Tu1634 Minor Effects of Rifaximin-α Modulating Luminal and Wall-Adhered Gut Commensal Microbiota...
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Tu1633 Effect of Phytoestrogenic Dietary Supplementation on the Progression of Chronic Intestinal Inflammation to Cancer in Animal Model Mariabeatrice Principi, Nicola De Tullio, Katia Lofano, Maria Principia Scavo, Maria Pricci, Gennj Piacentino, Antonella Contaldo, Viviana Neve, Enzo Ierardi, Maria Cristina Comelli, Alfredo Di Leo Background and aim : Colorectal cancer (CRC) is increased in inflammatory bowel diseases (IBD). Chemopreventive agents could minimize this risk. Estrogens exert prevention against CRC throught Estrogen Receptor β (ERβ) with an inverse relation with the tumor. ER β induction may be a target in CRC prevention. Phytoestrogens are dietary compounds with higher binding affinity for ER β than Erα. A blend of the dietary phytoestrogens (silymarin and lignan) and non-starch insoluble fibers (Eviendep, CM&D Pharma) has been tested in an experimental mouse model of AOM/DSS induced colitis, to assess the anti-inflammatory and anti-carcinogenetic properties and to verify whether such effect is related to an increased ERβ expression. Material and methods : A known model of dextran sodium sulfate/azoxy- methane induced CRC was used to obtain the best simulation of IBD-related CRC pathogene- sis. Seventy-six C57BL/6J male mice were divided into three groups: 35 mice fed a Eviendep- modified diet during the whole carcinogenetic process, 35 fed a standard diet (positive control) and 6 mice with no treatment or modified diet (negative control). Monitoring of colitis and tumorigenesis was performed by a specific video-endoscopic system (Coloview Miniendoscopic System) at 21 weeks. Animals were sacrificed at 22 weeks for histogical and ERs immunohistochemical evaluation. Histological score was: 0 (no dysplasia), 1 (low grade dysplasia), 2 (high grade dysplasia) and 3 (carcinoma). Results : Mortality rate was of 40-50%. At sacrifice, treated animals showed a decrease in number/volume of polyps and in histological score (p,0.05 ANOVA-Bonferroni post hoc test). Only 30% of Eviendep- supplemented mice showed at least 1 CRC compared with the 100% of the positive controls. ERα expression was higher in neoplastic tissue than in normal mucosa both in treated and positive control groups, while ER β labeling index (LI%) showed a higher value in non- neoplastic tissue of the Eviendep-supplemented group (63.8±4.7) than in positive control (46.6±6.4), resulting similar to the ER β LI value of negative controls (54.9±7.9) (p ,0.05 Fisher Exact Test) Conclusions : Our results suggest a chemopreventive effect of Eviendep on colonic carcinogenesis arising from inflamed tissue, and such an effect associates with an increase ERβ content in the tissue. Tu1634 Minor Effects of Rifaximin-α Modulating Luminal and Wall-Adhered Gut Commensal Microbiota and Toll-Like Receptors (TLRS) Expression in Mice Marina Ferrer, Mònica Aguilera, Javier Estévez, Patri Vergara, Vicente Martinez BACKGROUND: Rifaximin is a broad-spectrum, non-absorbable antibiotic that ameliorates symptomatology in inflammatory bowel disease or irritable bowel syndrome patients. Although the mechanisms of action remain unclear both microbial dependent and indepen- dent mechanisms have been suggested. Indeed, Rifaximin's effects on gut microbiota are still unclear. AIMS: To assess changes in luminal and wall-adhered gut commensal microbiota (GCM) and the TLR-dependent bacterial recognition system associated to Rifaximin treatment in mice. METHODS: Adult C57BL/6 female mice were treated (1 or 2 weeks, PO) with Rifaximin (50 or 150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCM were characterized by fluorescent in situ hybridization (FISH). Microbial profiles were charac- terized by terminal restriction fragment length polymorphism (t-RFLP). Colonic expression of TLR-2, -4 and -5 was assessed by RT-qPCR. RESULTS: Regardless the period of treatment or the dose tested, Rifaximin did not alter total bacterial counts (x10 10 cells/ml; vehicle: 4.3±1.1; 50 mg: 4.1±0.4; 150 mg: 4.8±1.2; n=8-12; P .0.05). Similarly, Rifaximin had minor effects on the overall composition of the luminal microbiota, with only a slight increase in the counts of Bacteroides spp. observed at the 150 mg dose after a 1-week, but not a 2- week, treatment (x10 8 cells/ml; vehicle: 32.3±9.8; 1-week treatment: 71.5±12.4, P ,0.05 vs. vehicle; 2-week treatment: 47.1±5.2; n=8-12). In agreement, bacterial diversity, as assessed by t-RFLP, was not affected by Rifaximin. In control conditions, only Clostridium spp. (40% incidence) and Bifidobacterium spp. (13% incidence) were found attached to the colonic epithelium. Rifaximin showed a tendency to favour the adherence of both Bifidobacterium spp. (50% incidence) and Clostridium spp. (75% incidence) after a 1-week treatment; while no changes were observed at 2 weeks. Overall, regardless the dose or time of treatment, Rifaximin induced a minor increase (1 to 2 fold) in TLRs expression. Only the 50 mg dose for 1 week led to a significant increase (by 3-fold) in TLR-4 expression. In no case, structural alterations consistent with the induction of colonic inflammation were observed. CONCLU- SIONS: Results obtained show that Rifaximin, eventhough its antibacterial properties, induces very minor changes in luminal GCM and bacterial wall adherence in standard mice. Similarly, minor changes, with tendency to the up-regulation, in TLRs (2, 4 and 5)-dependent bacterial recognition systems were observed. More noticeable effects of Rifaximin on dysbiotic states, vs. a normal GCM, can not be discarded. Moreover, from the present data we can not exclude that the beneficial effects of Rifaximin in functional and inflammatory gastrointestinal disorders are associated to alternative mechanisms of action not directly related to the modulation of the microbiota. Tu1635 Protection of the Enteric Nervous System in Colitis: A Therapeutic Approach for IBD? Panagiotis Giannogonas, Charalabos Pothoulakis, Achille Gravanis, Katia P. Karalis There is close interaction between the immune and nervous systems in the regulation of the inflammation. The gastrointestinal tract is richly innervated by an intrinsic enteric nervous system (ENS). Emerging evidence \ suggests the contribution of the ENS in various pathological conditions, such as the irritable bowel syndrome and inflammatory bowel disease (IBD). Previous studies have shown increased vulnerability of genetically-induced "hyperinnervation" to IBD. Our aim was to characterize the specific role of the ENS in the development and progress of experimental IBD due to innate immune activation. We assessed S-811 AGA Abstracts the role of ENS throughout the progress of a model of IBD in mice, the DSS-induced colitis. As has been previously shown, DSS administration for 7 days results in the development of severe local inflammation, significant anorexia and weight loss. Repeated cycles of DSS administration mimic the human disease characterized by alternating remission and relapsing phases. We found reduced expression of both beta III - tubulin, a neuron specific cytoskeletal protein, and synaptophysin, a major synaptic vesicle protein, following DSS treatment in support of functional impairment of the ENS during colitis. To better understand the specific role of the ENS during DSS colitis we induced "pharmacological hyperinnervetion" at different time points throughout the progress of the disease. Co-administration of a synthetic neuroste- roid (BNN93, DHEA analog - 2mg per animal per day), a neuroprotective agent in the CNS, worsened the acute inflammatory response and increased the expression of proinflammatory cytokines such as TNFalpha, in agreement with a recent study (Margolis KG, 2011) . To assess the effects of the "neuroprotection" of ENS over the course of experimental colitis, we subjected the mice to the chronic, remission-relapsing disease model and administered BNN27 for 5 consecutive days in each treatment cycle, starting at the 4th day of DSS treatment, for 1, 2 and 4 cycles (chronic disease model). Histological analysis of the affected tissues revealed an active inflammatory response characterized by increased angiogenesis and fibrosis. All inflammatory parameters studied were significantly elevated compared to the control (vehicle-treated) mice following co-administration of DSS and BNN27 for 1 cycle. To our surprise repeated administration of BNN27 was protective from chronic DSS-induced colitis, as shown by reduced inflammation and increased tissue regeneration compared to vehicle-treated mice. These findings suggest the intriguing hypothesis that ENS may participate actively in the resolution of inflammation and repair of the epithelial barrier, via mechanisms that remain to be elucidated. The potential implication of these results for the development of novel, specific therapeutic approaches for IBD are under investigation. Tu1636 Immunomodulatory Effect of Enalapril on Peritoneal Macrophage and Chronic Colitis in Interleukin-10-Deficient Mice Changhyun Lee, Jaeyoung Chun, Jong Pil Im, Hyun Chae Jung, Joo Sung Kim Background/Aims: Interleukin (IL)-10 potently reduces the T helper type 1 response by preventing synthesis of IL-12 from antigen-presenting cells. Enalapril, an angiotensin-con- verting enzyme (ACE) inhibitor, blocked inflammatory response and prevented lung or renal injury in vivo. The aim of this study was to evaluate the immunomodulatory effect of enalapril on peritoneal macrophages from IL-10-deficient (IL-10-/-) mice and on chronic colitis in IL-10-/- mice. Methods: Peritoneal macrophages from IL-10-/- mice were elicited by intra-peritoneal injection of 4% thioglycolate. Peritoneal macrophages were collected and pretreated with enalapril, and then stimulated with lipopolysaccharide (LPS). Real-time RT- PCR and ELISA were performed for TNF- α and IL-12p40. Western blot analysis was done for phospho-IκBα and I κBα. For In vivo study, IL-10-/- mice were induced colitis with piroxicam and then treated with enalapril by oral gavage. The effects of 1 and 5 mg/kg enalapril for 2 weeks have been compared with that of vehicle. Colitis was quantified by the evaluation of histopathological findings, and the gene expression of proinflammatory cytokines was assessed in the colonic tissue. Results: Enalapril strongly inhibited LPS-induced TNF-α and IL-12p40 mRNA and protein expression in peritoneal macrophages from IL-10-/- mice. Enalapril also blocked LPS-induced I κBα phosphorylation/degradation in peritoneal macrophages. In IL-10-/- mice, administration of enalapril significantly reduced the severity of colitis as assessed by histology in a dose-dependent manner. Gene expression of TNF- α and IL-12p40 in the colonic tissue was significantly lower in enalapril-treated IL-10-/- mice. Conclusions: Enalapril may block the activation of peritoneal macrophages and ameliorates colitis in IL-10-/- mice, which suggesting that enalapril is a potential therapeutic agent for inflammatory bowel disease. Tu1637 Transplantaion of Human Fetal Membrane-Derived Mesenchymal Stem Cells Improves Severe Colitis Induced by Dextran Sulfate Sodium in Rats Reizo Onishi, Shunsuke Ohnishi, Ryosuke Higashi, Michiko Watari, Waka Kobayashi, Takehiko Katsurada, Hiroshi Takeda, Naoya Sakamoto Aims: Mesenchymal stem cells (MSCs) have been reported to be a valuable cell source in cell therapy, and bone marrow (BM) represents a major source of MSCs; however, there are several limitations when using BM-MSCs, including inadequate cell numbers, invasiveness and donor site morbidity. Recently, several studies have shown that MSCs can be easily isolated from human fetal membrane (FM), and a large amount of cells can be obtained. We thus examined the therapeutic effects of transplantation of human FM-derived MSCs (hFM-MSCs) in dextran sulfate sodium (DSS)-induced colitis in rats. Methods: The Medical Ethical Committee approved this examination and all pregnant women gave written informed consent. FM was obtained at Cesarean delivery, and hFM-MSCs were isolated and expanded by digestion with collagenase, followed by culturing in uncoated plastic dishes. Severe colitis was induced in 7-week-old male Sprague-Dawley rats by administration of 8% DSS in drinking water ad libitum from day 0 to day 5. At day 1, hFM-MSCs (1×10 6 cells) were transplanted intravenously. Changes in body weight and disease activity index (DAI) were evaluated daily for 5 days. Rats were sacrificed on day 5, and the entire colon was extracted. Colon length, histological colitis score were evaluated, and mRNA expression of inflammatory mediators were measured by quantitative RT-PCR. Infiltration of T cells (CD3) and mono- cytes/macrophages (CD68) was investigated by immunohistochemistry. Effect of hFM-MSCs on induction of TNF-α by lipoplysaccahride (LPS) in mouse macrophage cell line RAW264.7 was evaluated by quantitative RT-PCR. Results: Transplantation of hFM-MSCs significantly ameliorated the DAI score, weight loss, colon shortening and histological colitis score. mRNA expression of TNF- α, IL-1β and MIF was significantly decreased in the rectum of hFM- MSC-treated rats. In addition, infiltration of CD68-positive cells was significantly decreased in hFM-MSC-treated rats. Furthermore, induction of TNF- α from RAW264.7 cells by LPS was significantly attenuated by co-culture with hFM-MSCs, or by culture with conditioned medium obtained from hFM-MSCs. Conclusion: Transplantation of hFM-MSCs provided significant improvement in a rat model of severe colitis, possibly through inhibition of AGA Abstracts
Transcript of Tu1634 Minor Effects of Rifaximin-α Modulating Luminal and Wall-Adhered Gut Commensal Microbiota...
Tu1633
Effect of Phytoestrogenic Dietary Supplementation on the Progression ofChronic Intestinal Inflammation to Cancer in Animal ModelMariabeatrice Principi, Nicola De Tullio, Katia Lofano, Maria Principia Scavo, MariaPricci, Gennj Piacentino, Antonella Contaldo, Viviana Neve, Enzo Ierardi, Maria CristinaComelli, Alfredo Di Leo
Background and aim : Colorectal cancer (CRC) is increased in inflammatory bowel diseases(IBD). Chemopreventive agents could minimize this risk. Estrogens exert prevention againstCRC throught Estrogen Receptor β (ERβ) with an inverse relation with the tumor. ER βinduction may be a target in CRC prevention. Phytoestrogens are dietary compounds withhigher binding affinity for ERβ than Erα. A blend of the dietary phytoestrogens (silymarinand lignan) and non-starch insoluble fibers (Eviendep, CM&D Pharma) has been tested inan experimental mouse model of AOM/DSS induced colitis, to assess the anti-inflammatoryand anti-carcinogenetic properties and to verify whether such effect is related to an increasedERβ expression. Material and methods : A known model of dextran sodium sulfate/azoxy-methane induced CRC was used to obtain the best simulation of IBD-related CRC pathogene-sis. Seventy-six C57BL/6J male mice were divided into three groups: 35 mice fed a Eviendep-modified diet during the whole carcinogenetic process, 35 fed a standard diet (positivecontrol) and 6 mice with no treatment or modified diet (negative control). Monitoring ofcolitis and tumorigenesis was performed by a specific video-endoscopic system (ColoviewMiniendoscopic System) at 21 weeks. Animals were sacrificed at 22 weeks for histogicaland ERs immunohistochemical evaluation. Histological score was: 0 (no dysplasia), 1 (lowgrade dysplasia), 2 (high grade dysplasia) and 3 (carcinoma). Results : Mortality rate wasof 40-50%. At sacrifice, treated animals showed a decrease in number/volume of polypsand in histological score (p,0.05 ANOVA-Bonferroni post hoc test). Only 30% of Eviendep-supplemented mice showed at least 1 CRC compared with the 100% of the positive controls.ERα expression was higher in neoplastic tissue than in normal mucosa both in treated andpositive control groups, while ERβ labeling index (LI%) showed a higher value in non-neoplastic tissue of the Eviendep-supplemented group (63.8±4.7) than in positive control(46.6±6.4), resulting similar to the ERβ LI value of negative controls (54.9±7.9) (p ,0.05Fisher Exact Test) Conclusions : Our results suggest a chemopreventive effect of Eviendepon colonic carcinogenesis arising from inflamed tissue, and such an effect associates withan increase ERβ content in the tissue.
Tu1634
Minor Effects of Rifaximin-α Modulating Luminal and Wall-Adhered GutCommensal Microbiota and Toll-Like Receptors (TLRS) Expression in MiceMarina Ferrer, Mònica Aguilera, Javier Estévez, Patri Vergara, Vicente Martinez
BACKGROUND: Rifaximin is a broad-spectrum, non-absorbable antibiotic that amelioratessymptomatology in inflammatory bowel disease or irritable bowel syndrome patients.Although the mechanisms of action remain unclear both microbial dependent and indepen-dent mechanisms have been suggested. Indeed, Rifaximin's effects on gut microbiota arestill unclear.AIMS: To assess changes in luminal and wall-adhered gut commensal microbiota(GCM) and the TLR-dependent bacterial recognition system associated to Rifaximin treatmentin mice. METHODS: Adult C57BL/6 female mice were treated (1 or 2 weeks, PO) withRifaximin (50 or 150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCMwere characterized by fluorescent in situ hybridization (FISH). Microbial profiles were charac-terized by terminal restriction fragment length polymorphism (t-RFLP). Colonic expressionof TLR-2, -4 and -5 was assessed by RT-qPCR. RESULTS: Regardless the period of treatmentor the dose tested, Rifaximin did not alter total bacterial counts (x1010 cells/ml; vehicle:4.3±1.1; 50 mg: 4.1±0.4; 150 mg: 4.8±1.2; n=8-12; P .0.05). Similarly, Rifaximin hadminor effects on the overall composition of the luminal microbiota, with only a slight increasein the counts of Bacteroides spp. observed at the 150 mg dose after a 1-week, but not a 2-week, treatment (x108 cells/ml; vehicle: 32.3±9.8; 1-week treatment: 71.5±12.4, P,0.05vs. vehicle; 2-week treatment: 47.1±5.2; n=8-12). In agreement, bacterial diversity, as assessedby t-RFLP, was not affected by Rifaximin. In control conditions, only Clostridium spp. (40%incidence) and Bifidobacterium spp. (13% incidence) were found attached to the colonicepithelium. Rifaximin showed a tendency to favour the adherence of both Bifidobacteriumspp. (50% incidence) and Clostridium spp. (75% incidence) after a 1-week treatment; whileno changes were observed at 2 weeks. Overall, regardless the dose or time of treatment,Rifaximin induced a minor increase (1 to 2 fold) in TLRs expression. Only the 50 mg dosefor 1 week led to a significant increase (by 3-fold) in TLR-4 expression. In no case, structuralalterations consistent with the induction of colonic inflammation were observed. CONCLU-SIONS: Results obtained show that Rifaximin, eventhough its antibacterial properties,induces very minor changes in luminal GCM and bacterial wall adherence in standard mice.Similarly, minor changes, with tendency to the up-regulation, in TLRs (2, 4 and 5)-dependentbacterial recognition systems were observed. More noticeable effects of Rifaximin on dysbioticstates, vs. a normal GCM, can not be discarded. Moreover, from the present data wecan not exclude that the beneficial effects of Rifaximin in functional and inflammatorygastrointestinal disorders are associated to alternative mechanisms of action not directlyrelated to the modulation of the microbiota.
Tu1635
Protection of the Enteric Nervous System in Colitis: A Therapeutic Approachfor IBD?Panagiotis Giannogonas, Charalabos Pothoulakis, Achille Gravanis, Katia P. Karalis
There is close interaction between the immune and nervous systems in the regulation ofthe inflammation. The gastrointestinal tract is richly innervated by an intrinsic entericnervous system (ENS). Emerging evidence \ suggests the contribution of the ENS in variouspathological conditions, such as the irritable bowel syndrome and inflammatory boweldisease (IBD). Previous studies have shown increased vulnerability of genetically-induced"hyperinnervation" to IBD. Our aim was to characterize the specific role of the ENS in thedevelopment and progress of experimental IBD due to innate immune activation. We assessed
S-811 AGA Abstracts
the role of ENS throughout the progress of a model of IBD in mice, the DSS-induced colitis.As has been previously shown, DSS administration for 7 days results in the developmentof severe local inflammation, significant anorexia and weight loss. Repeated cycles of DSSadministration mimic the human disease characterized by alternating remission and relapsingphases. We found reduced expression of both beta III - tubulin, a neuron specific cytoskeletalprotein, and synaptophysin, a major synaptic vesicle protein, following DSS treatment insupport of functional impairment of the ENS during colitis. To better understand the specificrole of the ENS during DSS colitis we induced "pharmacological hyperinnervetion" at differenttime points throughout the progress of the disease. Co-administration of a synthetic neuroste-roid (BNN93, DHEA analog - 2mg per animal per day), a neuroprotective agent in the CNS,worsened the acute inflammatory response and increased the expression of proinflammatorycytokines such as TNFalpha, in agreement with a recent study (Margolis KG, 2011) . Toassess the effects of the "neuroprotection" of ENS over the course of experimental colitis,we subjected the mice to the chronic, remission-relapsing disease model and administeredBNN27 for 5 consecutive days in each treatment cycle, starting at the 4th day of DSStreatment, for 1, 2 and 4 cycles (chronic disease model). Histological analysis of the affectedtissues revealed an active inflammatory response characterized by increased angiogenesisand fibrosis. All inflammatory parameters studied were significantly elevated compared tothe control (vehicle-treated) mice following co-administration of DSS and BNN27 for 1cycle. To our surprise repeated administration of BNN27 was protective from chronicDSS-induced colitis, as shown by reduced inflammation and increased tissue regenerationcompared to vehicle-treated mice. These findings suggest the intriguing hypothesis that ENSmay participate actively in the resolution of inflammation and repair of the epithelial barrier,via mechanisms that remain to be elucidated. The potential implication of these results forthe development of novel, specific therapeutic approaches for IBD are under investigation.
Tu1636
Immunomodulatory Effect of Enalapril on Peritoneal Macrophage and ChronicColitis in Interleukin-10-Deficient MiceChanghyun Lee, Jaeyoung Chun, Jong Pil Im, Hyun Chae Jung, Joo Sung Kim
Background/Aims: Interleukin (IL)-10 potently reduces the T helper type 1 response bypreventing synthesis of IL-12 from antigen-presenting cells. Enalapril, an angiotensin-con-verting enzyme (ACE) inhibitor, blocked inflammatory response and prevented lung or renalinjury in vivo. The aim of this study was to evaluate the immunomodulatory effect ofenalapril on peritoneal macrophages from IL-10-deficient (IL-10-/-) mice and on chroniccolitis in IL-10-/- mice. Methods: Peritoneal macrophages from IL-10-/- mice were elicitedby intra-peritoneal injection of 4% thioglycolate. Peritoneal macrophages were collected andpretreated with enalapril, and then stimulated with lipopolysaccharide (LPS). Real-time RT-PCR and ELISA were performed for TNF-α and IL-12p40. Western blot analysis was donefor phospho-IκBα and IκBα. For In vivo study, IL-10-/- mice were induced colitis withpiroxicam and then treated with enalapril by oral gavage. The effects of 1 and 5 mg/kgenalapril for 2 weeks have been compared with that of vehicle. Colitis was quantified bythe evaluation of histopathological findings, and the gene expression of proinflammatorycytokines was assessed in the colonic tissue. Results: Enalapril strongly inhibited LPS-inducedTNF-α and IL-12p40 mRNA and protein expression in peritoneal macrophages from IL-10-/-mice. Enalapril also blocked LPS-induced IκBα phosphorylation/degradation in peritonealmacrophages. In IL-10-/- mice, administration of enalapril significantly reduced the severityof colitis as assessed by histology in a dose-dependent manner. Gene expression of TNF- αand IL-12p40 in the colonic tissue was significantly lower in enalapril-treated IL-10-/- mice.Conclusions: Enalapril may block the activation of peritoneal macrophages and amelioratescolitis in IL-10-/- mice, which suggesting that enalapril is a potential therapeutic agent forinflammatory bowel disease.
Tu1637
Transplantaion of Human Fetal Membrane-Derived Mesenchymal Stem CellsImproves Severe Colitis Induced by Dextran Sulfate Sodium in RatsReizo Onishi, Shunsuke Ohnishi, Ryosuke Higashi, Michiko Watari, Waka Kobayashi,Takehiko Katsurada, Hiroshi Takeda, Naoya Sakamoto
Aims: Mesenchymal stem cells (MSCs) have been reported to be a valuable cell source incell therapy, and bone marrow (BM) represents a major source of MSCs; however, thereare several limitations when using BM-MSCs, including inadequate cell numbers, invasivenessand donor site morbidity. Recently, several studies have shown that MSCs can be easilyisolated from human fetal membrane (FM), and a large amount of cells can be obtained.We thus examined the therapeutic effects of transplantation of human FM-derived MSCs(hFM-MSCs) in dextran sulfate sodium (DSS)-induced colitis in rats. Methods: The MedicalEthical Committee approved this examination and all pregnant women gave written informedconsent. FM was obtained at Cesarean delivery, and hFM-MSCs were isolated and expandedby digestion with collagenase, followed by culturing in uncoated plastic dishes. Severe colitiswas induced in 7-week-old male Sprague-Dawley rats by administration of 8% DSS indrinking water ad libitum from day 0 to day 5. At day 1, hFM-MSCs (1×106 cells) weretransplanted intravenously. Changes in body weight and disease activity index (DAI) wereevaluated daily for 5 days. Rats were sacrificed on day 5, and the entire colon was extracted.Colon length, histological colitis score were evaluated, and mRNA expression of inflammatorymediators were measured by quantitative RT-PCR. Infiltration of T cells (CD3) and mono-cytes/macrophages (CD68) was investigated by immunohistochemistry. Effect of hFM-MSCson induction of TNF-α by lipoplysaccahride (LPS) in mouse macrophage cell line RAW264.7was evaluated by quantitative RT-PCR. Results: Transplantation of hFM-MSCs significantlyameliorated the DAI score, weight loss, colon shortening and histological colitis score. mRNAexpression of TNF-α, IL-1β and MIF was significantly decreased in the rectum of hFM-MSC-treated rats. In addition, infiltration of CD68-positive cells was significantly decreasedin hFM-MSC-treated rats. Furthermore, induction of TNF-α from RAW264.7 cells by LPSwas significantly attenuated by co-culture with hFM-MSCs, or by culture with conditionedmedium obtained from hFM-MSCs. Conclusion: Transplantation of hFM-MSCs providedsignificant improvement in a rat model of severe colitis, possibly through inhibition of
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