Amelioration of dextran sulfate sodium-induced colitis in mice by oral administration of beta-caryophyllene, a sesquiterpene

beta-Caryophyllene (BCP), a naturally occurring plant sesquiterpene, was examined for anti-inflammatory activity in a mouse model of experimental colitis induced by dextran sulfate sodium (DSS). Colitis was induced by exposing male BALB/c mice to 5% DSS in drinking water for 7 days. BCP in doses of 30 and 300 mg/kg was administered orally once a day, beginning concurrently with exposure to DSS. The body weight and colon length were measured, and histological damage and myeloperoxidase (MPO) activity as well as inflammatory cytokines were assessed in both serum and colonic tissue after 7 days of treatment with DSS. The DSS treatment damaged the colonic tissue, increased MPO activity and inflammatory cytokines, lowered the body weight, and shortened the length of the colon. Oral administration of BCP at 300 mg/kg significantly suppressed the shortening of colon length and slightly offset the loss of body weight. BCP treatment (300 mg/kg) also significantly reduced the inflammation of colon and reversed the increase in MPO activity that had been induced by exposure to DSS. Further, BCP significantly suppressed the serum level of IL-6 protein (a 55% reduction) as well as the level of IL-6 mRNA in the tissue. These results demonstrate that BCP ameliorates DSS-induced experimental colitis, and may be useful in the prevention and treatment of colitis.     

Beneficial and preventive effect of chitin nanofibrils in a dextran sulfate sodium-induced acute ulcerative colitis model

Chitin nanofibrils, which are prepared from dried crab shells by a grinding method, are newly developed natural materials with uniform widths of approximately 10-20 nm. The bioactivities of chitin nanofibrils have not been investigated. In this study, we examined the preventive effects of chitin nanofibrils in a mouse model of dextran sulfate sodium (DSS)-induced acute ulcerative colitis. The results indicated that chitin nanofibrils improved clinical symptoms and suppressed ulcerative colitis. Furthermore, chitin nanofibrils suppressed myeloperoxidase activation in the colon and decreased serum interleukin-6 concentrations. Conversely, chitin powder did not suppress DSS-induced acute ulcerative colitis. Our results suggested that chitin nanofibrils have potential as a functional substance for inflammatory bowel disease patients.     

Dextran sulfate sodium enhances interleukin-1 beta release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages

Interleukin (IL)-1 beta is a pro-inflammatory cytokine that has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD), however, the molecular mechanisms underlying the production of IL-1 beta in IBD are not fully understood. We investigated dextran sulfate sodium (DSS)-induced IL-1 beta production and caspase-1 activities in murine peritoneal macrophages (pM phi). Further, the activation status of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun NH(2)-terminal kinase (JNK1/2), as well as their upstream target kinases, were examined by Western blotting. In addition, mRNA expression was assessed by RT-PCR and CXC chemokine ligand 16 (CXCL16) protein was detected by immunocytochemistry. DSS-treated pM phi released IL-1 beta protein in a time-dependent manner without affecting mRNA levels during 3-24 h, and caspase-1 activity peaked at 5 min (29-fold). IL-1 beta release and caspase-1 activity induced by DSS were significantly inhibited by a MAPK kinase 1/2 inhibitor, a p38 MAPK inhibitor, and NAC, however, not by JNK1/2 or a protein kinase C inhibitor. In addition, DSS strikingly induced the phosphorylation of p38 MAPK and ERK1/2 within 2 and 10 min, respectively. DSS also induced intracellular generation of reactive oxygen species (ROS). Pre-treatment with anti-CXCL16 for 24 h, but not anti-scavenger receptor-A, anti-CD36, or anti-CD68 antibodies, significantly suppressed DSS-induced IL-1 beta production. Our results suggest that DSS triggers the release of IL-1 beta protein from murine pM phi at a post-translational level through binding with CXCL16, ROS generation, and resultant activation of both p38 MAPK and ERK1/2 pathways, and finally caspase-1 activation.     

Apurinic/apyrimidinic endonuclease 1, the sensitive marker for DNA deterioration in dextran sulfate sodium-induced acute colitis

Abstract

Mutations in mismatch repair (MMR) genes are commonly associated with the development of colorectal cancer. Additionally, base excision repair, which involves apurinic/apyrimidinic endonuclease 1 (APE1), recognizes and eliminates oxidative DNA damage. Here, we investigated the possible roles of APE1 in dextran sulfate sodium (DSS)-induced acute colitis using the young rat model. Four-week-old Sprague–Dawley rats were administered 2% DSS in drinking water for 1 week. MMR and APE1 expression levels were assessed by western blotting and immunohistochemistry. Following DSS treatment, growth of young rats failed and the animals had loose stools. Together with the histological changes associated with acute colitis, APE1 and MSH2 levels increased significantly at 3 and 5 days after DSS treatment, respectively. The difference between APE1 and MSH2 expression was significant. DSS-induced DNA damage and subsequent repair activity were evaluated by staining for 8-hydroxy-deoxyguanosine (8-OHdG) and APE1, respectively; 8-OHdG immunoreactivity increased throughout the colonic mucosa, while APE1 levels in the surface epithelium increased at an earlier timepoint. Taken together, our data suggest that changes in APE1 expression after DSS treatment occurred earlier and were more widespread than changes in MMR expression, suggesting that APE1 is more sensitive for prediction of DNA deterioration in DSS-induced colitis.     

Transgenic overexpression of ITGB6 in intestinal epithelial cells exacerbates dextran sulfate sodium-induced colitis in mice

Integrins, as a large family of cell adhesion molecules, play a crucial role in maintaining intestinal homeostasis. In inflammatory bowel disease (IBD), homeostasis is disrupted. Integrin αvβ6, which is mainly regulated by the integrin β6 subunit gene (ITGB6), is a cell adhesion molecule that mediates cell-cell and cell-matrix interactions. However, the role of ITGB6 in the pathogenesis of IBD remains elusive. In this study, we found that ITGB6 was markedly upregulated in inflamed intestinal tissues from patients with IBD. Then, we generated an intestinal epithelial cell-specific ITGB6 transgenic mouse model. Conditional ITGB6 transgene expression exacerbated experimental colitis in mouse models of acute and chronic dextran sulphate sodium (DSS)-induced colitis. Survival analyses revealed that ITGB6 transgene expression correlated with poor prognosis in DSS-induced colitis. Furthermore, our data indicated that ITGB6 transgene expression increased macrophages infiltration, pro-inflammatory cytokines secretion, integrin ligands expression and Stat1 signalling pathway activation. Collectively, our findings revealed a previously unknown role of ITGB6 in IBD and highlighted the possibility of ITGB6 as a diagnostic marker and therapeutic target for IBD.     

Selective expression of detoxifying glutathione transferases in mouse colon: effect of experimental colitis and the presence of bacteria

Glutathione transferases (GSTs) play a central role in the cellular defense against harmful endogenous compounds and xenobiotics in mouse and man. The gastrointestinal channel is constantly exposed to bacteria, bacterial products, and xenobiotics. In the present study the distribution of alpha, mu, and pi class GSTs was examined immunohistologically in the colon of conventional and germ-free (GF) mice subjected to experimental colitis. The tissues samples were from conventional mice with and without colitis induced by dextran sulfate sodium (DSS); GF mice treated with DSS or carrageenan; and GF mice inoculated with normal mouse bacterial flora as well as with Lactobacillus GG. In conventional as well as in GF mice the mu and pi class GSTs showed reduced intestinal expression when colitis was induced. In contrast, the level of GSTs reacting with antibodies directed against the alpha class, in particular mGST A4-4, was elevated after induction of inflammation. Of special interest is mGST A4-4 because of its high catalytic activity with toxic products of lipid peroxidation. In the colon of conventionalized GF mice that were given mouse intestinal flora, the mGST A4-4 expression was increased with time for several weeks, but then showed a decrease to a normal level. Additionally, the inoculation of GF mice with Lactobacillus GG induced all the intestinal GSTs studied.     

Anti-inflammatory effect of Euphorbia supina extract in dextran sulfate sodium-induced colitis mice

The aim of this study is to examine the anti-inflammatory effect of Euphorbia supina (ES) ethanol extract in dextran sulfate sodium (DSS)-induced experimental colitis model. ES was per orally administered at different doses of 4 or 20 mg/kg body weight with 5% DSS in drinking water for 7 days. Twenty mg/kg of ES administration regulated body weight decrease, recovered colon length shortening, and increased disease activity index score and myeloperoxidase level in DSS-induced colitis. Histological features showed that 20 mg/kg of ES administration suppressed edema, mucosal damage, and the loss of crypts induced by DSS. Furthermore, ES suppressed the expressions of COX-2, iNOS, NF-kB, IkBα, pIkBα in colon tissue. These findings demonstrated a possible effect of amelioration of ulcerative colitis and could be clinically applied.     

Correlation of neutrophil and monocyte derived interleukin-1 receptor antagonist and interleukin-8 with colitis severity in the rabbit

Activated neutrophils and monocytes produce interleukin (IL)-8, a pro-inflammatory chemokine, but also IL-1 receptor antagonist (IL-1ra), which is an anti-inflammatory cytokine. We were interested to see the profiles of IL-8 and IL-1ra in the colonic tissue and in the peripheral blood leukocytes (PBL) during the development of immune complex induced colitis in rabbits. IL-1ra and IL-8 in PBL were measured in 26 rabbits at time 0 h, 24 h, and 48 h after induction of colitis. The colons were removed at 48 h for measuring myeloperoxidase (MPO), ulcer area, IL-1ra and IL-8. Epithelial damage, crypt abscess formation and leukocyte infiltration of the colonic tissue were major features of this colitis model. During the development of colitis, there was an increase in circulating neutrophils and monocytes (P < 0.0001), but not lymphocytes. Likewise, elevated amounts of IL-1ra (P = 0.0001) and IL-8 (P = 0.0219) production by PBL were observed following induction of colitis. Flow cytometry revealed major source of IL-1ra was monocytes, while the main sources of IL-8 were neutrophils and monocytes. There was correlation between MPO and ulcer area (R_s = 0.6327, P < 0.0001). At 24 h, PBL from MPO^High group (n = 11) showed increased IL-1ra (P = 0.027) and IL-8 (P = 0.0128) levels vs MPO^Low group (n = 15). IL-8 production by PBL showed correlation with tissue MPO (R_s = 0.4273, P = 0.0295). The colitis in this model was associated with an increase in circulating monocytes and neutrophils, which released increased amounts of IL-8 and IL-1ra. Further, IL-8 and IL-1ra showed correlation with the severity of colitis. These observations should significantly further understandings on the role of neutrophils and monocytes in the immunopathogenesis of ulcerative colitis.     

Interleukin-1beta induces macrophage inflammatory protein-1beta expression in human hepatocytes

The investigation of factors that regulate expression of CC-chemokines, the important mediators in immune responses and inflammation processes, has an important significance in understanding the immunopathogenesis of liver diseases. We examined the role of interleukin-1beta (IL-1beta), a multifunctional cytokine, in regulating the expression of macrophage inflammatory protein (MIP)-1beta in human hepatocytes (Huh7 and HepG2). IL-1beta significantly enhanced MIP-1beta expression in these cells at both the mRNA and protein levels. Cytokine-enriched supernatants from monocyte-derived macrophage (MDM) cultures also induced MIP-1beta expression. IL-1beta is responsible for MDM supernatant-mediated up-regulation of MIP-1beta since the antibody to IL-1beta abolished MDM supernatant action. Investigation of the mechanism involved in MIP-1beta induction by IL-1beta showed that IL-1beta activated the nuclear factor kappa B (NF-kappaB) promoter in Huh7 cells. In addition, caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-kappaB, not only abolished IL-1beta-mediated NF-kappaB promoter activation, but also blocked IL-1beta-induced MIP-1beta expression. These observations suggest that IL-1beta-mediated up-regulation of MIP-1beta production in the hepatic cells may contribute a critical mechanism for continuous recruitment of inflammatory cell to liver and maintenance of inflammation.     

Application of 2-aminopyridine fluorescence labeling in the analysis of in vivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography

The present study describes a size-exclusion high-performance liquid chromatographic method for the separation and quantification of sulfated polysaccharides, such as dextran sulfate sodium (DSS). Pyridylamination of DSS was achieved without difficulty using 2-aminopyridine as a fluorometric label. In addition, 0.1–0.2 M phosphate buffer (pH 3.0) was found to be the mobile phase which produced the best separation. In vitro enzymatic degradation of the pyridylamino-DSS (PA-DSS₅₀₀₀, M_r 5000) using α-amylase and the in vivo metabolism in the rat feces after oral administration of PA-DSS₅₀₀₀ were then evaluated. Two small peaks of approximately M_r 380 and 600 appeared after co-incubation with α-amylase, indicating PA-DSS₅₀₀₀ may be considerably depolymerized. In vivo, however, PA-DSS₅₀₀₀ excreted in the feces was mainly of PA-DSS₅₀₀₀ polymer. No peaks of less than M_r 5000 were not clearly detectable in the feces because of background fluorescence attributable to gut lumen contents. This method of fluorometric analysis allows fairly selective detection of sulfated polysaccharides in biological materials.     

Associations of interleukin-6 with interleukin-1beta, interleukin-8 and macrophage inflammatory protein-1beta in midlife women

Objective: The aim of the present study was to determine the associations of interleukin (IL)-6 with other cytokines and chemokines and to compare these associations in peri- and postmenopausal women. Methods: Ninety-nine perimenopausal and 92 postmenopausal women were enrolled in this study. Serum concentrations of IL-6, IL-1β, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, tumor necrosis factor (TNF)-α, interferon γ, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, macrophage inflammatory protein (MIP)-1β and monocyte chemotactic protein (MCP)-1 were measured simultaneously using a multiplexed cytokine assay. Results: Among the 17 cytokines, IL-6, IL-1β, IL-5, IL-7, IL-8, IL-10, MCP-1 and MIP-1β were detected in serum in more than 50% of the women. Serum levels of IL-4 and MCP-1 in postmenopausal women were significantly higher than those in perimenopausal women. Serum IL-6 concentrations showed significant and positive correlations with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, and these correlations were still significant after adjustment for age and body mass index. Conclusion: Serum IL-6 concentration was found to be closely associated with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, suggesting that these cytokines act in concert with the progression of several symptoms and various diseases.     

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated K_d in the 10^?4 M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.     

Proteomic profiling of dextran sulfate sodium induced acute ulcerative colitis mice serum exosomes and their immunomodulatory impact on macrophages

Macrophages are essential for the maintenance of intestinal homeostasis, and their activation has been proposed to be critical to the pathogenesis of inflammatory bowel disease (IBD). Although there are many recognized mediators of macrophage activation, increasing evidence suggests that macrophages respond to exosome stimulation. Exosomes are 40–150 nm microvesicles released from different cell types and are found in a variety of physiological fluids, including serum. As studies have shown that circulating exosomes participate in intercellular communication and can mediate the immune response, we hypothesized that exosomes may play a role in the pathogenesis of IBD though modulation of macrophage activity. In this study, we used the dextran sulfate sodium (DSS) induced acute colitis mice model to investigate the effect of serum exosomes on macrophages and identify exosome proteins potentially involved in macrophage activation. We treated RAW264.7 macrophages with serum exosomes isolated from dextran sulfate sodium induced mice and found that treatment induced phosphorylation of p38 and ERK and production of tumor necrosis factor α when compared to treatment with exosomes isolated from control mice. Subsequent proteomic analysis identified 56 differentially expressed proteins, a majority of which were acute‐phase proteins and immunoglobulins. Bioinformatics analysis suggested these proteins were mainly involved in the complement and coagulation cascade, which has been implicated in macrophage activation. Our findings provide new insight into the role of circulating serum exosomes in acute colitis and contribute to the understanding of macrophage activation in the pathogenesis of IBD.     

Intestinal epithelial cells related lncRNA and mRNA expression profiles in dextran sulphate sodium-induced colitis

Intestinal epithelial barrier damage caused by intestinal epithelial cells (IECs) dysfunction plays a crucial role in the pathogenesis and development of inflammatory bowel disease (IBD). Recently, some studies have suggested the emerging role of long non-coding RNAs (lncRNAs) in IBD. The aim of this study was to reveal lncRNAs and mRNA expression profiles in IECs from a mouse model of colitis and to expand our understanding in the intestinal epithelial barrier regulation. IECs from the colons of wild-type mice and dextran sulphate sodium (DSS)-induced mice were isolated for high-throughput RNA-sequencing. A total of 254 up-regulated and 1013 down-regulated mRNAs and 542 up-regulated and 766 down-regulated lncRNAs were detected in the DSS group compared with the Control group. Four mRNAs and six lncRNAs were validated by real-time quantitative PCR. Function analysis showed that dysregulated mRNAs participated in TLR7 signalling pathway, IL-1 receptor activity, BMP receptor binding and IL-17 signalling pathway. Furthermore, the possibility of indirect interactions between differentially expressed mRNAs and lncRNAs was illustrated by the competing endogenous RNA (ceRNA) network. LncRNA ENSMUST00000128026 was predicted to bind to mmu-miR-6899-3p, regulating Dnmbp expression. LncRNA NONMMUT143162.1 was predicted to competitively bind to mmu-miR-6899-3p, regulating Tnip3 expression. Finally, the protein-protein interaction (PPI) network analysis was constructed with 311 nodes and 563 edges. And the highest connectivity degrees were Mmp9, Fpr2 and Ccl3. These results provide novel insights into the functions of lncRNAs and mRNAs involved in the regulation of the intestinal epithelial barrier.     

Dihydroberberine,an isoquinoline alkaloid,exhibits protective effect against dextran sulfate sodium-induced ulcerative colitis in mice

BackgroundAs a chronic inflammatory disease, ulcerative colitis (UC) is relevant to a rising risk of colorectal cancer. Dihydroberberine (DHBB), a natural occurring isoquinoline alkaloid with various bioactivities, was found in many plants including Coptis chinensis Franch. (Ranunculaceae), Phellodendron chinense Schneid. (Rutaceae), and Chelidonium majus L. (Papaveraceae). However, its protective effect on UC is sparsely dissected out.PurposeTo explore the protective role and underlying mechanism of DHBB on a model of colitis.MethodsAcute colitis model was established by gavage with 3% dextran sulfate sodium (DSS) for 8 days. Influence of DHBB on DSS-induced clinical symptoms and disease activity index (DAI) was monitored and analyzed. Pathological injury of colon tissues was examined by hematoxylin-eosin and Alcian blue staining. The expression of intestinal mucosal barrier function proteins, immune-inflammation related biomarkers and signal pathway key targets were determined by ELISA kit, Western blot, immunohistochemistry and qRT-PCR.ResultsDHBB treatment effectively alleviated DSS-induced UC by relieving clinical manifestations, DAI scores and pathological damage, which exerted similar beneficial effect to azathioprine (AZA), and better than berberine (BBR). In addition, DHBB significantly improved the gut barrier function through up-regulating the levels of tight junction proteins and mucins. Furthermore, DHBB dramatically ameliorated colonic immune-inflammation state, which was related to the decrease of colonic pro-inflammatory cytokines and immunoglobulin through blocking TLR4/MyD88/NF-κB signal pathway.ConclusionThese results demonstrated that DHBB exerted a significant protective effect on DSS-induced experimental UC, at least partly through suppressing immune-inflammatory response and maintaining gut barrier function.     

Anti‐inflammatory effects of eriocitrin against the dextran sulfate sodium–induced experimental colitis in murine model

Inflammatory bowel disease (IBD) is a continual ailment condition which engrosses the entire alimentary canal. The IBD can be primarily distinguished into two forms, ulcerative colitis, and Crohn's disease. The major symptoms of IBD include pustules or abscesses, severe abdominal pain, diarrhea, fistula, and stenosis, which may directly affect the patient's quality of life. A variety of mediators can stimulate the circumstances of IBD, some examples include infections by microbes such as bacteria, perturbation of the immune system and the surrounding environment of the intestines. Severe colitis was stimulated in the experimental animals through administering 4% dextran sulfate sodium (DSS) which is mixed in water ad libitum for 6 days. Eriocitrin (30 mg/kg) was then administered to the experimental animals followed by the induction of severe colitis to evaluate the therapeutic prospective of eriocitrin against the colon inflammation stimulated by DSS. In this study, eriocitrin (30 mg/kg) demonstrated significant (P < .05) attenuation activity against the DSS‐stimulated severe colitis in experimental animals. Eriocitrin counteracted all of the clinical deleterious effects induced by DSS, such as body‐weight loss, colon shortening, histopathological injury, accretion of infiltrated inflammatory cells at the inflamed region and the secretion of inflammatory cytokines. The results clearly showed that eriocitrin effectively attenuated DSS‐induced acute colitis in experimental animals.