Colitis Tissue Research Articles

Characterization of candidate genes in inflammatory bowel disease-associated risk loci.

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn's disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.

Epidermal growth factor is a critical regulator of the cytokine IL-33 in intestinal epithelial cells.

IL-33 is a novel cytokine that is believed to be involved in inflammation and carcinogenesis. However, its source, its production and its secretion process remain unclear. Recently, we have reported that IL-33 is up-regulated in dextran sulfate sodium (DSS) colitis in mice. Production of IL-33 from intestinal tissue was studied in a murine cancer model induced by azoxymethane (AOM) and DSS in vivo and in cultures of IEC-6 epithelial cells. Cytokine levels were measured by real time PCR, immunohistochemistry and elisa. Mice with AOM/DSS-induced colitis expressed all the characteristic symptoms of colon cancer pathology. Immunohistochemical analysis demonstrated epithelial cell-derived IL-33 in colon tissues from mice with AOM/DSS colitis. Real time PCR and quantitative PCR analysis revealed that AOM/DSS colitis tissues expressed up-regulated IL-1β, IL-33, TGF-β, and EGF mRNA. Gefitinib, an EGFR inhibitor, inhibited IL-33 mRNA expression in AOM/DSS colitis mice. The pathophysiological role of IL-33 in the rat intestinal epithelial cell line (IEC-6 cells) was then investigated. We found that EGF, but not TGF-β1 or PDGF, greatly enhanced mRNA expression of IL-33 and its receptor ST2. In accordance with the gene expression and immunohistochemical analysis of IL-33 levels, elisa-based analysis of cytoplasmic and nuclear extracts showed increased IL-33 protein levels in IEC-6 cells after treatment with EGF. Our results suggest that EGF is a key growth factor that increased IL-33 production and ST2 receptor expression during intestinal inflammation and carcinogenesis. The EGF/IL-33/ST2 axis represents a novel therapeutic target in colon cancer.

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

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

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease.

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

PSMP, a novel chemotactic cytokine, triggers inflammatory Ly6ChiCCR2+ monocyte migration and promotes colitis in a CCR2-dependent manner

Abstract Chemokines plays a crucial role in the migration of leukocytes, which are involved in immune and inflammatory reactions with their receptors. To find novel chemotactic cytokines, we carried out genome-wide bioinformatics analysis and PBMC chemoattractant platform screen. Seven novel potential chemotactic cytokines were picked up, one of them was PC3-secreted microprotein (PSMP). The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, lymphocytes and THP-1 cells. CCR2 was identified as a high-affinity receptor for PSMP by in vitro chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays. Furthermore, we found that PSMP was extensively expressed at low levels in normal human tissues, but was significantly up-regulated in intestinal biopsies from patients with inflammatory bowel disease and mouse DSS-induced colitic tissue in the early stage prior to IL-6, TNF-α and CCL2 up-regulation. Moreover, the results showed that PSMP triggered inflammatory Ly6ChiCCR2+ monocyte migration from the circulation into the lamina propria in a CCR2-dependent manner and promoted the progression of inflammation. A PSMP neutralizing monoclonal antibody significantly mollified inflammation in a mouse DSS colitis model. Taken together, our study provides the first evidence that PSMP is a novel chemotactic cytokines whose receptor is CCR2 and PSMP plays a vital and irreplaceable role in triggering and promoting DSS colitis. Our finding not only reveals the function and regulating mechanism of PSMP, but also provides a promising therapeutic strategy for treating inflammatory bowel disease.

Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-α, and IL-1β, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

Fusobacterium Isolates Recovered From Colonic Biopsies of Inflammatory Bowel Disease Patients in Korea.

Dear Editor, Inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory disorders of the intestine that are characterized by abdominal pain and diarrhea [1,2]. The incidence of IBD is increasing globally and has also increased in Korea recently [2,3]. Although the etiology is unknown, the relationship between the colonic microbiota and IBD has been indicated as a possible contributor. Fusobacterium varium and F. nucleatum have emerged as compelling candidates responsible for IBD exacerbation [4,5]. The colonization by these strains is possibly a useful biomarker for diagnosing gastrointestinal diseases [5]. We aimed to determine the prevalence of Fusobacterium spp. from colonic biopsies of IBD patients in Korea. All 54 patients fulfilling the diagnostic criteria for IBD including 26 of CD, 25 of UC, or 3 of Behcet's disease (BD), at Hanyang University Guri Hospital between June 2014 and June 2015 were included in this study. Among them, 41 (76%) were male. The patient ages ranged from 14 to 75 yr (median: 31 yr). The Institutional Review Board of Hanyang University Guri Hospital approved this study. Biopsy material was obtained from patients undergoing colonoscopy for assessment of IBD status or for confirmation of gastrointestinal disease. Tissues were put into pre-reduced phosphate-buffered saline and immediately transported to the laboratory for bacterial culture. The specimens were ground, inoculated on crystal violet erythromycin (CVE) agar supplemented with tryptophan, and incubated at 37℃ for 48 hr in an anaerobic jar [6]. All isolates showing a different colony morphology were identified by 16S rRNA gene sequencing by using PCR predicted to yield a product of 1,300 bp [7]. The PCR conditions were as follows: 95℃ for 5 min; 30 cycles at 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 1 min; and a final 10-min step at 72℃. The sequencing of the amplification products were performed in a commercial facility (Macrogen, Seoul, Korea). The sequencing analysis was completed using the GenBank and EzTaxon databases. Some strains were identified using the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA, USA) and the Vitek MS system (bioMerieux, Marcy l'Etoile, France). Ten Fusobacterium isolates were recovered from nine of the 54 patients as follows: six F. mortiferum, two F. varium, and one F. nucleatum and F. mortiferum (Table 1). Table 1 Fusobacterium species and other bacteria isolated from nine patients, identified by using 16S rRNA gene sequencing Clinically, the most important Fusobacterium species are F. nucleatum and F. necrophorum, but F. ulcerans and the F. mortiferum–F. varium group have occasionally been isolated from human. Ohkusa et al. [4] suggested that F. varium might be one of the elusive pathogenic factors in UC. In addition, Strauss et al. [5] reported that an F. nucleatum strain isolated from IBD tissues was more invasive than strains isolated from normal tissues. A correlation between UC and colonization with F. varium has also been noted in a cohort of Japanese patients [4]. F. nucleatum has been linked to colorectal cancer as well as IBD, which has been recognized as a risk factor of colorectal cancer [8]. Fusobacterium spp. were isolated from 63.6% and 53.1% of IBD patients in Canada and Japan, where F. nucleatum was more prevalent in the Canadian patients (43%) than in the Japanese patients (4%) [5,9]. The prevalence of Fusobacterium in our study cohort was 17%, which was lower than that in Canada and Japan. There were limitations of our study that could have affected the low recovery rate. First, whereas we allowed only two days of incubation time for anaerobic culture, Strauss et al. [5] allowed five days. Hence, it was possible that some Fusobacterium spp. were lost owing to early termination of the incubation. Furthermore, with a longer incubation time, the anaerobic chamber may be more optimal than the anaerobic jar that we used. Second, for the specific recovery of Fusobacterium spp. from clinical specimens, the josamycin, vancomycin, and norfloxacin in JVN agar are better than the CVE agar that we used [10]. Further studies are necessary because of our small sample size and the absence of strains isolated from healthy controls. Although it is not possible to draw any strong conclusions from this small data subset, it is likely that there are differences of isolated species according to the country of origin, supporting the notion that no single bacterial species is a biomarker of IBD. In conclusion, this study is the first attempt to assess the prevalence of Fusobacterium spp. in colonic tissues of IBD patients in Korea, revealing that only 17% of the patients carried species of this genus.

Anti-inflammatory activity and mechanisms of a lipid extract from hard-shelled mussel (Mytilus coruscus) in mice with dextran sulphate sodium-induced colitis

The anti-inflammatory effect of a lipid extract from hard-shelled mussel (HMLE) on dextran sulphate sodium (DSS)-induced colitis in mice was investigated. Salicylazosulphapyridine (SASP) and different doses of HMLE were administered by gastric gavage. HMLE significantly attenuated DSS-induced colitis disease activity index scores, tissue damage, splenic enlargement and colon myeloperoxidase accumulation. In addition, HMLE improved colon oxidative stress and production and expression of anti-inflammatory cytokine, interleukin (IL)-10, while HMLE inhibited the abnormal productions and mRNA expressions of pro-inflammatory cytokines, namely tumour necrosis factor-α, IL-1β, and IL-6, as well as the expression of key molecules in the toll-like receptor (TLR)-4/nuclear factor (NF)-κB signalling pathway. These findings suggest that HMLE has an anti-inflammatory effect on DSS-induced colitis, equivalent to that of SASP, and this effect might be related to the regulation of inflammatory mediators and key molecules in the TLR-4/NF-κB pathway.

Rheumatoid Arthritis

Immune-mediated inflammatory disorders include a clinically diverse group of conditions sharing similar pathogenic mechanisms. Conditions such as rheumatoid arthritis, psoriasis, spondyloarthropathy, inflammatory bowel disease and connective tissue diseases are characterised by immune dysregulation and chronic inflammation. This review will focus immuno-pathogenic mechanisms, aspects of early disease, co-morbidity and therapy in rheumatoid arthritis.

MicroRNA 429 Regulates Mucin Gene Expression and Secretion in Murine Model of Colitis.

miRNAs are non-coding RNAs that play important roles in the pathogenesis of human diseases by regulating target gene expression in specific cells or tissues. We aimed to detect miRNAs related to ulcerative colitis [UC], identify their target molecules, and analyse the correlation between the miRNAs and their target genes in colorectal cells and dextran sulphate sodium [DSS]-induced mouse colitis. UC-associated miRNAs were identified by miRNA microarray analysis using DSS-induced colitis and normal colon tissues. The results were validated by quantitative real-time polymerase chain reaction [RT-PCR]. We identified target genes of MIR429, a colitis-associated miRNA, from our screen by comparing the mRNA microarray analysis in MIR429-overexpressed cells with predicted candidate target genes. We constructed luciferase reporter plasmids to confirm the effect of MIR429 on target gene expression. The protein expression of the target genes was measured by western blot,enzyme-linked immunosorbent assay [ELISA] analysis, or immunohistochemistry. We identified 37 DSS-induced colitis associated miRNAs. We investigated MIR429 that is down-regulated in DSS-induced colitis, and identified 41 target genes of MIR429. We show that the myristoylated alanine-rich protein kinase C substrate [MARCKS] is a direct target of MIR429. MARCKS mRNA and protein expression levels are down-regulated by MIR429, and MIR429 regulates the expression of MARCKS and MARCKS-mediated mucin secretion in colorectal cells and DSS-induced colitis. In addition, anti-MIR429 up-regulates MARCKS expression in colorectal cell lines. Our findings suggest that MIR429 modulates mucin secretion in human colorectal cells and mouse colitis tissues by up-regulating of MARCKS expression, thereby making MIR429 a candidate for anti-colitis therapy in human UC.

Combination Therapy for Ulcerative Colitis: Orally Targeted Nanoparticles Prevent Mucosal Damage and Relieve Inflammation

Combination therapy is an emerging strategy that is under intensive preclinical investigation for the treatment of various diseases. CD98 is highly overexpressed on the surfaces of epithelial cells and macrophages in the colon tissue with ulcerative colitis (UC), which is usually associated with mucosal damage and inflammation. We previously proved that CD98 siRNA (siCD98)-induced down-regulation of CD98 in colitis tissue decreased the severity of UC to a certain extent. In an effort to further improve the therapeutic efficacy, we aim to simultaneously deliver siCD98 in combination with a potent anti-inflammatory agent, curcumin (CUR), using hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant spherical HA-siCD98/CUR-NPs are featured by a desirable particle size (∼246 nm) and slightly negative zeta potential (∼-14 mV). The NPs functionalized with HA are able to guide the co-delivery of drugs to the targeted cells related to UC therapy (colonic epithelial cells and macrophages). Compared to either siCD98- or CUR-based monotherapy, co-delivery of siCD98 and CUR by HA-functionalized NPs can exert combinational effects against UC by protecting the mucosal layer and alleviating inflammation both in vitro and in vivo. This study shows the promising capability of the co-delivered siCD98 and CUR for boosting the conventional monotherapy via this novel nanotherapeutic agent, which offers a structurally simple platform for orally administered delivery of drugs to target cells in UC therapy.

Abstract 1030: Loss of PHLPPs protects against colitis by inhibiting epithelial apoptosis through upregulating AKT activity

Abstract Background & Aims: PHLPP, a novel family of protein phosphatases, plays an important role in balancing cellular signaling pathways that control cell death and proliferation. Loss of PHLPP expression promotes colorectal cancer (CRC) tumorigenesis and progression. Intestine epithelial cells (IECs) apoptosis contributes to the development of inflammatory bowel disease (IBD), and patients with IBD are at increased risk for CRC. We investigated the functional importance of PHLPP in intestinal inflammation. Methods: Both Phlpp1 and Phlpp2 genes were deleted in mice. The effect of PHLPP double knockout (DKO) on the development of acute colitis was examined in dextran sulfate sodium salt (DSS)-treated mice. Biochemical and histological analyses were conducted to determine the extent of tissue inflammation and colon epithelial cell apoptosis. The expression of PHLPP proteins was examined in DSS-treated mice and IBD patient samples. Results: In ulcerative colitis and Crohn's disease patient samples, both PHLPP1 and PHLPP2 protein expression was markedly decreased in colitis tissues compared to that in uninvolved tissues. In mice, PHLPP expression was significantly reduced in colonic epithelial cells following induction of colitis by DSS. Mechanistically, the inflammation-induced downregulation of PHLPP was mediated through proteasome-dependent protein degradation. Compared with wild-type mice, PHLPP DKO mice were protected from colitis induced by the DSS treatment as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in colonic epithelial cells. Furthermore, epithelial organoids derived from colon and small intestine of PHLPP DKO mice are more resistant to inflammation-induced apoptosis upon Akt activation. Conclusion: These results indicated that PHLPP contributes to the pathogenesis of colitis by controlling epithelial cell apoptosis. Downregulation of PHLPP at the onset of inflammation plays a role in protecting intestinal epithelial cell injury by inhibiting apoptosis. However, prolonged PHLPP-loss may promote colon cancer initiation in IBD patients. Citation Format: Yang-an Wen, Xin Li, Tatiana Goretsky, Terrence A. Barrett, Tianyan Gao. Loss of PHLPPs protects against colitis by inhibiting epithelial apoptosis through upregulating AKT activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2015-1030

Computational Prediction and Validation of BAHD1 as a Novel Molecule for Ulcerative Colitis.

Ulcerative colitis (UC) is a common inflammatory bowel disease (IBD) producing intestinal inflammation and tissue damage. The precise aetiology of UC remains unknown. In this study, we applied a rank-based expression profile comparative algorithm, gene set enrichment analysis (GSEA), to evaluate the expression profiles of UC patients and small interfering RNA (siRNA)-perturbed cells to predict proteins that might be essential in UC from publicly available expression profiles. We used quantitative PCR (qPCR) to characterize the expression levels of those genes predicted to be the most important for UC in dextran sodium sulphate (DSS)-induced colitic mice. We found that bromo-adjacent homology domain (BAHD1), a novel heterochromatinization factor in vertebrates, was the most downregulated gene. We further validated a potential role of BAHD1 as a regulatory factor for inflammation through the TNF signalling pathway in vitro. Our findings indicate that computational approaches leveraging public gene expression data can be used to infer potential genes or proteins for diseases, and BAHD1 might act as an indispensable factor in regulating the cellular inflammatory response in UC.

Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.

Interferon (IFN)-γ is a central pathogenesis factor in inflammatory bowel disease (IBD) with pleiotropic effects on many different cell types. However, as yet, the immune modulatory functions of IFN-γ in IBD have been predominantly investigated. Based on previous studies showing that IFN-γ acts antiangiogenic in colorectal carcinoma, we investigated the effects of IFN-γ on the vascular system in IBD. Colon tissues of patients with IBD and dextran sulfate sodium-induced colitis in mice were subjected to immunohistochemistry, quantitative real-time polymerase chain reactions, and in situ hybridization to quantify cell activation, angiogenesis, and immune responses. Vascular structure and permeability in mice were analyzed by ultramicroscopy and in vivo confocal laser endomicroscopy. We showed a significantly increased blood vessel density in IBD and dextran sulfate sodium colitis. In mice, this was associated with a disorganized blood vessel structure and profound vascular leakage. As compared with genes associated with angiogenesis, genes associated with inflammatory cell activation including IFN-γ were more strongly upregulated in colitis tissues. IFN-γ exerted direct effects on endothelial cells in IBD tissues in vivo, as indicated by the expression of IFN-γ-induced guanylate binding protein 1 (GBP-1). Neutralization of IFN-γ in the acute dextran sulfate sodium colitis model demonstrated that this cytokine exerts endogenous angiostatic activity in IBD and contributes to increased vascular permeability. The dissection of the pleiotropic activities of IFN-γ in IBD provides new insights to the pathological functions of this cytokine and may be of high relevance for the optimization of combination therapy approaches.

Expressions of E-cadherin, p120ctn, β-catenin and NF-κB in ulcerative colitis.

This study was aimed to investigate the expressions of E-cadherin, p120ctn, β-catenin and NF-κB in ulcerative colitis (UC) tissues and the implications of their expressions in the pathogenesis of UC. The expressions of E-cadherin, p120ctn, β-catenin and NF-κB were detected by immunohistochemistry, and those of p120ctn and NF-κB by Western blotting in 23 cases of UC and 17 cases of normal colonic tissues. The relationship between the expression of E-cadherin or NF-κB and that of p120ctn was analyzed by Spearman rank correlation analysis. The results showed that in UC and normal colonic groups, the abnormal expression rate of E-cadherin, p120ctn, β-catenin, and NF-κB was 52.2% vs. 0 (P<0.05), 73.9% vs. 23.5% (P<0.05), 65.2% vs. 17.6% (P<0.05) and 78.4% vs. 23.5% (P<0.05), respectively. p120ctn expression was positively correlated with E-cadherin expression (r=0.404, P<0.05), but negatively with nuclear NF-κB expression (r= - 0.347, P<0.05). Western blotting showed that as compared with the normal controls, the p120ctn protein level was significantly decreased (P<0.05), whereas the NF-κB protein level was increased (P<0.05) in UC tissues. It was concluded that in the colonic tissues of UC patients, the expressions of E-cadherin, p120ctn and β-catenin are decreased, suggesting the mucosal barrier is impaired in UC. Moreover, NF-κB is increased and activated in the UC tissues, resulting in the inflammation in UC. p120ctn may influence the UC development through modulating intercellular adhesion and inflammatory response.

대추의 궤양성 대장염에 대한 개선 효과

Objectives : Zizyphus jujube (ZJ) has been used as a traditional medicine for various diseases. However, the inhibitory effect of ZJ on intestinal inflammation has not been fully understood, yet. The aim of this study is to investigate anti-colitis activity of ZJ in dextran sulfate sodium (DSS)-induced colitis mouse model. Methods : To investigate the protective effects of ZJ,the colitis mice were induced by drinking water containing 5% DSS for 7 days. Mice were randomized into groups receiving ZJ (500 mg/kg), sulfasalazine (SFZ) (150 mg/kg) as a positive control, or water as a negative control. We assayed the effects of ZJ on DSS-induced the clinical signs, measuring weight loss, colon length and disease activity index (DAI). Additionally, to find a possible explanation for the anti-inflammatory effects of ZJ, we evaluated the effects of ZJ on the production of prostaglandin <TEX>$E_2$</TEX> (<TEX>$PGE_2$</TEX>) and expression of cyclooxygenase (COX)-2 in colitis tissue. Results : The results showed that mice treated with DSS showed considerable clinical signs, including weight loss, and reduced colon length. However, administration of ZJ significantly reduced the weight loss, shortens colon length, and improved DAI as clinical symptoms. Moreover, ZJ inhibited the <TEX>$PGE_2$</TEX> production and COX-2 expression levels in DSS-treated colon tissues. Conclusions : Collectively, the findings of this study provide us with novel insights into the pharmacological actions of ZJ as a potential molecule for use in the treatment of intestinal inflammation including ulcerative colitis.

The improving effect of Zizyphus jujube on dextran sulfate sodium-induced colitis in mice

Objectives : Zizyphus jujube (ZJ) has been used as a traditional medicine for various diseases. However, the inhibitory effect of ZJ on intestinal inflammation has not been fully understood, yet. The aim of this study is to investigate anti-colitis activity of ZJ in dextran sulfate sodium (DSS)-induced colitis mouse model. Methods : To investigate the protective effects of ZJ,the colitis mice were induced by drinking water containing 5% DSS for 7 days. Mice were randomized into groups receiving Z J (500 mg/kg), sulfasalazine (SFZ) (150 mg/kg) as a positive control, or water as a negative control. W e assayed the effects of ZJ on DSS-induced the clinical signs, measuring weight loss, colon length and disease activity index (DAI). Additionally, to find a possible explanation for the anti-inflammatory effects of ZJ, we evaluated the effects of ZJ on the production of prostaglandin E2 (PGE2) and expression of cyclooxygenase (COX)-2 in colitis tissue. Results : The results showed that mice treated with DSS showed considerable clinical signs, including weight loss, and reduced colon length. However, administration of ZJ significantly reduced the weight loss, shortens colon length, and improved DAI as clinical symptoms. Moreover, ZJ inhibited the PGE2 production and COX-2 expression levels in DSS-treated colon tissues. Conclusions : Collectively, the findings of this study provide us with novel insights into the pharmacological actions of ZJ as a potential molecule for use in the treatment of intestinal inflammation including ulcerative colitis.

Expression of Wnt and Notch signaling pathways in inflammatory bowel disease treated with mesenchymal stem cell transplantation: evaluation in a rat model.

IntroductionThe purpose of this study was to investigate the expression of Wnt and Notch signaling pathway-related genes in inflammatory bowel disease (IBD) treated with mesenchymal stem cell transplantation (MSCT).MethodsTNBS (2,4,6-trinitrobenzene sulfonic acid) was used to establish IBD in a rat model. Mesenchymal stem cells (MSCs) were transplanted via tail vein transfusion. Saline water was used in a control group. The expression of Wnt and Notch main signaling molecules was screened by gene chips and verified by quantitative reverse transcription-polymerase chain reaction in the IBD rat model on day 14 and day 28 after transplantation.ResultsThe IBD rat models were successfully established and MSCs were transplanted into those models. Genome-wide expression profile chips identified a total of 388 differentially expressive genes, of which 191 were upregulated and 197 were downregulated in the MSC-transplanted group in comparison with the IBD control group. Real-time quantitative polymerase chain reaction results showed that the level of Olfm4 mRNA expression in the IBD group (2.54±0.20) was significantly increased compared with the MSCT group (1.39±0.54) and the normal group (1.62±0.25) (P <0.05). The Wnt3a mRNA was more highly expressed in IBD rats (2.92±0.94) and decreased in MSCT rats (0.17±0.63, P <0.05). The expression of GSK-3β mRNA was decreased in the setting of inflammation (0.65±0.04 versus 1.00±0.01 in normal group, P <0.05) but returned to normal levels after MSCT (0.81±0.17). The expression of β-catenin was observed to increase in IBD tissues (1.76±0.44) compared with normal tissues (1.00±0.01, P <0.05), but no difference was found in the MSCT group (1.12±0.36). Wnt11 declined at 14 days and returned to normal levels at 28 days in the IBD group; in comparison, a significantly lower expression was found in MSCT rats. There were no differences in the expression of Fzd3, c-myc, TCF4, and Wnt5a in inflammation, but all of those genes declined after MSCT treatment.ConclusionsThe canonical Wnt and Notch signaling pathways are activated in IBD and may be suppressed by stem cell transplantation to differentiate into intestinal epithelium after MSCT. Moreover, the non-canonical Wnt signaling may be inhibited by canonical Wnt signaling in the setting of inflammation and may also be suppressed by MSCT.

Erythropoietin attenuates intestinal inflammation and promotes tissue regeneration

Background. The prevalence of inflammatory bowel disease (IBD) is increasing. Since patients usually need long-term treatment and suffer from reduced quality of life, there is a need to develop new therapeutic strategy. The aim of this study was to investigate the therapeutic potential of erythropoietin (EPO) for the treatment of IBD. Methods. Murine colitis was induced by 3.0% Dextran Sulfate Sodium (DSS). Recombinant human EPO (rhEPO) was given to evaluate the anti-inflammatory and regenerative effects on intestinal inflammation. The effect of rhEPO on human colon epithelial cells was also evaluated. Immunohistochemical analysis of EPO receptor was performed in human IBD tissues. Results. While about 62% of control mice with severe colitis induced by 5-day DSS died, 85% of mice treated with rhEPO survived. Histological analysis confirmed that EPO treatment reduced the colonic inflammation. Furthermore, EPO treatment significantly downregulated the local expressions of IFN-γ, TNF-α and E-selectin in the colon, suggesting that the effect was associated with inhibiting local immune activation. In a 4-day DSS-induced colitis model, rhEPO significantly improved the recovery of body weight loss compared to controls. Furthermore, proliferating cell nuclear antigen expression was significantly upregulated in the colon tissue from mice treated with rhEPO compared to controls. In addition, rhEPO increased the growth of cultured human colon epithelial cells in a dose-dependent manner. Furthermore, EPO-receptor expression was confirmed in human IBD colon tissues. Conclusion. Three major functions of EPO, hematopoiesis, anti-inflammation and regeneration, may produce significant effects on intestinal inflammation, therefore suggesting that rhEPO might be useful for IBD.

Mo1750 Genetic Associations With Preferential 6TGN Metabolizers Reveal Novel Pathways Involved in Purine Metabolism

INTRODUCTION: The prevalence of inflammatory bowel disease (IBD) is increasing globally whilst genetic factors account for only a small portion of overall disease variance. Accumulating evidence has highlighted the importance of epigenetic regulation in IBD pathogenesis. We aimed to identify key epigenetic modulators of IBD in a population of increasing disease incidence and characterize the mechanisms involved. METHODS: Customized PCR array containing 128 well-defined epigenetic modulators were performed and candidate genes were validated in 96 tissues from patients with Crohn's disease (CD), ulcerative colitis (UC) andmatched healthy controls using quantitative RT-PCR,Western blot and immunohistochemistry. Functional assays were performed using normal colonic epithelium cell line, NCM460, in which histone modification status and expressions of inflammatory cytokines were monitored upon small-molecule inhibition or RNA interference. RESULTS: Seven epigenetic modulators were differentially expressed in inflamed IBD tissues compared with controls. Amongst them, lysine acetyltransferase 2B (KAT2B) mRNA and protein levels were significantly down regulated in CD and UC inflamed tissues in a validation cohort (p< 0.05). KAT2B proteins were localized abundantly in nuclei of epithelial cells of non-inflamed colonic tissues but not in paired inflamed tissues. Inhibition of KAT2B by anacardic acid (AA), a specific small-molecule KAT2B antagonist, or small-interfering RNA (siRNA) in NCM460 cells reduced acetylation level of histone H4, but not histone H3 lysine 9 and lysine 14. Moreover, siRNA knockdown of KAT2B significantly decreased IL-10 but increased IL-1β and IL-18 expression in NCM460 cells, these changes were dose-dependent and influenced by AA treatment. CONCLUSION: Our findings demonstrated for the first time a novel epigenetic link between histone modification and IBD through KAT2B. Downregulation of KAT2B may promote histone deacetylation that leads to suppression of IL-10, a key anti-inflammatory cytokine critical for innate and adaptive inflammatory responses. Ongoing functional characterization of tissue or disease-specific epigenetic variations in IBD will provide insight into biological pathways that may translate into new therapeutic approaches.