene sulfonic acid (TNBS) for establishing the IBD model. The tail-amputated, LPS-, and TNBS-induced models were subjected to RPQE (ethanol fraction, 10-20 µ g/mL) administration for 12 and 24 h, respectively. Anti-inflammatory activity of RPQE was evaluated by detecting migration and aggregation of leukocytes and expression of inflammation-related genes. Meanwhile, TNBS-induced fish were immersed in 0.2% (W/V) calcein for 1.5 h and RPQE for 12 h before photographing to analyze the intestinal efflux efficiency (IEE). Moreover, the expression of inflammation-related genes in these fish was detected by quantitative polymerase chain reaction.@*RESULTS@#Subject to RPQE administration, the migration and aggregation of leukocytes were significantly alleviated in 3 zebrafish models (P<0.01). Herein, RPQE ameliorated TNBS-induced IBD with respect to a significantly reduced number of leukocytes, improved IEE, and inhibited gene expression of pro-inflammatory factors (P<0.05 or P<0.01).@*CONCLUSION@#RPQE exhibited therapeutic effects on IBD by inhibiting inflammation.

Oral supplementation with selected Lactobacillus acidophilus triggers IL-17-dependent innate defense response, activation of innate lymphoid cells type 3 and improves colitis.

Live biotherapeutic products constitute an emerging therapeutic approach to prevent or treat inflammatory bowel diseases. Lactobacillus acidophilus is a constituent of the human microbiota with probiotic potential, that is illustrated by improvement of intestinal inflammation and antimicrobial activity against several pathogens. In this study, we evaluated the immunomodulatory properties of the L. acidophilus strain BIO5768 at steady state and upon acute inflammation. Supplementation of naïve mice with BIO5768 heightened the transcript level of some IL-17 target genes encoding for protein with microbicidal activity independently of NOD2 signaling. Of these, the BIO5768-induced expression of Angiogenin-4 was blunted in monocolonized mice that are deficient for the receptor of IL-17 (but not for NOD2). Interestingly, priming of bone marrow derived dendritic cells by BIO5768 enhanced their ability to support the secretion of IL-17 by CD4+ T cells. Equally of importance, the production of IL-22 by type 3 innate lymphoid cells is concomitantly heightened in response to BIO5768. When administered alone or in combination with Bifidobacterium animalis spp. lactis BIO5764 and Limosilactobacillus reuteri, BIO5768 was able to alleviate at least partially intestinal inflammation induced by Citrobacter rodentium infection. Furthermore, BIO5768 was also able to improve colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). In conclusion, we identify a new potential probiotic strain for the management of inflammatory bowel diseases, and provide some insights into its IL-17-dependent and independent mode of action.

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).

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.

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.

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.