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Analysis of Intestinal Fibrosis in Chronic Colitis in Mice Induced by Dextran Sulfate Sodium
AGA Abstracts
the chronic colitis model, IL-4-/- mice tended to have lower weight gain than WT mice, but this was not different significantly. In addition, both IL-4-/- and WT mice had similar DAI, colon length and histologic severity of colitis on the end point. The expression of TNFα and MIP-2, and levels of MPO in colonic tissue also did not show significant difference between two groups. Conclusions: Our results suggest that IL-4 is not an important independent factor on regulation of acute and chronic DSS colitis.
and ulcerative colitis. Here, we examined the biological function and the therapeutic effect of antisense MIF/SPG complex against colitis. Method: C57BL/6 mice were given 2 % dextran sodium sulfate (DSS) drinking water for 5 days. Severity of colitis and MIF expression was evaluated on day 14. The function of CD11b+ macrophages isolated from DSS-treated mice was analyzed. Immunofluorescence was performed to confirm whether CD11b+ macrophages uptake this complex. The inhibitory effect of antisense MIF/SPG complex was evaluated In Vitro. In addition, antisense MIF/SPG complex was intraperitoneally administered in DSS treated mice, and the severity of colitis was evaluated. Result: By 2% DSS administration, severe colitis was induced, and the expression of MIF was increased in the serum, colon, and MLN. MIF was mainly produced from CD11b+ macrophages in DSS-treated mice. Immunofluorescence showed that antisense MIF/SPG complex but not antisense MIF alone was uptake into macrophage. Further, Dectin-1, which was known as a receptor of SPG, was significantly increased in CD11b+ macrophages of DSS-treated mice compared with control mice by FACS analysis. MIF production both In Vitro and In Vivo was significantly suppressed by antisense MIF/SPG complex. Administration of antisense MIF/SPG complex ameliorated intestinal inflammation. Conclusion: Administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated the inflammation of colon. Our result demonstrated the possibility of new therapeutic approach against the inflammatory bowel disease.
Su1958 MicroRNA-146b Activates the NF-kB Pathway and Improves Intestinal Injury in a Mouse Enteritis Model Toshie Nata, Mikihiro Fujiya, Nobuhiro Ueno, Yuhei Inaba, Kentaro Moriichi, Yusuke Mizukami, Kazuya Sato, Yutaka Kohgo Background: microRNAs comprise approximately 20 nucleotides which modulate the level of their target proteins by either translational arrest or transcript degradation. While an abnormal miRNA expression is a common feature of various tumors, the precise role that microRNAs play in the regulation of inflammatory disease has just begun to be explored. Methods: 1. Differential expression of microRNAs in intestinal tissues of IL-10 deficient mice was examined with mirVanaTM miRNA Bioarray (Filgen). 2. Expression vectors containing a whole sequence of miR-146b or each siRNA wrapped with HVJ envelope were intraperitoneally injected to mice. The protein samples obtained from the mice 48 hours after the transfection, and the phosphorylation of NFkB was examined using western blotting. 3. The survival rate were compared between two groups, 4% dextran sodium sulfate (DSS)-treated mice and control mice, both were transfected with miR-146b vectors. Results: 1. In colonic tissues of IL-10 deficient mice, 19 microRNAs were downregulated and 26 microRNAs including miR-146b were upregulated more than twice. 2. NFkB phosphorylation was increased in the mice colon overexpressing either miR-146b. 3. The rate of survival was improved with the DSS-treated mice, when transfecting the vector. In addition, an inhibitor of NFkB (PDCT) eliminated the effect of miR-146b on relieving the intestinal inflammation. Discussion: miR-146b possibly improves the injury due to the intestinal inflammation through the regulation of NFkB pathway.
Su1961 Analysis of Intestinal Fibrosis in Chronic Colitis in Mice Induced by Dextran Sulfate Sodium Xiaomei Sun, Kenji Suzuki, Masaki Nagata, Kawase Tomoyuki, Hana Yamaguchi, Yusuke Kawauchi, Xiufen Tang, Xu Ren, Mitsuhiro Anzai, Takayoshi Nishino, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura
Su1959 Antibiotics Suppress Intestinal ICAM-1 Expression Independent of a Reducing Effect on Gut Microbial Density Ramadass Balamurugan, Julie Bard, Henry C. Lin
Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in the pathophysiology of intestinal fibrosis, however, their precise role in the development of fibrosis is largely unknown. To investigate their role in the intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. Clinical symptoms peaked at day 8 and were gradually ameliorated with the only apparent symptom of loose feces at day 19. Histopathological changes after DSS cessation in the chronic phase were dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of cytokines [interleukin (IL)-1β, IL-17a, and tumor necrosis factor (TNF)-α] are correlated with the extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-β, and matrix metalloproteinases (MMP-2, and -9) were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin+, α-smooth muscle actin(α-SMA)-) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin+, α-SMA+) were increased in mucosal but not in submucosal layers. A possible involvement of TGF-βin the fibrosis was further examined by primary mouse subcutaneous fibroblasts cultures. Exogenously added TGF-β1 substantially augmented the expressions of both vimentin and α-SMA proteins with increased production of collagen. Therefore, intestinal fibrosis in chronic DSS-induced colitis could be explained at least in part by TGF-β-dependent transition of mesenchymal progenitor fibroblasts into the profibrogenic fibroblasts and myofibroblasts with increased production of collagens. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in chronic colitis in B6 mice induced by a single cycle DSS administration.
A recent report showed that the gut microbial biomass need not be reduced in response to an oral antibiotic (Gut Microbes 2010:1(4):279-284). Since antibiotics have been used successfully to treat a variety of inflammatory disorders involving the intestine, could antibiotics have a direct effect on the host immune response even without a decontaminating effect on gut microbiota? Inflammation may be initiated when integrins bind to endothelium via intercellular adhesion molecule-1 (ICAM-1) leading to transmigration of the integrinexpressing leukocytes into tissues. However, whether or not ICAM-1 expression is changed by antibiotics independent of a reduction in the density of the indigenous gut microbial population is not known. A diet containing red kidney beans (RKB) is known to increase gut microbial density (DDS 2010;55(10):2778-84). Aim: Our aim was to test the hypothesis that antibiotics may alter ICAM-1 expression independent of a decontaminating effect on the indigenous microbes of intestinal mucosa. Methods: Twenty-four rats were randomized to 1 of 3 groups: 1) a standard rat chow alone (Control), 2) a standard rat chow with antibiotics (Antibiotics) or 3) a chow containing 26% crude red kidney beans with antibiotics (RKB+ Antibiotics). Antibiotic treatment consisted of 200μl mixture of kanamycin (4 mg/ ml), gentamicin (0.35 mg/ml), colistin (8500 U/ml), metronidazole (2.15 mg/ml), and vancomycin (0.45 mg/ml). Antibiotics were administered by gavage daily for 7 days. At the end of 7 days, the animals were sacrificed and intestinal tissue was collected. Total microbial load attached to the mucosa of the proximal, middle and distal 1/3 of the small intestine and cecum was quantitated by qPCR targeting 16s rRNA genes. Intestinal ICAM-1 gene expression was quantitated by qPCR. Results: When compared to controls, RKB+Antibiotic rats showed a significant increase in bacterial load (p<0.0001). Antibiotic group showed an intermediate result. However, there was a significant decrease in ICAM-1 expression (mean+/SE) in both RKB+Antibiotic group (0.00002± 3.64E-06) and Antibiotic group (0.00003± 6.93E-06) when compared to Controls (0.00089± 9 E-05)(p<0.05). There was an inverse relationship between ICAM-1 expression and microbial density in the proximal small intestine (p<0.004). Conclusion: Antibiotics decreased intestinal ICAM-1 expression independent of a reducing effect on microbial density demonstrating a potentially direct effect of antibiotics on host immune response. The suppression of ICAM-1 expression by some antibiotics may contribute to the beneficial response to antimicrobial agents in inflammatory gut disorders.
Su1962 Postnatal Changes in Monoamine Transporter Function in the Guinea Pig Ileum Hong Zhao, Xiaochun Bian, James Galligan, Greg M. Swain 5-hydroxytryptamine (5-HT) is a paracrine signaling molecule in the gut mucosa and a transmitter in the enteric nervous system. After release, 5-HT is cleared by the serotonin transporter (SERT). Previous work showed that SERT expression is low in the ileum of neonatal guinea pigs. In this study we examined the potential role of the dopamine and norepinephrine transporters (DAT and NET) in clearance of extracellular 5-HT in the mucosa of guinea pigs at 1 day, 1, 3 and 6 weeks postnatal. Methods. Extracellular 5-HT was monitored In Vitro in the ileum of guinea pigs as an oxidation current (continuous amperometry at 0.6V vs. Ag|AgCl reference electrode) using a boron-doped diamond microelectrode positioned near the mucosal surface. Sensitivity of the 5-HT oxidation current to citalopram (1 μM, SERT blocker), desmethylimpramine (DMI, 1 μM, NET blocker), GBR12909 (0.1 μM DAT blocker) and cocaine (10 μM, non-selective monoamine transport inhibitor) was tested. Tissue 5-HT and 5-hydroxy indole acetic acid (5-HIAA, 5-HT metabolite formed after clearance by SERT) levels were measured using HPLC. Results. 5-HT oxidation currents at 1 day postnatal were unaffected by all monoamine transport inhibitors. 5-HT oxidation currents were insensitive to citalopram at 1 week postnatal but thereafter showed a maturation dependent increase in citalopram sensitivity. GBR 12909 increased 5-HT oxidation currents at 1 and 3 weeks but not at 6 weeks. 5-HT oxidation currents were increased by DMI at 3 and 6 weeks postnatal. Cocaine increase 5-HT oxidation current at 1, 3 and 6 weeks. Steady state tissue levels of 5-HT were stable throughout the time course of these studies while 5-HIAA levels increased at 1 week and remained elevated at 3 and 6 weeks. Conclusion.
Su1960 The New Therapeutic Approach for Inflammatory Bowel Disease Using Antisense Macrophage-Migration Inhibitory Factor (MIF) / Schizophyllan (SPG) Complex Hidetoshi Takedatsu, Keiichi Mitsuyama, Shinichi Mochizuki, Kazuo Sakurai, Tetsuharu Oriishi, Jun Nishihira, Michio Sata Background and Aim: Schizophyllan (SPG), a polysaccharide that belongs to beta-(1-3) glucan family, adopts a triple helix formation. The triple helix formation of SPG is dissociated to three single chains of SPG (s-SPG) by dimethyl sulfoxide (DMSO) or alkali solution. These s-SPGs re-form triple helix formation in normal condition (renaturation). Interestingly, we found that a macromolecular complex was formed consisting of two s-SPG chains and one polynucleotide chain, during this renaturation process was carried out in a mixture containing s-SPG and a single-stranded polynucleotide (Figure 1). We applied to this complex to deliver functional oligonucleotides as antisense DNA to treat the inflammation of intestine. The pathogenesis role of Macrophage-migration inhibitory factor (MIF) mainly produced by macrophages has been shown in the inflammatory bowel disease, such as Crohn's disease
AGA Abstracts
S-520
the chronic colitis model, IL-4-/- mice tended to have lower weight gain than WT mice, but this was not different significantly. In addition, both IL-4-/- and WT mice had similar DAI, colon length and histologic severity of colitis on the end point. The expression of TNFα and MIP-2, and levels of MPO in colonic tissue also did not show significant difference between two groups. Conclusions: Our results suggest that IL-4 is not an important independent factor on regulation of acute and chronic DSS colitis.
and ulcerative colitis. Here, we examined the biological function and the therapeutic effect of antisense MIF/SPG complex against colitis. Method: C57BL/6 mice were given 2 % dextran sodium sulfate (DSS) drinking water for 5 days. Severity of colitis and MIF expression was evaluated on day 14. The function of CD11b+ macrophages isolated from DSS-treated mice was analyzed. Immunofluorescence was performed to confirm whether CD11b+ macrophages uptake this complex. The inhibitory effect of antisense MIF/SPG complex was evaluated In Vitro. In addition, antisense MIF/SPG complex was intraperitoneally administered in DSS treated mice, and the severity of colitis was evaluated. Result: By 2% DSS administration, severe colitis was induced, and the expression of MIF was increased in the serum, colon, and MLN. MIF was mainly produced from CD11b+ macrophages in DSS-treated mice. Immunofluorescence showed that antisense MIF/SPG complex but not antisense MIF alone was uptake into macrophage. Further, Dectin-1, which was known as a receptor of SPG, was significantly increased in CD11b+ macrophages of DSS-treated mice compared with control mice by FACS analysis. MIF production both In Vitro and In Vivo was significantly suppressed by antisense MIF/SPG complex. Administration of antisense MIF/SPG complex ameliorated intestinal inflammation. Conclusion: Administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated the inflammation of colon. Our result demonstrated the possibility of new therapeutic approach against the inflammatory bowel disease.
Su1958 MicroRNA-146b Activates the NF-kB Pathway and Improves Intestinal Injury in a Mouse Enteritis Model Toshie Nata, Mikihiro Fujiya, Nobuhiro Ueno, Yuhei Inaba, Kentaro Moriichi, Yusuke Mizukami, Kazuya Sato, Yutaka Kohgo Background: microRNAs comprise approximately 20 nucleotides which modulate the level of their target proteins by either translational arrest or transcript degradation. While an abnormal miRNA expression is a common feature of various tumors, the precise role that microRNAs play in the regulation of inflammatory disease has just begun to be explored. Methods: 1. Differential expression of microRNAs in intestinal tissues of IL-10 deficient mice was examined with mirVanaTM miRNA Bioarray (Filgen). 2. Expression vectors containing a whole sequence of miR-146b or each siRNA wrapped with HVJ envelope were intraperitoneally injected to mice. The protein samples obtained from the mice 48 hours after the transfection, and the phosphorylation of NFkB was examined using western blotting. 3. The survival rate were compared between two groups, 4% dextran sodium sulfate (DSS)-treated mice and control mice, both were transfected with miR-146b vectors. Results: 1. In colonic tissues of IL-10 deficient mice, 19 microRNAs were downregulated and 26 microRNAs including miR-146b were upregulated more than twice. 2. NFkB phosphorylation was increased in the mice colon overexpressing either miR-146b. 3. The rate of survival was improved with the DSS-treated mice, when transfecting the vector. In addition, an inhibitor of NFkB (PDCT) eliminated the effect of miR-146b on relieving the intestinal inflammation. Discussion: miR-146b possibly improves the injury due to the intestinal inflammation through the regulation of NFkB pathway.
Su1961 Analysis of Intestinal Fibrosis in Chronic Colitis in Mice Induced by Dextran Sulfate Sodium Xiaomei Sun, Kenji Suzuki, Masaki Nagata, Kawase Tomoyuki, Hana Yamaguchi, Yusuke Kawauchi, Xiufen Tang, Xu Ren, Mitsuhiro Anzai, Takayoshi Nishino, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura
Su1959 Antibiotics Suppress Intestinal ICAM-1 Expression Independent of a Reducing Effect on Gut Microbial Density Ramadass Balamurugan, Julie Bard, Henry C. Lin
Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in the pathophysiology of intestinal fibrosis, however, their precise role in the development of fibrosis is largely unknown. To investigate their role in the intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. Clinical symptoms peaked at day 8 and were gradually ameliorated with the only apparent symptom of loose feces at day 19. Histopathological changes after DSS cessation in the chronic phase were dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of cytokines [interleukin (IL)-1β, IL-17a, and tumor necrosis factor (TNF)-α] are correlated with the extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-β, and matrix metalloproteinases (MMP-2, and -9) were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin+, α-smooth muscle actin(α-SMA)-) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin+, α-SMA+) were increased in mucosal but not in submucosal layers. A possible involvement of TGF-βin the fibrosis was further examined by primary mouse subcutaneous fibroblasts cultures. Exogenously added TGF-β1 substantially augmented the expressions of both vimentin and α-SMA proteins with increased production of collagen. Therefore, intestinal fibrosis in chronic DSS-induced colitis could be explained at least in part by TGF-β-dependent transition of mesenchymal progenitor fibroblasts into the profibrogenic fibroblasts and myofibroblasts with increased production of collagens. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in chronic colitis in B6 mice induced by a single cycle DSS administration.
A recent report showed that the gut microbial biomass need not be reduced in response to an oral antibiotic (Gut Microbes 2010:1(4):279-284). Since antibiotics have been used successfully to treat a variety of inflammatory disorders involving the intestine, could antibiotics have a direct effect on the host immune response even without a decontaminating effect on gut microbiota? Inflammation may be initiated when integrins bind to endothelium via intercellular adhesion molecule-1 (ICAM-1) leading to transmigration of the integrinexpressing leukocytes into tissues. However, whether or not ICAM-1 expression is changed by antibiotics independent of a reduction in the density of the indigenous gut microbial population is not known. A diet containing red kidney beans (RKB) is known to increase gut microbial density (DDS 2010;55(10):2778-84). Aim: Our aim was to test the hypothesis that antibiotics may alter ICAM-1 expression independent of a decontaminating effect on the indigenous microbes of intestinal mucosa. Methods: Twenty-four rats were randomized to 1 of 3 groups: 1) a standard rat chow alone (Control), 2) a standard rat chow with antibiotics (Antibiotics) or 3) a chow containing 26% crude red kidney beans with antibiotics (RKB+ Antibiotics). Antibiotic treatment consisted of 200μl mixture of kanamycin (4 mg/ ml), gentamicin (0.35 mg/ml), colistin (8500 U/ml), metronidazole (2.15 mg/ml), and vancomycin (0.45 mg/ml). Antibiotics were administered by gavage daily for 7 days. At the end of 7 days, the animals were sacrificed and intestinal tissue was collected. Total microbial load attached to the mucosa of the proximal, middle and distal 1/3 of the small intestine and cecum was quantitated by qPCR targeting 16s rRNA genes. Intestinal ICAM-1 gene expression was quantitated by qPCR. Results: When compared to controls, RKB+Antibiotic rats showed a significant increase in bacterial load (p<0.0001). Antibiotic group showed an intermediate result. However, there was a significant decrease in ICAM-1 expression (mean+/SE) in both RKB+Antibiotic group (0.00002± 3.64E-06) and Antibiotic group (0.00003± 6.93E-06) when compared to Controls (0.00089± 9 E-05)(p<0.05). There was an inverse relationship between ICAM-1 expression and microbial density in the proximal small intestine (p<0.004). Conclusion: Antibiotics decreased intestinal ICAM-1 expression independent of a reducing effect on microbial density demonstrating a potentially direct effect of antibiotics on host immune response. The suppression of ICAM-1 expression by some antibiotics may contribute to the beneficial response to antimicrobial agents in inflammatory gut disorders.
Su1962 Postnatal Changes in Monoamine Transporter Function in the Guinea Pig Ileum Hong Zhao, Xiaochun Bian, James Galligan, Greg M. Swain 5-hydroxytryptamine (5-HT) is a paracrine signaling molecule in the gut mucosa and a transmitter in the enteric nervous system. After release, 5-HT is cleared by the serotonin transporter (SERT). Previous work showed that SERT expression is low in the ileum of neonatal guinea pigs. In this study we examined the potential role of the dopamine and norepinephrine transporters (DAT and NET) in clearance of extracellular 5-HT in the mucosa of guinea pigs at 1 day, 1, 3 and 6 weeks postnatal. Methods. Extracellular 5-HT was monitored In Vitro in the ileum of guinea pigs as an oxidation current (continuous amperometry at 0.6V vs. Ag|AgCl reference electrode) using a boron-doped diamond microelectrode positioned near the mucosal surface. Sensitivity of the 5-HT oxidation current to citalopram (1 μM, SERT blocker), desmethylimpramine (DMI, 1 μM, NET blocker), GBR12909 (0.1 μM DAT blocker) and cocaine (10 μM, non-selective monoamine transport inhibitor) was tested. Tissue 5-HT and 5-hydroxy indole acetic acid (5-HIAA, 5-HT metabolite formed after clearance by SERT) levels were measured using HPLC. Results. 5-HT oxidation currents at 1 day postnatal were unaffected by all monoamine transport inhibitors. 5-HT oxidation currents were insensitive to citalopram at 1 week postnatal but thereafter showed a maturation dependent increase in citalopram sensitivity. GBR 12909 increased 5-HT oxidation currents at 1 and 3 weeks but not at 6 weeks. 5-HT oxidation currents were increased by DMI at 3 and 6 weeks postnatal. Cocaine increase 5-HT oxidation current at 1, 3 and 6 weeks. Steady state tissue levels of 5-HT were stable throughout the time course of these studies while 5-HIAA levels increased at 1 week and remained elevated at 3 and 6 weeks. Conclusion.
Su1960 The New Therapeutic Approach for Inflammatory Bowel Disease Using Antisense Macrophage-Migration Inhibitory Factor (MIF) / Schizophyllan (SPG) Complex Hidetoshi Takedatsu, Keiichi Mitsuyama, Shinichi Mochizuki, Kazuo Sakurai, Tetsuharu Oriishi, Jun Nishihira, Michio Sata Background and Aim: Schizophyllan (SPG), a polysaccharide that belongs to beta-(1-3) glucan family, adopts a triple helix formation. The triple helix formation of SPG is dissociated to three single chains of SPG (s-SPG) by dimethyl sulfoxide (DMSO) or alkali solution. These s-SPGs re-form triple helix formation in normal condition (renaturation). Interestingly, we found that a macromolecular complex was formed consisting of two s-SPG chains and one polynucleotide chain, during this renaturation process was carried out in a mixture containing s-SPG and a single-stranded polynucleotide (Figure 1). We applied to this complex to deliver functional oligonucleotides as antisense DNA to treat the inflammation of intestine. The pathogenesis role of Macrophage-migration inhibitory factor (MIF) mainly produced by macrophages has been shown in the inflammatory bowel disease, such as Crohn's disease
AGA Abstracts
S-520