Effects of garlicin on apoptosis in rat model of colitis

AIM:To investigate the effects of garlicin on apoptosis and expression of bcl-2 and bax in lymphocytes in rat model of ulcerative colitis (UC). METHODS:Healthy adult Sprague-Dawley rats of both sexes, weighing 180±30 g, were employed in the present study. The rat model of UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) enema. The experimental animals were randomly divided into garlicin treatment group (including high and low concentration), model control group, and normal control group. Rats in garlicin treatment group and model control group received intracolic garlicin daily at doses of 10.0 and 30.0 mg/kg and equal amount of saline respectively 24 h after colitis model was induced by alcohol and TNBS co-enema. Rats in normal control group received neither alcohol nor only TNBS but only saline enema in this study. On the 28~(th) d of the experiment, rats were executed, the expression of bd-2 and bax protein was determined immunohistochemically and the apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. At the same time, the rat colon mucosal damage index (CMDI) was calculated. RESULTS:In garlicin treatment group, the positive expression of bd-2 in lymphocytes decreased and the number of apoptotic cells was more than that in model control group, CMDI was lower than that in model control group. The positive expression of bax in lymphocytes had no significant difference. CONCLUSION:Garlicin can protect colonic mucosa against damage in rat model of UC induced by TNBS enema.     

Effects of Rheum tanguticum polysaccharide on TNBS -induced colitis and CD4^＋T cells in rats

AIM: To study the effects of Rheum tanguticum polysaccharide-1 (RTP-1) on ulcerative colitis in rats induced by 2, 4, 6-trinitrophene sulphonic acid (TNBS) and their possible mechanism.METHODS: RTP1 (200 mg@kg-1, ig) extracted from Rheum tanguticum Maxim. Ex Regel was administrated to rats with colitis induced by TNBS for 5 d, 7 d, 10 d and 14 d,respectively. The effects of RTP1 and dexamethasone (DX,0.2 mg@kg-1, ig) were contrastively investigated. The MPO level and SOD activity were determined by chromatometry.The expansion and protein expression of CD4+T lymphocytes isolated from colon mucosae and mesenteric lymph nodes of colitis rats were performed by immunohistochemical analysis and Western-blot methods.RESULTS: Treatments of RTP1 (200 mg@kg-1, ig) significantly reduced diarrhea, mortality, colon mass, ulcer areas and MPO level in colon mucosae on days 5, 7, 10 and 14 (5.2±1.4,5.4±0.7, 5.2±1.8, P＜0.05.3.4±0.8, P＜0.01. 16.1±12.1,P＜0.01.31.8±8.6, 17.7±5.3, 12.7±4.1, P＜0.05). The effectsof RTP1 were similar to those noted above in DX group, but there were no immunosupressive effects of DX in RTP-1group, such as body mass loss, thymus and spleen atrophy.The decreased number and down-regulated protein levels of CD4+T cells isolated from the colon of colitis rats treated with RTP1 were found.CONCLUSION: RTP1 shows significantly protective effects but lower side effects on rats with colitis induced by TNBS.The mechanism may be due to the resistance to over expansion of CD4.     

Establishment of a trinitrobenzene sulfonic acid‐induced rat colitis model

OBJECTIVE : To set up a trinitrobenzene sulfonic acid (TNBS)‐induced colitis model in the rat and to assess its value in research studies of inflammatory bowel disease (IBD). METHODS : A 0.85‐mL enema containing 30 mg TNBS dissolved in 50% ethanol was instilled into the rat colon to induce distal colitis. Control rats were instilled with 50% ethanol or TNBS/saline alone. Macroscopic histological changes of the colon and myeloperoxidase (MPO) activity of the mucosa were evaluated. RESULTS : One week after the TNBS/ethanol enema, hyperemia, edema and ulceration of the colonic mucosa appeared with predominant infiltration of polymorphonuclear leukocytes in the distal colon. Four weeks after TNBS/ethanol enema, ulcers had healed macroscopically and lymphocytes and plasma cells became predominant. The tissue MPO activity increased on the first day after the TNBS/ethanol enema and this increase in MPO activity lasted for 3 weeks, but declined 4 weeks after the TNBS/ethanol enema. In contrast, macroscopic, histological changes and MPO activity returned to normal levels in control rats within the first week. CONCLUSIONS : Trinitrobenzene sulfonic acid/ ethanol‐induced rat distal colitis is an ideal model of IBD and may serve as a tool for the investigation of the pathogenesis of and effects of pharmaceuticals on IBD.     

Protective effect of angelica sinensis polysaccharide on experimental immunological colon injury in rats

AIM: To study the effect of angelica sinensis polysaccharide (ASP) on immunological colon injury and its mechanisms in rats.METHODS: Immunological colitis model of rats was induced by intracolon enema with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) and ethanol. The experimental animals were randomly divided into normal control, model control, 5-aminosalicylic acid therapy groups and three doses of ASP therapy groups. The 6 groups were treated intracolonically with normal saline, normal saline, 5-aminosalicylic acid (100 mg.kg-1), and ASP daily (8:00 am) at the doses of 200, 400 and 800 mg.kg-1 respectively for 21 days 7 d following induction of colitis. The rat colon mucosa damage index (CMDI), the histopathological score (HS), the score of occult blood test (OBT), and the colonic MPO activity were evaluated. The levels of SOD, MDA, NO, TNF-α, IL-2 and IL-10 in colonic tissues were detected biochemically and immunoradiometrically. The expressions of TGF-β and EGF in colonic tissues were also determined immunochemically.RESULTS: Enhanced colonic mucosal injury, inflammatory response and oxidative stress were observed in colitis rats,which manifested as significant increases of CMDI, HS, OBT,MPO activity, MDA and NO contents, as well as the levels of TNF-α and IL-2 in colonic tissues, although colonic TGF-β protein expression, SOD activity and TL-10 content were significantly decreased compared with the normal control (P＜0.01). However, these parameters were found to be significantly ameliorated in colitis rats treated intracolicly with ASP at the doses of 400 and 800 mg@kg-1 (P＜0.05-0.01).Meantime, colonic EGF protein expression in colitis rats was remarkably up-regulated.CONCLUSION: ASP has a protective effect on immunological colon injury induced by TNBS and ethanol enema in rats,which was propably due to the mechanism of antioxidation,immunomodulation and promotion of wound repair.     

生长抑素对溃疡性结肠炎模型大鼠肠道炎性损伤的治疗作用

目的: 观察生长抑素(奥曲肽)对溃疡性结肠炎(ulcerative colitis,UC)大鼠模型的作用,初步探讨其可能机制.方法: ♂SD大鼠随机分为正常对照组、奥曲肽对照组、模型组、治疗组,每组7只. 模型组、治疗组大鼠用三硝基苯磺酸(TNBS)/乙醇溶液灌肠复制UC模型. 观察各组实验大鼠体质量变化、大体及组织病理学改变. 采用酶联免疫吸附法检测细胞因子(IL-6、IL-10、TNF-α)的含量、蛋白质印迹杂交法检测结肠组织NF-κB p65蛋白的表达.结果: 生长抑素可以缓解大鼠体质量的减轻,减少腹泻及便血的发生,并且能够显著改善结肠组织大体和组织学评分. 与正常组比较,模型组大鼠结肠黏膜IL-6、TNF-α表达明显升高(188.27±11.65 ng/L vs 102.13±7.12 ng/L,87.39±6.74 ng/L vs 121.51±8.56 ng/L,均P<0.01);IL-10表达明显下降(71.40±8.28 ng/L vs 202.97±12.26 ng/L,P<0.01);与模型组比较,治疗组大鼠结肠黏膜IL-6、TNF-α表达均明显降低(142.03±12.68 ng/L,90.87±9.26ng/L,均P<0.01),IL-10表达明显升高(124.07±10.05 ng/L,P<0.01). 模型组结肠组织中NF-κB的蛋白含量明显高于治疗组(1059.60±96.35vs 471.23±11.61,P<0.01).结论: 生长抑素对TNBS诱导的大鼠溃疡性结肠炎具有显著治疗作用,其作用机制可能是通过影响炎症反应的信号通路NF-κB的活化,进而下调促炎细胞因子及上调抗炎细胞因子的产生和表达.     

三黄汤灌肠对溃疡性结肠炎大鼠治疗作用研究

[目的]探讨三黄汤灌肠对三硝基苯磺酸(TNBS)诱导溃疡性结肠炎(UC)模型大鼠的疗效。[方法]采用TNBS诱导制备UC大鼠模型45只,随机分为模型组、阳性对照组及中药治疗组,各15只,并选未造模大鼠15只作为正常对照组。各组均灌肠给药,模型组和正常对照组予0.9%氯化钠注射液,阳性对照组予柳氮磺吡啶0.5 g/kg,中药治疗组予三黄汤浓煎剂60 g/kg,各组均按10 ml/kg每日灌肠1次。给药时间从造模后第3天开始,连续7 d。第11天观察各组大鼠结肠黏膜损伤指数(CMDI)及黏膜病理组织学情况。[结果]中药治疗组结肠黏膜病理损伤明显改善,CMDI为(1.5±0.535)分,与模型组的(3.55±1.214)分比较差异有统计学意义(P0.05);与阳性对照组的(2.63±1.188)分比较差异有统计学意义(P0.05)。在肉眼及电镜下观察发现,中药治疗组大鼠结肠黏膜较模型组、阳性对照组明显改善。[结论]三黄汤灌肠对TNBS诱导的大鼠UC疗效确切,能显著改善结肠黏膜的充血水肿、溃疡、糜烂等。     

Effectiveness of a hydroxynaphthoquinone fraction from Arnebia euchroma in rats with experimental colitis

AIM:To evaluate the potential effectiveness of hydroxynaphthoquinone mixture(HM)in rats with 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:Colitis was induced by intracolonic administration of TNBS(80 mg/kg,dissolved in 50%ethanol).Rats were treated daily for 7 d with HM(2.5,5,10 mg/kg)and mesalazine 100 mg/kg 24 h after TNBS instillation.Disease progression was monitored daily by observation of clinical signs and body weight change.At the end of the experiment,macroscopic and histopathologic lesions of rats were scored,and myeloperoxidase(MPO)activity was determined.We also determined inflammatory cytokine tumor necrosis factor(TNF)-αlevel by ELISA,Western blotting and immunochemistry to explore the potential mechanisms of HM.RESULTS:After intracolonic instillation of TNBS,animals developed colitis associated with soft stool,diarrhea and marked colonic destruction.Administration of HM significantly attenuated clinical and histopathologic severity of TNBS-induced colitis in a dose-dependent manner.It abrogated body weight loss,diarrhea and inflammation,decreased macroscopic damage score,and improved histological signs,with a significant reduction of inflammatory infiltration,ulcer size and the severity of goblet cell depletion(all P<0.05 vs TNBS alone group).HM could reduce MPO activity.In addition,it also decreased serum TNF-αlevel and down-regulated TNF-αexpression in colonic tissue.This reduction was statistically significant when the dose of HM was 10 mg/kg(P<0.05 vs TNBS alone group),and the effect was comparable to that of mesalazine and showed no apparent adverse effect.The underlying mechanism may be associated with TNF-αinhibition.CONCLUSION:These findings suggest that HM possesses favourable therapeutic action in TNBS-induced colitis,which provides direct pharmacological evidence for its clinical application.     

Role of nitric oxide in the impairment of circular muscle contractility of distended, uninflamed mid-colon in TNBS-induced acute distal colitis in rats

AIM: To evaluate the role of nitric oxide (NO) in the motor disorders of the dilated uninflamed mid-colon (DUMC) from trinitrobenzene sulfonic acid (TNBS)-induced acute distal colitis in rats. METHODS: Colitis was induced in male Sprague-Dawley rats by a single intracolonic administration of TNBS. Control rats received an enema of 0.9% saline. The rats were killed 48 h after TNBS or saline administration. Macroscopic and histologic lesions of the colon were evaluated. Myeloperoxidase (MPO) and nitric oxide synthase (NOS) activity were measured on the colonic tissue. In TNBS rats, we evaluated spontaneous and evoked contractile activity in circular muscle strips derived from DUMC in comparison to the same colonic segment of control rats, both in the presence and in the absence of a non-selective NOS isoforms inhibitor N-nitro-L-arginine (L-NNA). Pharmacological characterization of electric field stimulation (EFS)-evoked contractile responses was also performed. RESULTS: In TNBS rats, the distal colon showed severe histological lesions and a high MPO activity, while the DUMC exhibited normal histology and MPO activity. Constitutive NOS activity was similar in TNBS and control rats, whereas inducible NOS activity was significantly increased only in the injured distal colon of TNBS rats. Isometrically recorded mechanical activity of circular muscle strips from DUMC of TNBS rats showed a marked reduction of the force and frequency of spontaneous contractions compared to controls, as well as of the contractile responses to a contracting stimulus. In the presence of L-NNA, the contractile activity and responses displayed a significantly greater enhancement compared to controls. The pharmacological characterization of EFS contractile responses showed that a cooperative-like interaction between cholinergic muscarinic and tachykinergic neurokinin 1 and 2 receptors mediated transmission in DUMC of TNBS rats vs a simple additive interaction in controls. CONCLUSION: The results of this study show that, during TNBS-induced acute distal colitis, circular muscle intrinsic contractile mechanisms and possible enteric neural excitatory activity are inhibited in the distended uninfiamed mid-colon. Suppression of NO synthesis markedly improves spontaneous and evokes muscle contractions, in spite of any evident change in local NO activity.     

Inflammatory bowel disease in rats： Bacterial and chemical interaction

AIM：To develop a novel model of colitis in rats, using a combination of iodoacetamide and enteropathogenic E. coli（EPEC）, and to elucidate the pathophysiologic processes implicated in the development of ulcerative colitis （UC）.
METHODS： Hale Sprague-Dawley rats （/7 = 158） were inoculated intrarectally on a weekly basis with 4 different combinations： （a） 1% methylcellulose （HC）, （b） 100 μL of 6% iodoacetamide （IA） in 1% HC, （c） 200 p.L containing 4×10^8 colony factor units （CFU） of EPEC, and （d） combined treatment of （IA） followed by bacteria （13） after 2 d. Thirty days post treatment, each of the four groups was divided into two subgroups; the inoculation was stopped for one subgroup and the other subgroup continued with biweekly inoculation until the end of the experiment. Colitis was evaluated by the clinical course of the disease, the macroscopic and microscopic alterations, activity of myeloperoxidase （HPO）, and by TNF-α gene expression. RESULTS： Findings indicative of UC were seen in the combined treatment （IA ＋ B） as well as the IA continued treatment groups： the animals showed slow rate of increase in body weight, diarrhea, bloody stools, high colonic ulcer score, as well as histological alterations characteristic of UC, with an extensive inflammatory reaction. During the course of the experiment, the MPO activity was consistently elevated and the TNF-α gene expression was upregulated compared to the control animals.
CONCLUSION： The experimental ulcerative colitis model used in the present study resembles, to a great extent, the human disease. It is reproducible with characteristics indicative of chronicity.     

Protective Effect of Lactulose on Dextran Sulfate Sodium-Induced Colonic Inflammation in Rats

Promising results have recently been obtained with pre- and probiotic therapy in ulcerative colitis (UC). The prebiotic potential of lactulose is well established, but it has not yet been investigated in experimental colitis models. The purpose of the study was to examine the effect of lactulose on an UC model induced by 3% dextran sulfate sodium (DSS) solution added to drinking water for 7 days in male Wistar rats. Lactulose (300-1000 mg/kg) or 5-aminosalicylic acid (5-ASA; 150 mg/kg) was administered orally twice daily for 6 days. Colonic ulceration area, colon length, body weight changes, diarrhea/bloody feces, colonic mucosal myeloperoxidase activity (MPO), thiobarbituric acid reactive substances (TBARS), and histology were examined. Treatment of animals with DSS for 7 days resulted in severe colonic lesions accompanied by diarrhea, bloody feces, a decrese in body weight, shortening of the colon length, and an increase in MPO activity as well as TBARS, compared to normal rats. Lactulose treatment ameliorated DSS-induced colitis in a dose-dependent manner, and at 1000 mg/kg all of the parameters examined, except TBARS, were shown to improve significantly as compared to controls. Daily administration of 5-ASA also significantly reduced the severity of colonic lesions following DSS treatment. These results demonstrated the protective effect of lactulose in this rat colitis model and suggested that the background of this lactulose effect may be due to alterations of colonic microflora.     

复方黄柏液对大鼠TNBS结肠炎的治疗机制探讨

背景：临床上采用复方黄柏液保留灌肠辅助治疗溃疡性结肠炎（UC）疗效满意,但其作用机制尚不清楚。目的：探讨复方黄柏液对三硝基苯磺酸（TNBS）诱发的大鼠结肠炎模型炎症损伤的治疗作用及其可能机制。方法：40只成年雌性Sprague-Dawley大鼠随机分成四组,正常对照组不予处理,其余三组以TNBS／乙醇溶液灌肠制作结肠炎模型后．分别予0．9％NaCl溶液1ml、5-氨基水杨酸（5-ASA）200mg／kg和复方黄柏液1ml灌肠,连续14d。治疗后评估大鼠疾病活动指数（DAI）以及结肠大体、组织学损伤情况;检测结肠组织髓过氧化物酶（MPO）活性和白三烯B4（LTB4）含量;心脏采血,流式细胞术检测中性粒细胞凋亡率。结果：与正常对照组相比,TNBS模型组DAI、结肠大体和组织学评分、结肠组织MPO活性和LTB。含量均显著升高,血中性粒细胞凋亡率显著降低（P〈0．01）;5-ASA治疗组和复方黄柏液治疗组上述指标均较TNBS模型组显著改善（P〈0．01）,两组间差异则无统计学意义。结论：复方黄柏液灌肠对大鼠TNBS结肠炎具有明显治疗作用,促进中性粒细胞凋亡、清除结肠局部损伤因子（MPO、LTB。）可能为其作用机制之一。     

Effects of melatonin on the expression of iNOS and COX－2 in rat models of colitis

AIM: To investigate the effects of melatonin (MT) on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in rat models of colitis. METHODS: Healthy adult Sprague-Dawlay (SD) rats of both sexes, weighing 280+/-30 g, were employed in the present study. The rat models of colitis were induced by either acetic acid or 2,4,6-trinitrobenzene sulfonic acid (TNBS) enemas. The experimental animals were randomly divided into melatonin treatment and model control group that were intracolicly treated daily with melatonin at doses of 2.5, 5.0, 10.0 mg/kg(-1) and equal amount of saline respectively from 24 h following induction of colitis in rats inflicted with acetic acid enema and the seventh day in rats with TNBS to the end of study. A normal control group of rats treated with neither acetic acid nor TNBS but saline enema was also included in the study. On the 28(th) day of the experiment, the rat colon mucosal damage index (CDMI) was calculated, and the colonic prostaglandin E(2) (PGE(2)), nitric oxide (NO), as well as the iNOS and COX-2 expression were also determined biochemically or immunohistochemically. RESULTS: CDMI increased to 2.87+/-0.64 and 3.12+/-1.12 respectively in rats treated with acetic acid and TNBS enema, which was in accordance with the significantly elevated colonic NO and PGE(2) contents, as well as the up-regulated colonic iNOS and COX-2 expression in both of the two rat models of colitis. With treatment by melatonin at the doses of 5.0 and 10.0 mg/kg(-1), CDMI in both models of rat colitis was significantly decreased (P<0.05-0.01), which accorded synchronously and unanimously with the reduced colonic NO and PGE(2) content, as well as the down-regulated expression of colonic iNOS and COX-2. CONCLUSION: Melatonin has a protective effect on colonic injury induced by both acetic acid and TNBS enemas, which is probably via a mechanism of local inhibition of iNOS and COX-2 expression in colonic mucosa.     

一氧化氮合酶抑制剂对实验性大鼠结肠炎疗效的研究

背景：炎症性肠病（IBD）中，诱生型一氧化氮合酶（iNOS）合成的高浓度一氧化氮（NO）与肠道炎症和疾病活动度相关。因此抑制iNOS为肠道炎症提供了新的治疗选择。目的：评估NOS抑制剂治疗进展期结肠炎的疗效。方法：予雌性Sprague．Dawlev大鼠含30mg三硝基苯磺酸（TNBS）的50％乙醇溶液0．85ml灌肠，诱导实验性结肠炎。7天后，将大鼠随机分成4组，每组7只，每天分别腹腔注射0．9％NaCl溶液1ml（造模组）、0．2mg／kg地塞米松（DX）、5mg／kg氨基胍（AG）和35mg／kgN-硝基-L-精氨酸甲酯（L-NAME）。另取4只正常大鼠作为正常对照组。通过肠道重量指数、溃疡面积、大体形态和组织学评分以及肠组织髓过氧化物酶（MPO）、丙二醛（MDA）、NOS和iNOS活性、血浆CD62P活性、血清NO含量测定评估疗效。结果：与造模组相比，DX组和AG组肠道重量指数、溃疡面积、大体形态和组织学评分以及肠组织MPO、MDA、NOS和iNOS活性、血浆CD62P活性、血清NO含量均显著降低，L-NAME组肠组织NOS和iNOS活性以及血清NO含量显著降低，但大体形态和组织学评分无改善。结论：选择性iNOS抑制剂AG能通过其抗炎作用减轻进展期肠道炎症，提示AG可作为IBD新的治疗选择，其疗效与DX相似。     

The Effect of Melatonin on TNBS-Induced Colitis

Ulcerative colitis is a multifactorial inflammatory disease of the colon and rectum with an unknown etiology. The present study was undertaken to investigate the effect of melatonin administration on oxidative damage and apoptosis in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Rats were divided into four groups as follows: Group 1 (n=8)—TNBS colitis; Group 2 (n=8)—melatonin, 10 mg/kg/day ip, for 15 days in addition to TNBS; Group 3 (n=8)—melatonin alone, 10 mg/kg/day ip, for 15 days; and Group 4 (n=8)—isotonic saline solution, 1ml/rat ip, for 15 days (sham control group). Colonic myeloperoxidase (MPO) activities, malondialdehyde (MDA) levels, and glutathione (GSH) levels are indicators of oxidative damage, while caspase-3 activities reveal the degree of apoptosis of the colonic tissue. In all TNBS-treated rats, colonic MPO activity and MDA levels were found to be increased significantly compared to those in the sham group. Colonic MPO activity and MDA levels were significantly lower in the melatonin treatment group compared to TNBS-treated rats. GSH levels of colonic tissues were found to be significantly lower in TNBS-treated rats compared to the sham group. Treatment with melatonin significantly increased GSH levels compared to those in TNBS-treated rats. Caspas-3 activity of colonic tissues was found to be significantly higher in TNBS-treated rats compared to the sham group. Treatment with melatonin significantly decreased caspase-3 activity compared to that in TNBS-treated rats. These results imply a reduction in mucosal damage due to anti-inflammatory and anti-apoptotic effects of melatonin.     

Juvenile ferric iron prevents microbiota dysbiosis and colitis in adult rodents

AIM: To assess whether juvenile chronic ferric iron ingestion limit colitis and dysbiosis at adulthood in rats and mice.METHODS: Two sets of experiments were designed. In the first set, recently weaned mice were either orally administered ferrous (Fe²⁺) iron salt or ferric (Fe³⁺) microencapsulated iron for 6 wk. The last week of experiments trinitrobenzene sulfonic acid (TNBS) colitis was induced. In the second set, juvenile rats received the microencapsulated ferric iron for 6 wk and were also submitted to TNBS colitis during the last week of experiments. In both sets of experiments, animals were sacrificed 7 d after TNBS instillation. Severity of the inflammation was assessed by scoring macroscopic lesions and quantifying colonic myeloperoxidase (MPO) activity. Alteration of the microflora profile was estimated using quantitative polymerase chain reaction (qPCR) by measuring the evolution of total caecal microflora, Bacteroidetes, Firmicutes and enterobacteria.RESULTS: Neither ferrous nor ferric iron daily exposures at the juvenile period result in any effect in control animals at adulthood although ferrous iron repeated administration in infancy limited weight gain. Ferrous iron was unable to limit the experimental colitis (1.71 ± 0.27 MPO U/mg protein vs 2.47 ± 0.22 MPO U/mg protein in colitic mice). In contrast, ferric iron significantly prevented the increase of MPO activity (1.64 ± 0.14 MPO U/mg protein) in TNBS-induced colitis. Moreover, this positive effect was observed at both the doses of ferric iron used (75 and 150 mg/kg per day po - 6 wk). In the study we also compared, in both rats and mice, the consequences of chronic repeated low level exposure to ferric iron (75 mg/kg per day po - 6 wk) on TNBS-induced colitis and its related dysbiosis. We confirmed that ferric iron limited the TNBS-induced increase of MPO activity in both the rodent species. Furthermore, we assessed the ferric iron incidence on TNBS-induced intestinal microbiota dysbiosis. At first, we needed to optimize the isolation and quantify DNA copy numbers using standard curves to perform by qPCR this interspecies comparison. Using this approach, we determined that total microflora was similar in control rats and mice and was mainly composed of Firmicutes and Bacteroidetes at a ratio of 10/1. Ferric juvenile administration did not modify the microflora profile in control animals. Total microflora numbers remained unchanged whichever experimental conditions studied. Following TNBS-induced colitis, the Firmicutes/Bacteroidetes ratio was altered resulting in a decrease of the Firmicutes numbers and an increase of the Bacteroidetes numbers typical of a gut inflammatory reaction. In parallel, the subdominant population, the enterobacteria was also increased. However, ferric iron supplementation for the juvenile period prevented the increase of Bacteroidetes and of enterobacteria numbers consecutive to the colitis in both the studied species at adulthood.CONCLUSION: Rats and mice juvenile chronic ferric iron ingestion prevents colitis and dysbiosis at adulthood as assessed by the first interspecies comparison.     

中药清肠栓对溃疡性结肠炎大鼠结肠黏膜紧密连接蛋白ZO-1、occludin的影响

目的:探讨复方中药清肠栓对三硝基苯磺酸(TNBS)诱导的UC大鼠结肠黏膜上皮紧密连接相关蛋白-l(ZO-1)、闭锁蛋白(occludin)的修复作用.方法:清洁级♂SD大鼠36只,随机分为正常组、模型组、SASP组、清肠栓高、中、低剂量组,每组6只.选用TNBS诱导的UC大鼠模型.采用免疫荧光的方法观察各组大鼠结肠黏膜...     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats.METHODS: Thirty-two healthy adult Sprague-Dawley (SD)rats were randomly divided into four groups of eight each:normal, NS, model I, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks,the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-κB p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-αand ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κBp65 and other parameters were analyzed.RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04,respectively), which were in accordance with the significantly elevated MPO activity (1.69±0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control (9.7±1.96 vs1.7±0.15, 84.09±14.52 vs16.03±6.21,77.69±8.09 vs13.41±4.91 P＜0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities,macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P＜0.05).CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-~B p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α (and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25&#177;1.39 and 6.24&#177;1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control(9.7&#177;1.96 vs 1.7&#177;0.15, 84.09&#177;14.52 vs 16.03&#177;6.21,77.69&#177;8.09 vs 13.41&#177;4.91 P&lt;0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P&lt;0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Acutely administered melatonin is beneficial while chronic melatonin treatment aggravates the evolution of TNBS-induced colitis

The aim of this study was to evaluate the effects of melatonin on the inflammatory response and hydroxyproline production in an experimental acute and chronic model of trinitrobenzene sulfonic (TNBS) acid-induced colitis in Wistar rats. In the acute model, melatonin (0.5, 1, and 2 mg/kg, i.p.) was applied 48, 24, and 1 hr prior to the induction of colitis and 24 and 48 hr after; the severity of colitis was less evident in melatonin-treated animals with significant response in the group treated with 2 mg/kg. All doses investigated significantly reduced the myeloperoxidase activity (MPO). In the chronic studies, melatonin (1 and 2 mg/kg, i.p.) was administered daily 24 hr before hapten instillation and for 7 or 21 days after TNBS; melatonin (2 mg/kg) worsened colitis evolution in the 21-day study with a significant increase in MPO activity and tumor necrosis factor-alpha production with respect to TNBS group. Histological slides were in concordance with macroscopic data where areas of extensive necrosis and edema, fibrosis, and absence of regenerated epithelium were observed. Moreover, the hydroxyproline determination, used as indicator of collagen production and fibrosis, also showed a marker increase. The results obtained in this experimental model showed that short-term administration is protective while in the long term it negatively influences evolution of inflammatory colitis; therefore, the immunostimulatory effect of melatonin in some situations when given chronically, such as during inflammatory bowel disease, might lead to negative consequences.