Efficacy profiles for different concentrations of Lactobacillus acidophilus in experimental colitis

AIM:To determine the efficacy profiles of different concentrations of Lactobacillus acidophilus(L.acidophilus)for treating colitis using an experimental murine model.METHODS:Colitis was established in 64 BALB/c mice by adding 5%dextran sodium sulfate(DSS)to the drinking water and allowing ad libitum access for 7 d.The mice were then randomly divided into the following control and experimental model groups(n=8 each;day 0):untreated model control;negative-treatment model control(administered gavage of 1 mL/10 g normal saline);experimental-treatment models C4-C8(administered gavage of 104,105,106,107,or 108CFU/10 g L.acidophilus,respectively);positive-treatment model control(administration of the anti-inflammatory agent prednisone acetate at 45 g/10 g).Eight mice given regular water(no DSS)and no subsequent treatments served as the normal control group.Body weight,fecal traits,and presence of fecal occult blood were assessed daily.All animals were sacrificed on post-treatment day7 to measure colonic length,perform histological scoring,and quantify the major bacteria in the proximal and distal colon.Intergroup differences were determined by one-way ANOVA and post-hoc Student-Newman-Keuls comparison.RESULTS:All treatments(L.acidophilus and prednisone acetate)protected against colitis-induced weight loss(P<0.05 vs model and normal control groups).The extent of colitis-induced colonic shortening was significantly reduced by all treatments(prednisone acetate>C4>C5>C7>C8>C6;P<0.05 vs untreated model group),and the C6 group showed colonic length similar to that of the normal control group(P>0.05).The C6 group also had the lowest disease activity index scores among the model groups.The bacterial profiles in the proximal colon were similar between all of the experimental-treatment model groups(all P>0.05).In contrast,the bacterial profile in the distal colon of the C6 group showed the distinctive features(P<0.05 vs all other experimental-treatment model groups)of Lactobacillus sp.and Bifidobact     

Antioxidative potential of a combined therapy of anti TNFα and Zn acetate in experimental colitis

AIM:To evaluate whether combination therapy with antitumour necrosis factor α (TNFα) antibody and Zn acetate is beneficial in dextran sodium sulphate (DSS) colitis.METHODS:Colitis was induced in CD1-Swiss mice with 5% DSS for 7 d.The experimental mice were then randomised into the following subgroups:standard diet + DSS treated (induced colitis group);standard diet + DSS + subcutaneous 25 μg anti-TNFα treated group;Zn acetate treated group + DSS + subcutaneous 25 μg anti-TNFα;standard diet + DSS + subcutane...     

The Role of Zinc and Metallothionein in the Dextran Sulfate Sodium-Induced Colitis Mouse Model

Zinc (Zn) and its binding protein metallothionien (MT) have been proposed to suppress the disease activity in ulcerative colitis. To determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice, a DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT−/−) mice by supplementing 2%, 3%, and 4% DSS in the drinking water for 6 days. In the intervention study, colitis was induced with 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) was scored daily. Histology, MT levels, and myeloperoxidase (MPO) activity were determined. DAI was increased (P<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively, compared to 2%, evident after 5 days of DSS administration. MPO activity was increased in MT+/+ compared to MT−/− mice and those receiving DSS. Zn administration had a 50% (P<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damage compared to MT−/− mice. MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.     

A recombinant cystatin from Ascaris lumbricoides attenuates inflammation of DSS‐induced colitis

Helminthiasis may ameliorate inflammatory diseases, such as inflammatory bowel disease and asthma. Information about immunomodulators from Ascaris lumbricoides is scarce, but could be important considering the co‐evolutionary relationships between helminths and humans. We evaluated the immunomodulatory effects of a recombinant cystatin from A. lumbricoides on an acute model of dextran sodium sulphate (DSS)‐induced colitis in mice. From an A. lumbricoides cDNA library, we obtained a recombinant cystatin (rAl‐CPI). Protease activity inhibition was demonstrated on cathepsin B and papain. Immunomodulatory effects were evaluated at two intraperitoneal doses (0.5 and 0.25 μg/G) on mice with DSS‐induced colitis. Body weight, colon length, Disease Activity Index (DAI), histological inflammation score, myeloperoxidase (MPO) activity, gene expression of cytokines and cytokines levels in colon tissue were analysed. Treatment with rAl‐CPI significantly reduced DAI, MPO activity and inflammation score without toxic effects. Also, IL‐10 and TGF‐B gene overexpression was observed in rAl‐CPI‐treated group compared to DSS‐exposed control and healthy mice. Furthermore, a reduction in IL‐6 and TNF‐A expression was found, and this was confirmed by the levels of these cytokines in colonic tissue. In conclusion, rAl‐CPI reduces inflammation in a mouse model of DSS‐induced colitis, probably by increasing the expression of anti‐inflammatory cytokines and reducing pro‐inflammatory ones.     

Polaprezinc (N-(3-aminopropionyl)-L-histidinato zinc) ameliorates dextran sulfate sodium-induced colitis in mice

OBJECTIVE: Polaprezinc (N-(3-Aminopropionyl)-L-histidinato zinc), an anti-ulcer drug, has been reported to have an anti-inflammatory action in several inflammatory diseases. The aim of this study was to investigate the effect of polaprezinc on dextran sulfate (DSS)-induced colitis in mice. MATERIAL AND METHODS: Mice with colitis induced by DSS were intrarectally treated with polaprezinc (15 mg/kg) or zinc sulfate (7.5 mg/kg) every day after the administration of DSS for 7 days. Disease activity index (DAI) and histological tissue damage were assessed. Levels of myeloperoxidase (MPO) activity, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in the colon were measured. Expression of heat shock protein (HSP) 25 and HSP70 in the colon was analyzed by Western blot analysis. RESULTS: DAI and histological scores were remarkably reduced in polaprezinc-treated mice with DSS-induced colitis. Polaprezinc suppressed the increase of MPO activity and the production of TNF-alpha and IFN-gamma in the colon tissues of mice with DSS-induced colitis. Expression of HSP25 and HSP70 was remarkably up-regulated in the colon tissues of polaprezinc-treated mice during DSS treatment. CONCLUSIONS: Polaprezinc suppresses DSS-induced colitis in mice, partly through inhibition of production of pro-inflammatory cytokine, suppression of neutrophils accumulation and cytoprotection by overexpression of HSPs. Polaprezinc could be useful in the treatment of inflammatory bowel diseases.     

Chymase inhibitor TY-51469 in therapy of inflammatory bowel disease

AIM: To investigate the effect of chymase inhibitor TY-51469 in the therapy of inflammatory bowel disease and the underlying mechanism. METHODS: Seventy-five healthy Sprague-Dawley rats were randomly assigned to one of the three groups(control group, model group and TY-51469 experiment group) and each group had twenty-five rats. The rats of the model group and experiment group were subjected to treatment with 3.5% dextran sulfate sodium(DSS) 10 mg/kg to induce colitis. The control group and model group were subjected to intraperitoneal injection of saline, while the experiment group was subjected to intraperitoneal injection of 10 mg/kg TY-51469 each day. Five rats of each group were sacrificed on 0, 7, 14, 21 and 28 d, respectively. The degree of inflammation was assessed by histopathological scoring; flow cytometry was performed to detect the proportion of CD4+CD25+ Tregs in peripheral blood; colon tissues of rats were collected to measure m RNA and protein expression by PCR, Western blot and immunohistochemistry; serum levels of interleukin(IL)-10, transforming growth factor(TGF)-β1 and IL-17A were detected by ELISA. RESULTS: The rats in the experiment group and model group had significantly more severe colitis than the ones in the control group(P 0.05) before treatment on day 0; no significant difference was observed between the experiment group and model group(P 0.05). After treatment with TY-51469, the rats in the experiment group had significantly less severe colitis compared with the model group on 7, 14, 21 and 28 d(P 0.05). The proportion of CD4+CD25+ Tregs was lower in the model group and experiment group than in the control group; the experiment group had a significantly higher proportion of CD4+CD25+ Tregs than that in the model group(P 0.05). The model group and experiment group demonstrated lower expression of Foxp3 than the control group; the experiment group had higher Foxp3 expression than the model group(P 0.05). Cytokines IL-10, TGF-β1 and IL-17 A were lower in the model group and experiment group than in the control group; the experiment group had higher expression than the model group(P 0.05). CONCLUSION: After treatment with chymase inhibitor TY-51469, the experiment group demonstrated more significantly reduced intestinal inflammation and higher expression of immune tolerance related cytokines(IL-10, TGF-β1, IL-17A) and Foxp3 which is specifically expressed in Tregs compared with the model group. Therefore, chymase inhibitor TY-51469 might ameliorate the progression of DSS-induced colitis possibly by increasing the expression of Tregs and cytokines.     

Angiopoietin and vascular endothelial growth factor expression in colorectal disease models

AIM:To investigate angiopoietin(Ang) and vascular endothelial growth factor(VEGF) expression in rats with ulcerative colitis(UC) and colorectal cancer(CRC).METHODS:Dysplasia and cancer were investigatedin rats that received three cycles of 3.5% dextran sulfate sodium(DSS) in drinking water for 7 d followed by distilled water for 14 d after intraperitoneal pretreatment with 20 mg/kg 1,2-dimethylhydrazine(DMH)(CRC group).Colitis was investigated in rats that received three cycles of 3.5% DSS in drinking water for 7 d followed by distilled water for 14 d after intraperitoneal pretreatment with saline(UC group).Rats without DSS or DMH treatment served as controls.Expression of the tyrosine kinase with immunoglobulinlike and EGF-like domains(Tie)-2 and its ligands,Ang-1 and Ang-2,as well as VEGF were evaluated in the colorectum by Western blotting.RESULTS:Compared with rats in the control group,rats in the CRC and UC groups developed the symptoms of acute colitis with diarrhea,rectal bleeding,wasting,and loss of body weight(P < 0.05).In addition,the mean length of colorectum of CRC and UC rats was significantly shorter than that of control rats(8.29 ± 0.21 and 8.31 ± 0.86,respectively,vs 12.34 ± 0.12 cm; P < 0.05).Furthermore,rats in the CRC group,but not in the UC or control groups,developed multiple tumors in the colorectal region.Western blot analysis revealed that rats in the CRC and UC groups had markedly increased protein levels of Ang-1,Ang-2,Tie-2,and VEGF in the colorectum compared to rats in the control group.CONCLUSION:Increased expression of Ang-1,Ang-2,Tie-2,and VEGF in ulcerative colitis-derived colorectal cancer might lead to chronic colitis and pathologic angiogenesis in rats.     

慢性应激对小鼠溃疡性结肠炎的影响

目的观察慢性束缚应激对葡聚糖酸钠(DSS)诱导的小鼠溃疡性结肠炎(UC)的影响。方法64只BALB/C小鼠分为6组,单纯应激组、正常对照组各16只,应激 2DSS组、应激 4DSS组、2DSS组、4DSS组各8只。采用自制的束缚笼对应激组小鼠建立慢性心理应激动物模型。采用小鼠自由饮用DSS溶液的方法建立UC模型。每日观察各组疾病活动指数(DAI)。并在实验结束后测量结肠组织损伤评分(HS)、髓过氧化物酶(MPO)活性。HS评分通过在光镜下观察结肠组织学改变得出。并用分光光度法测定结肠组织中MPO的活性。结果单纯应激组小鼠体重增长较正常对照组缓慢(P<0.05)。4DSS组小鼠DAI评分、HS评分及结肠组织MPO活性均较正常对照组增高(P<0.05)。应激 4DSS组上述指标较正常对照组变化更显著,与4DSS组比较亦增高(P<0.05)。2DSS组上述指标与正常对照组相比无明显区别(P>0.05)。应激 2DSS组上述指标较正常对照组增高(P<0.05)。结论慢性束缚应激可以使小鼠发生UC的易感性增加并加重DSS结肠炎的病理损伤。     

Protective role of metalloproteinase inhibitor (AE-941) on ulcerative colitis in rats

AIM: To evaluate the protective role of AE-941, a matrix metalloproteinase (MMP) inhibitor, on ulcerative colitis (UC) in rats.METHODS: Sprague Dawley (SD) rats were randomly divided into three groups: a control group, an AE-941 treatment group, and an UC model group. Rats were sacrificed on days 7, 21, or 56 following administration of treatment by enema and the disease activity index (DAI), colonic mucosa damage index (CMDI) and colonic expression of MMP-2 and MMP-9 were assessed.RESULTS: DAI and CDMI scores in the UC model group increased significantly compared to the control group at all timepoints (P < 0.001), and also increased significantly at the 21- and 56-d timepoints compared to the AE-941-treated group (DAI: 21- and 56-d = 2.09 ± 0.25, 1.52 ± 0.30 vs 1.55 ± 0.28, 0.59 ± 0.19, respectively, P = 0.040 and 0.007, CMDI: 21- and 56-d = 3.03 ± 0.42, 1.60 ± 0.35 vs 2.08 ± 0.46, 0.86 ± 0.37, respectively, P = 0.040 and 0.005). Furthermore, the colonic expression of MMP-2 and MMP-9 in the UC model group increased significantly compared to the control group (P < 0.001), and also increased compared to the AE-941-treated group on the 21- and 56-d timepoints (MMP-2: 21- and 56-d = 0.6048 ± 0.0522, 0.4163 ± 0.0330 vs 0.3983 ± 0.0218, 0.1093 ± 0.0072, respectively, P = 0.010; MMP-9: 21- and 56-d = 0.6873 ± 0.0472, 0.4328 ± 0.0257 vs 0.5179 ± 0.0305, 0.2673 ± 0.0210, respectively, P = 0.010 and 0.040).CONCLUSION: Expression of MMP-2 and MMP-9 increased significantly in rats with UC. AE-941 can reduce colonic mucosal damage by downregulating the expression of MMP-2 and MMP-9.     

低蛋氨酸饮食对实验性结肠炎大鼠紧密连接蛋白表达和功能的影响

目的 研究低蛋氨酸(methionine restriction,MetR)饮食对葡聚糖硫酸钠(DSS)诱导的大鼠结肠炎的结肠黏膜病理组织学、肠黏膜通透性以及结肠上皮紧密连接蛋白表达的影响,并探讨其可能机制.方法 SD大鼠随机分为常规饮食正常组(AA组)、低蛋氨酸饮食正常组(MetR组)、常规饮食模型组(DSS+ AA组)、低蛋氨酸饮食模型组(DSS+ MetR组),每组15只.DSS建模后第21天腹主动脉采血,分析血常规、肝肾功能和电解质水平,取结肠组织行HE染色分析肠黏膜病理学变化,检测肠组织髓过氧化物酶(MPO)活性,免疫组化染色检测增殖细胞核抗原(PCNA)分析肠上皮细胞的增殖状况,采用尤斯灌流室(Ussing chamber)检测肠黏膜通透性;采用蛋白质印迹法分析肠上皮紧密连接蛋白的表达.结果 结肠炎造模大鼠出现腹泻、便血、体重下降,炎症集中在远端结肠,表现为隐窝脓肿,炎性细胞的浸润.与DSS+ AA组比较,MetR饮食干预可显著降低模型大鼠结肠的组织病理学评分[(10.55 ±3.62)分比(15.00±4.89)分,P=0.003].DSS+ MetR组和DSS+ AA组大鼠血白细胞计数、肠组织MPO活性以及PCNA免疫组化结果的差异均无统计学意义.Ussing chamber检测显示,DSS+ AA组的跨膜电阻抗显著低于AA组[(28.40±6.78)Ω·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],MetR组显著高于AA组[(60.64 ±8.40)Ω·cm2比(46.53 ±4.03)Ω·cm2,P＜0.05],DSS+ MetR组的短路电流值显著高于DSS+ AA组[(35.01 ±2.19) μA/cm2比(29.61±1.19)μA/cm2,P＜0.05].蛋白质印迹结果显示,AA组和MetR组未见claudin2蛋白表达,MetR组的结肠上皮claudin3蛋白表达量明显高于AA组;与DSS+ AA组比较,DSS+ MetR组claudin2的表达量更高,claudin3表达量更高.结论 MetR饮食对DSS诱导的结肠炎模型大鼠有明显的治疗作用,其机制可能不是通过调节炎性细胞浸润以及促进肠细胞生长的途径来缓解炎症损伤,而可能是通过改变紧密连接蛋白结构和功能而改善其肠黏膜屏障的功能,促进受损肠黏膜的修复.     

Beneficial effect of butyrate,Lactobacillus casei and L-carnitine combination in preference to each in experimental colitis

AIM: To investigate the beneficial effect of the combination of butyrate, Lactobacillus casei, and L-carnitine in a rat colitis model.METHODS: Rats were divided into seven groups. Four groups received oral butyrate, L-carnitine, Lactobacillus casei and the combination of three agents for 10 consecutive days. The remaining groups included negative and positive controls and a sham group. Macroscopic, histopathological examinations, and biomarkers such as tumor necrosis factor-alpha (TNF-α) and interlukin-1β (IL-1β), myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), and ferric reduced ability of plasma (FRAP) were determined in the colon.RESULTS: The combination therapy exhibited a significant beneficial effect in alleviation of colitis compared to controls. Overall changes in reduction of TNF-α (114.66 ± 18.26 vs 171.78 ± 9.48 pg/mg protein, P < 0.05), IL-1β (24.9 ± 1.07 vs 33.06 ± 2.16 pg/mg protein, P < 0.05), TBARS (0.2 ± 0.03 vs 0.49 ± 0.04 μg/mg protein, P < 0.01), MPO (15.32 ± 0.4 vs 27.24 ± 3.84 U/mg protein, P < 0.05), and elevation of FRAP (23.46 ± 1.2 vs 15.02 ± 2.37 μmol/L, P < 0.05) support the preference of the combination therapy in comparison to controls. Although the monotherapies were also effective in improvement of colitis markers, the combination therapy was much better in improvement of colon oxidative stress markers including FRAP, TBARS, and MPO.CONCLUSION: The present combination is a suitable mixture in control of experimental colitis and should be trialed in the clinical setting.     

Promising effect of Magliasa,a traditional Iranian formula,on experimental colitis on the basis of biochemical and cellular findings

AIM: To investigate the efficacy of Magliasa, a traditional Iranian formula, on experimental colitis. METHODS: After botanical authentication of herbal ingredients, formulation of Magliasa, quantitative determination of total glucosinolates and total phenolic content, and analysis of the thin layer chromatography profile were performed. Colitis was then induced in male rats by instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in all groups, aside from the Sham group.The experimental groups consisted of: the Sham group that received only normal saline; the Mag-50, Mag-100 and Mag-200 groups, which received 50, 100 and 200 mg/kg per day of Magliasa, respectively; the control group, which received vehicle water orally; the infliximab group, which received infliximab (5 mg/kg per day, subcutaneously); and the Dexa group, which received dexamethasone (1 mg/kg per day, orally). After completing the treatment period (2 wk), the rats were sacrificed, the colon was removed, its macroscopic and microscopic changes were recorded, and tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1b), total antioxidant capacity, myeloperoxidase (MPO), and lipid peroxidation (LPO) were assessed in colon homogenate.RESULTS: The mean value of total glucosinolates in one gram of Magliasa was 19 ± 1 μmol. The mean value of the total phenolic content was 293.8 ± 17.6 mg gallic acid equivalents per 100 gram of Magliasa. Macroscopic scores were significantly decreased in Mag-100 (1.80 ± 0.58, P = 0.019) and Mag-200 (1.20 ± 0.20, P = 0.001) compared to the control group (3.40 ± 0.24), although some inflammation and hyperemia were evident. Treatment of rats by dexamethasone (0.33 ± 0.21, P < 0.001) and infliximab (0.83 ± 0.31, P < 0.001) remarkably attenuated scores where mild hyperemia was observed macroscopically. In comparison to the control group (4.00 ± 0.32), only Mag-200 (1.60 ± 0.40) showed a significant decrease in colonic histopathological scores (P = 0.005). Minimal mucosal inflammation was observed i     

Protective effect of cigarette smoke on the course of dextran sulfate sodium-induced colitis is accompanied by lymphocyte subpopulation changes in the blood and colon

Background

Cigarette smoke (CS) exerts protective effect against ulcerative colitis. The mechanism of this phenomenon remains unknown. One of the possible explanation by which CS exerts its anti-inflammatory action is modulation of immune system. Therefore, the aim of the study was to evaluate the effect of CS on the course of inflammation and subpopulations of lymphocytes in the blood and colon in mice with dextran sulfate sodium (DSS)-induced colitis.

Methods

C57BL6/cmdb mice were exposed to CS for 4 weeks. Colitis was induced with 3.5% DSS given for 10 days. Severity of colitis was determined by disease activity index (DAI), body weight changes, and macro- and microscopic characteristics of inflammation. Peripheral subpopulations of lymphocytes were assessed by flow cytometry (blood) or immunohistochemistry (colonic tissue).

Results

Mice treated with 3.5% DSS developed severe colitis with significantly decreased body weight, increased DAI, and macroscopic and histological features of colonic inflammation. These findings were diminished after concomitant exposure to CS. Mice exposed to DSS alone demonstrated significantly decreased percentage of total CD4⁺ cells (73.1 vs. 52%, p = 0.0007), accompanied by increase of CD8⁺ cells (18.4 vs. 39.5%, p = 0.0001). Concomitant CS exposure reversed inappropriate CD4⁺/CD8⁺ ratio both in the blood and colon and significantly increased B cell presence in the colon.

Conclusions

Our study has demonstrated that CS exposure decreases severity of DSS-induced colitis. This phenomenon was accompanied by changes in CD4/CD8 ratio and B cell level in the peripheral blood and colon. These mechanisms may be responsible for protective effect of smoking in ulcerative colitis.

     

Mitofusin-2 ameliorates high-fat diet-induced insulin resistance in liver of rats

AIM:To investigate the effects of mitofusin-2(MFN2) on insulin sensitivity and its potential targets in the liver of rats fed with a high-fat diet(HFD).METHODS:Rats were fed with a control or HFD for 4 or 8 wk,and were then infected with a control or an MFN2 expressing adenovirus once a week for 3wk starting from the 9th wk.Blood glucose(BG),plasma insulin and insulin sensitivity of rats were determined at end of the 4th and 8th wk,and after treatment with different amounts of MFN2 expressing adenovirus(108,109 or 1010 vp/kg body weight).BG levels were measured by Accu-chek Active Meter.Plasma insulin levels were analyzed by using a Rat insulin enzymelinked immunosorbent assay kit.Insulin resistance was evaluated by measuring the glucose infusion rate(GIR) using a hyperinsulinemic euglycemic clamp technique.The expression or phosphorylation levels of MFN2 and essential molecules in the insulin signaling pathway,such as insulin receptor(INSR),insulin receptor substrate 2(IRS2),phosphoinositide-3-kinase(PI3K),protein kinase beta(AKT2) and glucose transporter type 2(GLUT2) was assayed by quantitative real-time polymerase chain reaction and Western-blotting.RESULTS:After the end of 8wk,the body weight of rats receiving the normal control diet(ND) and the HFD was not significantly different(P>0.05).Compared with the ND group,GIR in the HFD group was significantly decreased(P<0.01),while the levels of BG,triglycerides(TG),total cholesterol(TC) and insulin in the HFD group were significantly higher than those in the ND group(P<0.05).Expression of MFN2 mRNA and protein in liver of rats was significantly downregulated in the HFD group(P<0.01) after 8 wk of HFD feeding.The expression of INSR,IRS2 and GLUT2 were down-regulated markedly(P<0.01).Although there were no changes in PI3K-P85 and AKT2 expression,their phosphorylation levels were decreased significantly(P<0.01).After intervention with MFN2 expressing adenovirus for 3wk,the expression of MFN2 mRNA and protein levels were up-regulated(P<0.01).Th     

Plecanatide and dolcanatide,novel guanylate cyclase-C agonists,ameliorate gastrointestinal inflammation in experimental models of murine colitis

AIM: To evaluate the effect of orally administeredplecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models.METHODS: The cyclic guanosine monophosphate(cG MP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cellbased assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid(5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium(DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic(TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout(TCRα-/-) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity.RESULTS: Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C(GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cG MP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs(0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity(P 0.05) and disease activity index(P 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα-/- mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. CONCLUSION: This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.     

Enterocyte dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin expression in inflammatory bowel disease

AIM: To investigate dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin(DC-SIGN) expression in intestinal epithelial cells(IECs) in inflammatory bowel disease(IBD).METHODS: The expression of DC-SIGN in IECs was examined by immunohistochemistry of intestinal mucosal biopsies from 32 patients with IBD and 10 controls.Disease activity indices and histopathology scores were used to assess the tissue lesions and pathologic damage.Animal studies utilized BALB/c mice with dextran sodium sulfate(DSS)-induced colitis treated with anti-P-selectin lectin-EGF domain monoclonal antibody(PsL-EGFmA b).Controls,untreated and treated mice were sacrificed after 7 d,followed by isolation of colon tissue and IECs.Colonic expression of DC-SIGN,CD80,CD86 and MHC Ⅱ was examined by immunohistochemistry or flow cytometry.The capacity of mouse enterocytes or dendritic cells to activate T cells was determined by coculture with naive CD4+ T cells.Culture supernatant and intracellular levels of interleukin(IL)-4 and interferon(IFN)-γ were measured by enzyme-linked immunosorbent assay and flow cytometry,respectively.The ability of IECs to promote T cell proliferation was detected by flow cytometry staining with carboxyfluorescein diacetate succinimidyl ester.RESULTS: Compared with controls,DC-SIGN expression was significantly increased in IECs from patients with Crohn's disease(P 0.01) or ulcerative colitis(P 0.05).DC-SIGN expression was strongly correlated with disease severity in IBD(r = 0.48; P 0.05).Similarly,in the DSS-induced colitis mouse model,IECs showed upregulated expression of DC-SIGN,CD80,CD86 and MHC,and DC-SIGN expression was positively correlated with disease activity(r = 0.62: P 0.01).IECs from mouse colitis stimulated naive T cells to generate IL-4(P 0.05).Otherwise,dendritic cells promoted a T-helper-1-skewing phenotype by stimulating IFN-γ secretion.However,DC-SIGN expression and T cell differentiation were suppressed following treatment of mice with DSS-induced colitis with Ps L-EGFm Ab.The proliferation cycles of CD4+ T cells from mice with DSS-induced colitis appeared as five cycles,which was more than in the control and treated groups.These results suggest that IECs can promote T cell proliferation.CONCLUSION: IECs regulate tissue-associated immune compartments under the control of DC-SIGN in IBD.     

Geranylgeranylacetone protects mice from dextran sulfate sodium-induced colitis

Objective. Geranylgeranylacetone (GGA) has recently been reported to induce heat shock protein (HSP) 70, which has a protective function against inflammation. We investigated the therapeutic effects of oral administration of GGA on dextran sulfate sodium (DSS)-induced colitis in mice.Material and methods. BALB/c mice were given 3% DSS solution orally for 7?days to induce colitis. The disease activity of colitis was assessed clinically every day, and histology in the colon was evaluated at 7?days post-DSS. The levels of myeloperoxidase (MPO) activity, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the colon tissues were also examined. In addition, expression of HSPs 25, 40, 70 and 90 in the colon tissue was determined by Western blot analysis. Mice were orally administered GGA (50–500?mg/kg) when treatment of DSS started.Results. It was found that GGA significantly reduced the clinical severity of colitis and suppressed the levels of MPO activity, TNF-α and IFN-γ induced by DSS in the colon. On the other hand, GGA enhanced the expression of HSP70 in the colon of mice given DSS. HSP70-positive cells were identified in the epithelial cells of the colon from mice treated with GGA and DSS.Conclusions. Taken together, these results suggest that GGA is a new anti-inflammatory drug that could be useful in the treatment of colitis such as inflammatory bowel disease.