Murine Colitis Research Articles

Extracellular vesicles package dsDNA to aggravate Crohn\u2019s disease by activating the STING pathway

Crohn’s disease (CD) is an intestinal immune-dysfunctional disease. Extracellular vesicles (EVs) are membrane-enclosed particles full of functional molecules, e.g., nuclear acids. Recently, EVs have been shown to participate in the development of CD by realizing intercellular communication among intestinal cells. However, the role of EVs carrying double-strand DNA (dsDNA) shed from sites of intestinal inflammation in CD has not been investigated. Here we isolated EVs from the plasma or colon lavage of murine colitis and CD patients. The level of exosomal dsDNA, including mtDNA and nDNA, significantly increased in murine colitis and active human CD, and was positively correlated with the disease activity. Moreover, the activation of the STING pathway was verified in CD. EVs from the plasma of active human CD triggered STING activation in macrophages in vitro. EVs from LPS-damaged colon epithelial cells were also shown to raise inflammation in macrophages via activating the STING pathway, but the effect disappeared after the removal of exosomal dsDNA. These findings were further confirmed in STING-deficient mice and macrophages. STING deficiency significantly ameliorated colitis. Besides, potential therapeutic effects of GW4869, an inhibitor of EVs release were assessed. The application of GW4869 successfully ameliorated murine colitis by inhibiting STING activation. In conclusion, exosomal dsDNA was found to promote intestinal inflammation via activating the STING pathway in macrophages and act as a potential mechanistic biomarker and therapeutic target of CD.

Therapeutic potential of isobavachalcone, a natural flavonoid, in murine experimental colitis by inhibiting NF-κB p65.

The incidence of ulcerative colitis (UC), one of the two types of inflammatory bowel disease, is increasing in many countries. Various natural products have been demonstrated with therapeutic potentials for UC. Herein, the therapeutic effects and mechanisms of isobavachalcone (IBC), a natural chalcone, were evaluated in dextran sulfate sodium (DSS)-induced colitis mice and lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The results demonstrated that IBC treatment significantly improved the clinical symptoms, assessed by the disease activity index (DAI) scores and the histological changes of the colon. The levels of myeloperoxidase (MPO), TNF-α, IL-6, IL-1β, and prostaglandin E2 (PGE2) in colon tissues were suppressed by IBC. The upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB p65 in colon tissues were reversed by IBC as well. Furthermore, IBC significantly inhibited LPS-triggered secretion of TNF-α, IL-6, and nitrite, and nuclear translocation of NF-κB p65, in RAW264.7 cells. The luciferase reporter assay indicated that IBC significantly inhibited LPS-triggered transcription of toll-like receptor 4 (TLR4). Molecular docking results showed that the binding pocket of IBC was adjacent to Ser276 of p65-p50 heterodimer and IBC could form H-bond with Thr191. Collectively, these results demonstrated that IBC ameliorated colitis in mice possibly through inhibition of NF-κB p65.

Short-Chain Naphthoquinone Protects Against Both Acute and Spontaneous Chronic Murine Colitis by Alleviating Inflammatory Responses

Ulcerative colitis (UC) is characterised by chronic, relapsing, idiopathic, and multifactorial colon inflammation. Recent evidence suggests that mitochondrial dysfunction plays a critical role in the onset and recurrence of this disease. Previous reports highlighted the potential of short-chain quinones (SCQs) for the treatment of mitochondrial dysfunction due to their reversible redox characteristics. We hypothesised that a recently described potent mitoprotective SCQ (UTA77) could ameliorate UC symptoms and pathology. In a dextran sodium sulphate- (DSS-) induced acute colitis model in C57BL/6J mice, UTA77 substantially improved DSS-induced body weight loss, disease activity index (DAI), colon length, and histopathology. UTA77 administration also significantly increased the expression of tight junction (TJ) proteins occludin and zona-occludin 1 (ZO-1), which preserved intestinal barrier integrity. Similar responses were observed in the spontaneous Winnie model of chronic colitis, where UTA77 significantly improved DAI, colon length, and histopathology. Furthermore, UTA77 potently suppressed elevated levels of proinflammatory cytokines and chemokines in colonic explants of both DSS-treated and Winnie mice. These results strongly suggest that UTA77 or its derivatives could be a promising novel therapeutic approach for the treatment of human UC.

The Selective 5-HT1A Agonist SR57746A Protects Intestinal Epithelial Cells and Enteric Glia Cells and Promotes Mucosal Recovery in Experimental Colitis.

Neurotrophic growth factors can stabilize the intestinal barrier by preventing the apoptosis of enteric glial cells (EGCs) and enterocytes. We reasoned that a selective 5-HT1A receptor agonist may have neuroprotective properties in the gut and that topical application of SR57746A might be an effective treatment strategy in inflammatory bowel disease (IBD). The therapeutic potential of 5-HT1A receptor agonist SR57746A in IBD was evaluated in vitro (nontransformed NCM460 colonic epithelial cells, SW480 colorectal carcinoma cells) and in vivo (murine dextran sulfate sodium [DSS] colitis and CD4-T-cell transfer colitis). In vitro, we analyzed the effect of SR57746A on apoptosis in intestinal epithelial cells (IECs) and EGCs, and upon proliferation, migration, and intracellular signaling in IECs. In vivo, the effect of topical application of SR57746 on disease activity and on histological and endoscopic findings was compared with intraperitoneal infliximab and placebo, respectively. The SR57746A activates PI3-K/AKT- and ERK-signaling in IECs. Depending on ERK- and AKT activation, SR57746A potently prevents apoptosis of IECs without inducing proliferation or migration in these cells. Moreover, SR57746A prevented apoptosis in EGCs in vitro. Topical SR57746A treatment significantly reduced mucosal injury in 2 experimental murine colitis models and was as effective as intraperitoneal infliximab treatment. Treatment with SR57746A prevents inflammatory cell damage and apoptosis in IECs and EGCs, similar to the neurotrophic effects of EGCs on IECs. Topical treatment with SR57746A could be a candidate for clinical evaluation in the treatment of IBD.

Lactobacillus paracasei R3 protects against dextran sulfate sodium (DSS)-induced\xa0colitis\xa0in mice via regulating Th17/Treg cell balance

Inflammatory bowel diseases (IBD), mainly comprising ulcerative colitis (UC) and Crohn's Disease, are most often a polygenic disorder with contributions from the intestinal microbiome, defects in barrier function, and dysregulated host responses to microbial stimulation. Strategies that target the microbiota have emerged as potential therapies and, of these, probiotics have gained the greatest attention. Herein, we isolated a strain of Lactobacillus paracasei R3 (L.p R3) with strong biofilm formation ability from infant feces. Interestingly, we also found L.p R3 strain can ameliorate the general symptoms of murine colitis, alleviate inflammatory cell infiltration and inhibit Th17 while promote Treg function in murine dextran sulfate sodium (DSS)-induced colitis. Overall, this study suggested that L.p R3 strain significantly improves the symptoms and the pathological damage of mice with colitis and influences the immune function by regulating Th17/Treg cell balance in DSS-induced colitis in mice.

Protease inhibitory activity of secretory leukocyte protease inhibitor ameliorates murine experimental colitis by protecting the intestinal epithelial barrier.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine, and the dysfunction of intestinal epithelial barrier (IEB) may trigger the onset of IBD. Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that has been implicated in the tissue-protective effect in the skin and lung. We found that SLPI was induced in lipopolysaccharides-treated colon carcinoma cell line and in the colon of dextran sulfate sodium (DSS)-treated mice. SLPI-deficient mice were administered DSS to induce colitis and sustained severe inflammation compared with wild-type mice. The colonic mucosa of SLPI-deficient mice showed more severe inflammation with neutrophil infiltration and higher levels of proinflammatory cytokines compared with control mice. Moreover, neutrophil elastase (NE) activity in SLPI-deficient mice was increased and IEB function was severely impaired in the colon, accompanied with the increased number of apoptotic cells. Importantly, we demonstrated that DSS-induced colitis was ameliorated by administration of protease inhibitor SSR69071 and recombinant SLPI. These results suggest that the protease inhibitory activity of SLPI protects from colitis by preventing IEB dysfunction caused by excessive NE activity, which provides insight into the novel function of SLPI in the regulation of gut homeostasis and therapeutic approaches for IBD.

A Retinoid X Receptor Agonist Directed to the Large Intestine Ameliorates T-Cell-Mediated Colitis in Mice.

Retinoid X receptor (RXR) is a nuclear receptor that heterodimerizes with several nuclear receptors, integrating ligand-mediated signals across the heterodimers. Synthetic RXR agonists have been developed to cure certain inflammatory diseases, including inflammatory bowel diseases (IBDs). However, pre-existing RXR agonists, which are lipophilic and readily absorbed in the upper intestine, cause considerable adverse effects such as hepatomegaly, hyperlipidemia, and hypothyroidism. To minimize these adverse effects, we have developed an RXR agonist, NEt-3IB, which has lipophilic and thus poorly absorptive properties. In this study, we evaluated the effects of NEt-3IB in an experimental murine colitis model induced through the adoptive transfer of CD45RBhighCD4+ T cells. Pharmacokinetic studies demonstrated that the major portion of NEt-3IB was successfully delivered to the large intestine after oral administration. Notably, NEt-3IB treatment suppressed the development of T cell-mediated chronic colitis, as indicated by improvement of wasting symptoms, inflammatory infiltration, and mucosal hyperplasia. The protective effect of NEt-3IB was mediated by the suppression of IFN-γ-producing Th1 cell expansion in the colon. In conclusion, NEt-3IB, a large intestine-directed RXR agonist, is a promising drug candidate for IBDs.

Butyrate functions in concert with myeloid-derived suppressor cells recruited by CCR9 to alleviate DSS-induced murine colitis

Ulcerative colitis (UC) is a precancerous disease caused mainly by a combination of genetic susceptibility, environmental factors and microbiota dysbiosis. As a kind of short-chain fatty acid (SCFA), butyrate has been shown to be closely related to the progression of colitis. However, the exact regulatory mechanism of butyrate in colitis needs to be further elucidated. In our current research, the effects of butyrate were examined in a dextran sulfate sodium (DSS)–induced murine colitis model, which simulates human UC. The administration of butyrate significantly reversed the signs of colitis and alleviated colonic histological damage in DSS‑induced colitis. The transcription levels of the main proinflammatory mediators, including tumor necrosis factor–α, interleukin–6 and interleukin–12, were also reduced, as determined by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). This indicates that butyrate could alleviate DSS-induced colitis by inhibiting proinflammatory mediators. In addition, we found that myeloid-derived suppressor cells (MDSCs), which have an inflammation-relieving effect, did not effectively alleviate DSS‑induced colitis but showed a compensatory increase in the DSS group. However, the compensatory increase in MDSCs in the DSS group significantly decreased after butyrate treatment. Moreover, the chemokine receptor CCR9, which mediates the homing of intestinal immune cells, also showed consistent changes similar to MDSCs. Butyrate alone did not have the aforementioned effects on mice. Thus, butyrate may effectively relieve DSS‑induced colitis by synergistic regulatory effects with MDSCs, which migrate and gather through CCR9 recruitment.

An Escherichia coli strain with extra catalase activity protects against murine colitis by scavenging hydrogen peroxide and regulating regulatory t cell/interleukin-17 pathways

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract whose occurrence is attributed to various factors, including genetic factors, immune response, microbial changes, and oxidative stress. Microbial-targeted therapy has emerged as an alternative to immunosuppressive therapy for IBD. MethodsThe effects of an atypical commensal Escherichia coli strain harboring an additional catalase gene (compared to typical E. coli strain) on dextran sulfate sodium (DSS)-induced colitis were explored in mice. ResultsThe atypical E. coli (atEc) significantly restored body weight, reduced disease activity score, and improved histological scores in mice with colitis. Hydrogen peroxide levels in colitis mice were noticeably decreased when the mice were administered atEc. The proinflammatory cytokine levels were decreased and regulatory T cell numbers were increased after the administration of atEc. The abundance of Firmicutes was significantly recovered, while that of Proteobacteria decreased in atEc -treated mice compared with that in vehicle-treated wild-type mice. To investigate the role of interleukin (IL)-17A in mediating the anti-inflammatory effects of the atEc, IL-17A‒knockout mice were orally administered atEc. Clinical and immune responses and microbial composition were significantly reduced in IL-17A‒knockout mice compared with those in wild-type mice. ConclusionsatEc ameliorates colonic inflammation by controlling hydrogen peroxide levels, immune responses (including regulatory T cells and IL-17A), and microbial composition. atEc could be a novel candidate of probiotic for IBD treatment.

IL-10 Receptor Neutralization-Induced Colitis in Mice: A Comprehensive Guide.

Interleukin-10 (IL-10) and its receptor (IL-10R) have been foremost targets to understand inflammatory bowel disease (IBD) pathogenesis. For the past several decades, IL-10-deficient (Il10-/- ) mice were considered one of the best models to study immune-mediated colitis. Several physiologic limitations with this model, e.g., delayed and varied disease onset, have hindered investigators in testing new clinical therapies for IBD. In this article, we provide comprehensive guidance for using anti-IL-10R monoclonal antibody (αIL-10R mAb) neutralization as a superior alternative model to study IBD. This article describes the feasibility of using αIL-10R mAb to induce chronic colitis (within 4 weeks), perform time-dependent mechanistic studies, and assess the efficacy of IBD therapeutics. This article also delineates protocols for in-house assays to critically assess colitis and associated inflammatory parameters. Overall, we underscore αIL-10R mAb neutralization as a relevant immune-mediated murine colitis model to study human Crohn's disease. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Induction of chronic colitis in mice via αIL-10R mAb neutralization Basic Protocol 2: Biochemical evaluation of αIL-10R mAb neutralization-induced chronic colitis Support Protocol 1: Stool analysis and scoring Support Protocol 2: Swiss roll method.

Dioscin ameliorates murine ulcerative colitis by regulating macrophage polarization

Restoring immune balance by targeting macrophage polarization is a potentially valuable therapeutic strategy for ulcerative colitis (UC). Dioscin is a steroidal saponin with potent anti-inflammatory, immunoregulatory, and hypolipidemic effects. This study examined the protective effect of Dioscin on UC in mice and explored the underlying mechanisms. Mice were induced colitis by dextran sulfate sodium (DSS) and concurrently treated with Dioscin oral administration. RAW264.7 cells were skewed to M1 macrophage polarization by lipopolysaccharide (LPS) and interferon-γ (INF-γ) in vitro, and received Dioscin treatment. The results showed that Dioscin ameliorated colitis in mice, reduced macrophage M1 polarization, but markedly promoted M2 polarization in mice colon. Dioscin inhibited mammalian target rapamycin complex 1 (mTORC1)/hypoxia-inducible factor-1α (HIF-1α) signaling and restrained glycolysis in RAW264.7; however, it activated mammalian target rapamycin complex 2 (mTORC2)/peroxisome proliferator-activated receptor-γ (PPAR-γ) signal and facilitated fatty acid oxidation (FAO). The modulation of mTORs signaling may inhibit M1, but promote M2 polarization. Furthermore, the effect of Dioscin on M2 polarization was neutralized by the FAO inhibitor Etomoxir and the mTORC2 inhibitor JR-AB2-011. In parallel, the inhibitory effect of Dioscin on M1 polarization was mitigated by the mTORC1 agonist L-leucine. Both JR-AB2-011 and L-leucine blocked the therapeutic effect of Dioscin in mice with UC. Therefore, Dioscin ameliorated UC in mice, possibly by restraining M1, while skewing M2 polarization of macrophages. Regulation of mTORC1/HIF-1α and mTORC2/PPAR-γ signals is a possible mechanism by which Dioscin inhibited aerobic glycolysis and promoted FAO of macrophages. In summary, Dioscin protected mice against DSS-induced UC by regulating mTOR signaling, thereby adjusting macrophage metabolism and polarization.

Fermented and Germinated Processing Improved the Protective Effects of Foxtail Millet Whole Grain Against Dextran Sulfate Sodium-Induced Acute Ulcerative Colitis and Gut Microbiota Dysbiosis in C57BL/6 Mice.

This study investigated the effects of foxtail millet whole grain flours obtained through different processing methods on alleviating symptoms and gut microbiota dysbiosis in a dextran sulfate sodium (DSS)-induced murine colitis model. Sixty C57BL/6 mice were divided into six groups (n = 10 in each group), including one control group (CTRL) without DSS treatment and five DSS-treated groups receiving one of the following diets: AIN-93M standard diet (93MD), whole grain foxtail millet flour (FM), fermented (F-FM), germinated (G-FM), and fermented-germinated foxtail millet flour (FG-FM). A comparison of the disease activity index (DAI) demonstrated that foxtail millet whole grain-based diets could alleviate the symptoms of enteritis to varying degrees. In addition, 16S rRNA gene sequencing revealed that FG-FM almost completely alleviated DSS-induced dysbiosis. Mice on the FG-FM diet also had the lowest plasma IL-6 levels and claudin2 expression levels in the colon, indicating reduced systemic inflammation and improved gut barrier function. This study suggested that foxtail millet whole grain is an attractive choice for the intervention of IBD and gut microbiota dysbiosis, and its prebiotic properties are highly affected by the processing methods.

New Therapeutic Approach for Intestinal Fibrosis Through Inhibition of pH-Sensing Receptor GPR4.

Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of pH-sensing receptors compared with non-IBD controls. Acidification leads to angiogenesis and extracellular matrix remodeling. We aimed to determine the expression of pH-sensing G protein-coupled receptor 4 (GPR4) in fibrotic lesions in Crohn's disease (CD) patients. We further evaluated the effect of deficiency in Gpr4 or its pharmacologic inhibition. Paired samples from fibrotic and nonfibrotic terminal ileum were obtained from CD patients undergoing ileocaecal resection. The effects of Gpr4 deficiency were assessed in the spontaneous Il-10-/- and the chronic dextran sodium sulfate (DSS) murine colitis model. The effects of Gpr4 deficiency and a GPR4 antagonist (39c) were assessed in the heterotopic intestinal transplantation model. In human terminal ileum, increased expression of fibrosis markers was accompanied by an increase in GPR4 expression. A positive correlation between the expression of procollagens and GPR4 was observed. In murine disease models, Gpr4 deficiency was associated with a decrease in angiogenesis and fibrogenesis evidenced by decreased vessel length and expression of Edn, Vegfα, and procollagens. The heterotopic animal model for intestinal fibrosis, transplanted with terminal ileum from Gpr4-/- mice, revealed a decrease in mRNA expression of fibrosis markers and a decrease in collagen content and layer thickness compared with grafts from wild type mice. The GPR4 antagonist decreased collagen deposition. The GPR4 expression was also observed in human and murine intestinal fibroblasts. The GPR4 inhibition reduced markers of fibroblast activation stimulated by low pH, notably Acta2 and cTgf. Expression of GPR4 positively correlates with the expression of profibrotic genes and collagen. Deficiency of Gpr4 is associated with a decrease in angiogenesis and fibrogenesis. The GPR4 antagonist decreases collagen deposition. Targeting GPR4 with specific inhibitors may constitute a new treatment option for IBD-associated fibrosis.

Persistence of Lgr5+ colonic epithelial stem cells in mouse models of inflammatory bowel disease.

Intestinal mucosal healing is the primary therapeutic goal of medical treatments for inflammatory bowel disease (IBD). Epithelial stem cells are key players in the healing process. Lgr5+ stem cells maintain cellular turnover during homeostasis in the colonic crypt. However, they are lost and dispensable for repair in a wide variety of injury models, including dextran sulfate sodium (DSS) colitis, radiation, helminth infection, and T-cell activation. The direct loss of Lgr5+ cells activates a plasticity response in the epithelium in which other cell types can serve as stem cells. Whether this paradigm applies to mouse models of IBD remains unknown. In contrast to previously tested models, IBD models involve an inflammatory response rooted in the loss of immunologic tolerance to intestinal luminal contents including the microbiome. Here, we show the persistence of Lgr5+ cells in oxazolone, 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Il10-/-, and Il10-/- Tnfr1-/- IBD models. This contrasts with results obtained from DSS-induced injury. Through high-throughput expression profiling, we find that these colitis models were associated with distinct patterns of cytokine expression. Direct exposure of colonic epithelial organoids to DSS, oxazolone, or TNBS resulted in increased apoptosis and loss of Lgr5+ cells. Targeted ablation of Lgr5+ cells resulted in severe exacerbation of chronic, antibody-induced IL-10-deficient colitis, but had only modest effects in TNBS-induced colitis. These results show that distinct mouse models of IBD-like colitis induce different patterns of Lgr5+ stem cell retention and function.NEW & NOTEWORTHY Acute intestinal injury and epithelial repair are associated with the loss of fast-cycling Lgr5+ stem cells and plasticity in the activation of formerly quiescent cell populations. In contrast, here we show in murine inflammatory bowel disease the persistence of the Lgr5+ stem cell population and its essential role in restricting the severity of chronic colitis. This demonstrates a diversity of stem cell responses to colitis.

The Regulatory Effects of Licochalcone A on the Intestinal Epithelium and Gut Microbiota in Murine Colitis.

The gut epithelium is a mechanical barrier that protects the host from the luminal microenvironment and interacts with the gut microflora, which influences the development and progression of ulcerative colitis (UC). Licochalcone A (LA) exerts anti-inflammatory effects against UC; however, whether it also regulates both the gut barrier and microbiota during colitis is unknown. The current study was conducted to reveal the regulatory effects of LA on the intestinal epithelium and gut microflora in C57BL/6 mice subjected to dextran sodium sulfate (DSS). Sulfasalazine (SASP) was used as the positive control. Results of clinical symptoms evaluation, hematoxylin, and eosin (H&E) staining, and enzyme-linked immunosorbent (ELISA) assays showed that LA significantly inhibited DSS-induced weight loss, disease activity index (DAI) increase, histological damage, and gut inflammation. Additionally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunohistochemical (IHC) analysis showed that LA maintained the integrity of the intestinal barrier by suppressing cell apoptosis and preserving the expression of tight junction (TJ) proteins. Notably, the optimal dose of LA for gut barrier preservation was low, while that for anti-inflammatory effects was high, indicating that LA might preserve gut barrier integrity via direct effects on the epithelial cells (ECs) and TJ proteins. Furthermore, 16S rRNA analysis suggested that the regulatory effect of LA on the gut microbiota differed distinctly according to dose. Correlation analysis indicated that a low dose of LA significantly modulated the intestinal barrier-associated bacteria as compared with a moderate or high dose of LA. Western blot (WB) analysis indicated that LA exhibited anti-UC activity partly by blocking the mitogen-activated protein kinase (MAPK) pathway. Our results further elucidate the pharmacological activity of LA against UC and will provide valuable information for future studies regarding on the regulatory effects of LA on enteric diseases.

Targeting of the Tec Kinase ITK Drives Resolution of T Cell–Mediated Colitis and Emerges as Potential Therapeutic Option in Ulcerative Colitis

The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. We analyzed patients with inflammatory bowel disease (n= 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.

Colon-targeted delivery of tacrolimus using pH-responsive polymeric nanoparticles for murine colitis therapy

The present study aimed at constructing an oral nanoparticle delivery system loaded with tacrolimus (FK506) for effective treatment of inflammatory bowel disease. A FK506/HP-β-CD inclusion compound was prepared by grinding to increase drug solubility. To address the side- effects in non-target organs and systemic toxicity of FK506, pH-responsive Eudragit S100 (ES100) and hyaluronic acid (HA) with high affinity to CD44 receptor were adsorbed onto the surface of chitosan (CS) nanoparticles loaded with FK506/HP-β-CD through electrostatic interactions to obtain FK506@ES100/HA/CS/HP-β-CD nanoparticles (FK506@EHCh NPs). Caco-2 cells and Raw 264.7 macrophages were used to confirm the lack of cytotoxicity and good uptake ability of the newly generated nanoparticles. FK506@EHCh NPs significantly suppressed secretion of TNF-α, IL-1β and IL-6 by LPS-activated Raw 264.7 macrophages. A dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) murine model was established to further confirm the colon targeting and in vivo efficacy of oral IR-775@EHCh NPs. Based on the collective results, we conclude that packaging FK506 into active targeting nanocarriers sensitive to pH facilitates concentration of the drug within the sites of intestinal inflammation and improves the drug levels in target tissues, thus avoiding systemic side-effects and improving efficacy. In view of the promising results obtained in this study, the potential of EHCh nanoparticles for drug delivery and targeted treatment of inflammatory bowel disease warrants further investigation.

Deguelin treatment attenuated the lipopolysaccharides-provoked inflammation in murine macrophage and dextran sulfate sodium-inflamed acute colitis in mice through suppression of inflammatory markers

Background: Inflammatory bowel disease was a chronic inflammatory disease associated with the gastrointestinal region along with its two main types, i.e., ulcerative colitis and Crohn's disease. The occurrences of colitis were quickly expanding in recent decades worldwide. Objectives: In this investigation, we anticipated to examine the curative usefulness of deguelin, a natural herbal rotenoid against the lipopolysaccharide (LPS)-provoked inflammatory responses in murine macrophages and dextran sulfate sodium (DSS)-inflamed acute colitis in replica of mice. Materials and Methods: The inflammation response in RAW-264.7 cells was triggered with the LPS administration. The acute colitis was stimulated in BALB/c mice through administering the DSS. The colon length and disease activity index score was evaluated. The statuses of inflammatory arbitrators such as interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and nitric oxide (NO) and enzymatic function of myeloperoxidase were inspected via commercial kits. The matrix metalloproteinase-2 expression in the colon tissues and colon histological examination were done to assess the deleterious effects of DSS in mice. Results: The deguelin treatment markedly alleviated the proinflammatory markers augmentation such as IL-6, TNF-α, and NO in the murine macrophages, as well as in DSS-provoked colitic mice. Deguelin markedly reversed the colon shortening and also improved the colon weight. The histopathological investigation of colon tissues exposed the protective effect of deguelin. A marked alleviation in DSS-incited colon inflammation was noted in the deguelin-supplemented mice. Conclusion: The findings of this research were established the remedial values of deguelin against the DSS-inflamed colitis in mice. It was clinched that the deguelin can be a promising therapeutic agent to treat the colitis.

L-arginine as a novel target for clinical intervention in inflammatory bowel disease

Arginase-1 (Arg1) and the inducible nitric oxide synthase 2 (NOS2) compete for the common substrate L-arginine, semi-essential amino acid, and central intestinal metabolite. Both enzymes exhibit various, sometimes opposing effects on immune responses, tissue regeneration, or microbial growth and replication. In sub-mucosal tissues of patients suffering from inflammatory bowel disease (IBD), similar as in experimental colitis, the expression and activity of both enzymes, Arg1 and NOS2 are more prominent than in respective controls. Accordingly, the metabolism of L-arginine is altered in IBD patients. Thus, L-arginine represents a promising medical target for clinical intervention in these devastating diseases. Previous studies primarily focused on the host side of L-arginine metabolism. Initial reports using Arg1 inhibitors generated conflicting results in murine colitis models. Subsequently, only the generation of conditional Arg1 knockout mice allowed reliable functional analyses of Arg1 and the L-arginine metabolism in the immune system. Utilizing cell-specific conditional Arg1 knockouts, we have recently reported that Arg1, surprisingly, hampered the resolution of experimental colitis due to the restriction of the intraluminal availability of L-arginine. Reduced levels of L-arginine restrained the compositional diversity of the intestinal microbiota and subsequently the mutual metabolism between the microbiota and the host. Thus, the intraluminal microbiota represents a potential therapeutic target for L-arginine metabolism aside from host-dependent L-arginine consumption.

Aureusidin derivative CNQX inhibits chronic colitis inflammation and mucosal barrier damage by targeting myeloid differentiation 2 protein.

Our previous study has found that aureusidin can inhibit inflammation by targeting myeloid differentiation 2 (MD2) protein. Structural optimization of aureusidin gave rise to a derivative named CNQX. LPS was used to induce inflammation in intestinal macrophages; flow cytometry, PI staining and Hoechst 33342 staining were used to detect the apoptotic level of macrophages; enzyme‐linked immunosorbent assay (ELISA) was utilized to detect the expression level of inflammatory factors (including IL‐1β, IL‐18 and TNF‐α); immunofluorescence staining was used to investigate the expression of MD2; Western blot was employed to measure the protein level of TLR4, MD2, MyD88 and p‐P65. As a result, CNQX with IC50 of 2.5 μM can significantly inhibit the inflammatory damage of macrophages, decrease apoptotic level, reduce the expression level of inflammatory factors and simultaneously decrease the expression level of TLR4, MD2, MyD88 as well as p‐P65. Caco‐2 cell line was used to simulate the intestinal mucosal barrier in vitro, LPS was employed to induce cell injury in Caco‐2 (to up‐regulate barrier permeability), and CNQX with IC50 of 2.5 μl was used for intervention. Flow cytometry was used to detect the apoptotic level of Caco‐2 cells, trans‐epithelial electric resistance (TEER) was measured, FITC‐D was used to detect the permeability of the intestinal mucosa, and Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2). As a result, CNQX decreased the apoptotic level of Caco‐2 cells, increased TEER value, decreased the expression levels of MyD88, TLR4 and MD2, and increased the protein levels of tight junction proteins (including occludin and claudin‐1). C57BL/6 wild‐type mice were treated with drinking water containing Dextran sulphate sodium (DSS) to establish murine chronic colitis model. After CQNX intervention, we detected the bodyweight, DAI score and H&E tissue staining to evaluate the life status and pathological changes. Immunohistochemistry (IHC) staining was used to detect the expression of MD2 protein, tight junction protein (including occludin and claudin‐1). Transmission electron microscopy and FITC‐D were used to detect intestinal mucosal permeability. Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2) in the intestinal mucosa tissue. Consequently, CNQX can inhibit the intestinal inflammatory response in mice with colitis, inhibit the mucosal barrier injury, increase the expression of tight junction proteins (including occludin and claudin‐1) and decrease the expression levels of MyD88, TLR4 and MD2. Mechanistically, pull‐down and immunoprecipitation assays showed that CNQX can inhibit the activation of TLR4/MD2‐NF‐κB by binding to MD2 protein.Collectively, in this study, we found that CNQX can suppress the activation of TLR4 signals by targeting MD2 protein, thereby inhibiting inflammation and mucosal barrier damage of chronic colitis.