Constitutive androstane receptor and pregnane X receptor cooperatively ameliorate DSS-induced colitis

Background

Nuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated.

Prebiotic and Synbiotic Fructooligosaccharide Administration Fails to Reduce the Severity of Experimental Colitis in Rats

Opposing effects of the prebiotic, fructooligosaccharide, have been reported in experimental colitis. We compared the effects of the prebiotic, fructooligosaccharide, alone and in synbiotic combination with Lactobacillus fermentum BR11, on the development of dextran sulfate sodium-induced colitis in rats. Rats consumed an 18 percent casein-based diet or diet supplemented with 6 percent fructooligosaccharide or maltodextrin for 14 days. The synbiotic group was gavaged 1 ml of L. fermentum BR11 (1 × 10⁹ cfu/ml) twice daily. From Days 7 to 14, colitis was induced via 3 percent dextran sulfate sodium in drinking water. Disease activity was assessed daily, and at killing, gastrointestinal organs were measured, weighed, and examined by quantitative histology, proliferating cell nuclear antigen immunohistochemistry, and colonic myeloperoxidase activity. Administration of dextran sulfate sodium resulted in an increased colitic disease activity, and an increased colon and cecum weight compared with normal controls. Colon and cecum weights were further increased in dextran sulfate sodium+fructooligosaccharide (colon: 19 percent; cecum: 48 percent) and dextran sulfate sodium+fructooligosaccharide/L. fermentum BR11-treated rats (16 and 62 percent) compared with dextran sulfate sodium+vehicle-treatment. Dextran sulfate sodium+fructooligosaccharide-treated rats displayed an 81 percent increase in colonic myeloperoxidase activity compared with dextran sulfate sodium-treated controls. Histologic damage severity scores increased in dextran sulfate sodium+vehicle, dextran sulfate sodium+fructooligosaccharide, and dextran sulfate sodium+fructooligosaccharide/L. fermentum BR11-treated rats compared with normal controls (P < 0.05). Crypt depth increased in all treatments compared with normal controls (P < 0.01). No protection from dextran sulfate sodium-colitis was accorded by fructooligosaccharide alone or in synbiotic combination with L. fermentum BR11, whereas fructooligosaccharide actually increased some indicators of colonic injury.     

小鼠实验性结肠炎中骨桥蛋白的变化

目的 观察骨桥蛋白(osteopontin,OPN)在右旋葡聚糖硫酸钠(dextran sodium sulfate,DSS)诱导的小鼠结肠炎中的变化,探讨OPN在炎症性肠病(inflammatory bowel disease,IBD)发病中的作用.方法 32只雄性BALB/C小鼠随机分为对照组(予蒸馏水饮用23 d)、模型组(5%DSS饮用9 d后2.5%DSS维持2周)、柳氮磺胺吡啶(SASP)治疗组(在模型组基础上从第10天起予SASP 600 mg/kg灌胃2周)和英夫利昔治疗组(在模型组基础上在第10天予尾静脉注射英夫利昔100 mg/kg 1次).酶联免疫法检测小鼠血浆OPN浓度,逆转录聚合酶链反应和Western印迹法检测小鼠结肠组织中OPN的mRNA水平和蛋白质水平表达,免疫组化法检测OPN在结肠组织中的定位表达.结果 各组小鼠的血浆OPN浓度分别为(5.26±1.93)、(10.21±2.37)、(4.58±1.83)和(4.82±1.83)ng/ml;结肠组织中OPN mRNA表达量分别为(0.36±0.16)、(0.71±0.17)、(0.32±0.07)和(0.42±0.22);结肠组织中OPN的蛋白表达量分别为(0.44±0.10)、(0.85±0.04)、(0.61±0.11)和(0.58±0.17);结肠黏膜固有层OPN阳性细胞数分别为(46.6±10.9)、(155.5±43.8)、(73.1±6.8)和(70.6±8.3).与对照组相比,模型组血浆和结肠组织中OPN的表达明显增高(P均<0.05),SASP治疗组和英夫利昔治疗组OPN的表达均较SASP治疗组下降(P均<0.05),SASP治疗组和英夫利昔治疗组OPN的表达差异无统计学意义.结论 OPN在DSS诱导的小鼠结肠炎中表达增加,经药物治疗后表达降低.OPN促进了DSS诱导的小鼠结肠炎的发生.     

T辅助细胞在右旋葡聚糖硫酸钠诱导的小鼠结肠炎中的作用研究

目的　探讨Th1/Th2细胞在右旋葡聚糖硫酸钠 (DSS)诱导小鼠结肠炎中的作用。方法　利用细胞内细胞因子流式细胞检测法 ,测定DSS诱导急、慢性结肠炎小鼠肠黏膜和脾脏单核细胞 (SMC)中Th1/Th2比例。结果　①结肠炎急性期 ,小鼠肠黏膜固有层单核细胞 (LPMC)中Th1细胞为 (8.90± 1.2 3) % ,较对照组升高 (P <0 .0 5 ) ;Th2细胞占 (3.2 5± 1.2 5 ) % ,与对照组差异无显著性 (P >0 .0 5 ) ;Th1/Th2比例为 3.0 9± 1.18,较对照组升高 (P <0 .0 5 )。②在慢性期 ,肠黏膜LPMC中Th1、Th2细胞分别占 (5 .5 2± 1.2 8) %和 (10 .0 8± 1.75 ) % ,均较对照组升高 (P <0 .0 5 ) ;Th1/Th2比例为 0 .5 2± 0 .2 1,较对照组降低 (P <0 .0 5 )。脾脏SMC中Th1、Th2百分比、Th1/Th2比例 ,各期无明显差异。结论　DSS结肠炎是一种以肠黏膜免疫功能紊乱为主的炎症性病变。Th1优势反应可能与急性期损伤有关 ,而Th2优势反应可能在炎症的慢性化过程中发挥重要作用。     

Cytomegalovirus in pediatric inflammatory bowel disease patients with acute severe colitis

BackgroundThe prevalence and significance of cytomegalovirus (CMV) colitis in pediatric acute severe colitis is unknown. The aim of this study was to determine the prevalence of CMV in colonic mucosa of children with acute severe refractory colitis and compare the clinical characteristics and outcomes of CMV positive and negative patients.MethodsIn a case-control study, colonic biopsy specimens from children with severe refractory colitis were tested for CMV, and matched with non-refractory IBD controls. We characterized CMV positive patients by assessing laboratory values, concurrent medications, and need for surgery as compared with CMV negative refractory colitis patients.ResultsColonic biopsies from 96 patients were evaluated for CMV; 48 with severe refractory colitis, and 48 non-refractory controls. There was an increased prevalence of CMV in severe refractory colitis [7/48 (14.6%), P < 0.0001]; all were previously CMV negative. Viral DNA burden on immunohistochemistry was not predictive of response to antiviral therapy or need for surgery at 12 months. Lymphopenia was seen in all CMV positive patients, but this did not demonstrate statistical significance (P = 0.09). We did not see an association between azathioprine or infliximab use and the need for surgery at 12 months.ConclusionsThere is an increased prevalence of CMV in colonic biopsies of pediatric patients with severe refractory colitis. Viral burden does not predict clinical outcomes or subsequent need for colectomy.     

葡聚糖硫酸钠结肠炎模型影响因素的研究进展

自1985年首次报道采用葡聚糖硫酸钠(dextran sulphate sodium,DSS)制备出仓鼠溃疡性结肠炎模型以来,已有大量研究证明DSS结肠炎模型与人类溃疡性结肠炎相似.该模型的病因、临床症状、病理改变及治疗应答均与人类UC相类似;对于研究UC病因、发病机制及UC相关增生和肿瘤,确定治疗手段有重要意义.虽然DSS模型制作简单;但该过程受到多个因素的影响:DSS浓度、给药时间、DSS相对分子质量和动物种属等.如不能正确处理这些因素,很难制造出成功的DSS结肠炎模型.本文主要针对DSS造模影响因素及尚需我们进一步研究和探讨的问题作综述如下.     

Bosentan, an endothelin receptor antagonist, reduces leucocyte adhesion and inflammation in a murine model of inflammatory bowel disease

Background and aims Endothelins, a group of polyfunctional cytokines, induce the adhesion of circulating leucocytes to venous endothelium, an initial step in the pathogenesis of a cellular infiltrate in inflammatory bowel disease (IBD). The effect of bosentan, a non-selective endothelin receptor antagonist, on leucocyte adhesion and inflammation in a murine model of IBD was studied.Materials and Methods Thirty BALB/c mice were divided into three groups of 10 animals: untreated controls, chronic colitis [dextran sodium sulphate (DSS), 3% w/v for 30 days], and treatment with bosentan (30 mg/kg i.p. daily on days 26–30). On day 30, adherent and rolling leucocytes and the average rolling velocity were assessed by intravital microscopy. Clinical and histological activity of inflammation were assessed by the disease activity index and modified Dieleman score, respectively.Statistics Kruskal–Wallis test was used, followed by Dunn's method. A value of p<0.05 was considered significant.Results Compared to healthy controls, mice treated with DSS showed pronounced clinical and histological inflammation, and a higher number of rolling and adhering leucocytes in colonic submucosal venules. Therapy with bosentan significantly reduced clinical and histological inflammation. Adherent leucocyte levels were markedly lower (1.2±0.3 vs 23.7±2.8 adherent cells per 0.01 mm², p<0.05). The number of rolling leucocytes was lower but not significantly different. However, rolling velocity was significantly higher (91.5±14.0 vs 19.0±1.6 μm/s, p<0.05).Conclusions Bosentan reduces the adhesion of leucocytes in colonic submucosal venules and reduces inflammation in this mouse model of IBD. By inhibiting leucocyte adhesion, a crucial step in the recruitment of leucocytes to the inflamed tissue, bosentan is a potent therapeutic drug in this animal model. Further studies are necessary to investigate the role of bosentan as a novel drug in human IBD.C. Anthoni and R.B. Mennigen contributed equally to this work     

The ICE Inhibitor Pralnacasan Prevents DSS-Induced Colitis in C57BL/6 Mice and Suppresses IP-10 mRNA but Not TNF-α mRNA Expression

Previously we demonstrated an ameliorating effect of the interleukin-1beta converting enzyme (ICE) inhibitor pralnacasan on dextran sulfate sodium (DSS)-induced colitis. This study investigates the effects of pralnacasan on cytokine expression in DSS-induced colitis. Colitis was induced by oral administration of DSS. Mice were treated intraperitoneally with the ICE inhibitor pralnacasan (50 mg/kg body weight twice daily). Body weight as well as the presence of occult blood or diarrhea was monitored daily. Subgroups were sacrificed at days 4, 8, and 11 after the beginning of DSS application. Cytokine profiles in colonic tissue were analyzed on the protein level by ELISA and on the mRNA level by real time RT-PCR. Administration of DSS led to an increase in IL-18, IL-12, TNF-alpha, and IFN-gamma protein as well as IP-10 and TNF-alpha mRNA. The increase in IL-18 and IFN-gamma was reduced by ICE inhibition. Pralnacasan prevented DSS-induced colitis in C57BL/6 mice. In C57BL/6 mice, the DSS-induced increase in IP-10 mRNA, but not TNF-alpha mRNA, was completely prevented by ICE inhibition. In conclusion, prevention of colitis in C57BL/6 mice was associated with a suppresion of IP-10 mRNA, but not TNF-alpha mRNA expression, indicating that IL-18-mediated cytokine production is a key element in the pathogenesis of DSS-induced colitis.     

Involvement of lymphocytes in dextran sulfate sodium-induced experimental colitis

AIM: To investigate the roles of lymphocytes in the development of dextran sulfate sodium-induced colitis. METHODS: Using various doses of dextran sulfate sodium (DSS), we induced colitis in wild-type B6 control and Rag-1 knockout (H-2b haplotype) mice, and evaluated the colitis in terms of symptomatic and histologic parameters, such as weight loss, survival, severity of diarrhea, shortage of colon length and histological changes. Symptomatic parameters were checked daily and histological changes were scored. RESULTS: Although development of colitis in Rag-1 knockout mice treated with high dose (5%) of DSS was comparable to that in B6 control mice, colitis progression was much more tolerable in Rag-1 knockout mice compared to than in B6 mice treated with low dose (1.5%) DSS. Symptomatic parameters as well as histopathologic changes were improved in Rag-1 knockout mice. CONCLUSION: These results indicate that the presence of lymphocytes contributes to colitis progression at low dose of DSS stimulation. Lymphocytes may play roles as an aggravating factor in DSS-induced colitis.     

Longitudinal analysis of inflammation and microbiota dynamics in a model of mild chronic dextran sulfate sodium-induced colitis in mice

AIM:To characterize longitudinally the inflammation and the gut microbiota dynamics in a mouse model of dextran sulfate sodium(DSS)-induced colitis.METHODS:In animal models,the most common method used to trigger colitis is based on the oral administration of the sulfated polysaccharides DSS.The murine DSS colitis model has been widely adopted to induce severe acute,chronic or semi-chronic colitis,and has been validated as an important model for the translation of mice data to human inflammatory bowel disease(IBD).However,it is now clear that models characterized by mild intestinal damage are more accurate for studying the effects of therapeutic agents.For this reason,we have developed a murine model of mild colitis to study longitudinally the inflammation and microbiota dynamics during the intestinal repair processes,and to obtain data suitable to support the recovery of gut microbiota-host homeostasis.RESULTS:All plasma cytokines evaluated,except IL-17,began to increase(P<0.05),after 7 d of DSS administration.IL-17 only began to increase 4 d after DSS withdrawal.IL-1βand IL-17 continue to increase during the recovery phase,even when clinical signs of colitis had disappeared.IL-6,IL-10 and IFN-γreached their maxima 4 d after DSS withdrawal and decreased during the late recovery phase.TNFαreached a peak(a three-fold increase,P<0.05),after which it slightly decreased,only to increase again close to the end of the recovery phase.DSS administration induced profound and rapid changes in the mice gut microbiota.After 3 d of DSS administration,we observed a major reduction in Bacteroidetes/Prevotella and a corresponding increase in Bacillaceae,with respect to control mice.In particular,Bacteroidetes/Prevotella decreased from a relative abundance of 59.42%-33.05%,while Bacillaceae showed a concomitant increase from 2.77%to 10.52%.Gut microbiota rapidly shifted toward a healthy profile during the recovery phase and returned normal 4 d after DSS withdrawal.Cyclooxygenase 2 expression started to increase 4 d after DSS withdrawal(P<0.05),when dysbiosis had recovered,and continued to increase during the recovery phase.Taken together,these data indicated that a chronic phase of intestinal inflammation,characterized by the absence of dysbiosis,could be obtained in mice using a single DSS cycle.CONCLUSION:Dysbiosis contributes to the local and systemic inflammation that occurs in the DSS model of colitis;however,chronic bowel inflammation is maintained even after recovery from dysbiosis.     

Cellular Localization,Binding Sites,and Pharmacologic Effects of TFF3 in Experimental Colitis in Mice

Trefoil factors (TFFs) are essential for protection and restitution of the gastrointestinal mucosa but many aspects of TFF biology are unclear. Our aim was to compare the localization of endogenous TFFs and binding sites for injected TFF3 in the colon of healthy and colitic mice and to study the effect of TFF3 on dextrane sulfate sodium (DSS)-induced colitis in mice. Expression of endogenous TFF1-3 was examined by in situ hybridization and immunohistochemistry, and the distribution of intravenously, intraperitoneally, and subcutaneously administered ¹²⁵I-TFF3 by autoradiography and gamma-counting. The effect of systemically administered TFF3 on DSS-induced colitis was assessed. We found increased expression of endogenous TFF3 and increased binding of injected ¹²⁵I-TFF3 in the colon of animals with DSS-induced colitis. The distribution of intraperitoneally and subcutaneously administered ¹²⁵I-TFF3 was comparable. Systemic administration of the peptides reduced the severity of colitis. Expression of endogenous TFF3 and binding of systemically administered TFF3 are increased in DSS-induced colitis. Systemic administration of TFF3 attenuates the disease. These findings suggest a role of TFF3 in mucosal protection.     

Intravenous vs intraperitoneal mesenchymal stem cells administration: What is the best route for treating experimental colitis?

AIM: To investigate the therapeutic effects of mesenchymal stem cells (MSCs) transplanted intraperitoneally and intravenously in a murine model of colitis.METHODS: MSCs were isolated from C57BL/6 mouse adipose tissue. MSC cultures were analyzed according to morphology, cellular differentiation potential, and surface molecular markers. Experimental acute colitis was induced in C57BL/6 mice by oral administration of 2% dextran sulfate sodium (DSS) in drinking water ad libitum from days 0 to 7. Colitis mice were treated with 1 × 10⁶ MSCs via intraperitoneal or intravenous injection on days 2 and 5. The disease activity index was determined daily based on the following parameters: weight loss, stool consistency and presence of blood in the feces and anus. To compare morphological and functional differences in tissue regeneration between different MSC injection modalities, mice were euthanized on day 8, and their colons were examined for length, weight, and histopathological changes. Inflammatory responses were determined by measuring the levels of different serum cytokines using a CBA Th1/Th2/Th17 kit. Apoptotic rates were evaluated by terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling assay.RESULTS: Intravenous infusion of MSCs was more effective than intraperitoneal treatment (P < 0.001) in reducing the clinical and histopathologic severity of colitis, which includes weight loss, diarrhea and inflammation. An histological evaluation demonstrated decreased colonic inflammation based on reduced crypt loss and reduced infiltration of inflammatory cells. This therapeutic effect was most likely mediated by the down-regulation of pro-inflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)]; and by the up-regulation of anti-inflammatory cytokines (IL-10 and IL-4). Intravenous transplantation also induced high levels of IFN that lead to activation of the immunosuppressive activity of the MSCs, which did not occur with intraperitoneal transplantation (P = 0.006). An increase in apoptotic T cells was observed after intravenous, but not intraperitoneal, MSC infusion, suggesting that MSCs can induce apoptosis in resistant T cells in colonic inflammation (P = 0.027).CONCLUSION: Our results demonstrate that intravenous treatment is a superior method for reducing colon inflammation compared with intraperitoneal therapy.     

Colitis-associated colon cancer: Is it in your genes?

Colitis-associated colorectal cancer(CA-CRC) is the cause of death in 10%-15% of inflammatory bowel disease(IBD) patients. CA-CRC results from the accumulation of mutations in intestinal epithelial cells and progresses through a well-characterized inflammation to dysplasia to carcinoma sequence. Quantitative estimates of overall CA-CRC risks are highly variable ranging from 2% to 40% depending on IBD severity, duration and location, with IBD duration being the most significant risk factor associated with CA-CRC development. Recently, studies have identified IBD patients with similar patterns of colonic inflammation, but that differ with respect to CA-CRC development, suggesting a role for additional non-inflammatory risk factors in CA-CRC development. One suggestion is that select IBD patients carry polymorphisms in various low penetrance disease susceptibility genes, which predispose them to CA-CRC development, although these loci have proven difficult to identify in human genomewide association studies. Mouse models of CA-CRC have provided a viable alternative for the discovery, validation and study of individual genes in CA-CRC pathology. In this review, we summarize the current CA-CRC literature with a strong focus on genetic predisposition and highlight an emerging role for mouse models in the search for CA-CRC risk alleles.     

老年发病炎症性肠病患者的临床特点和治疗方案选择

目的 了解老年发病炎症性肠病(IBD)患者的临床特点和治疗方案的选择。 方法 以1995年1月至2014年12月在北京大学第一医院住院治疗IBD患者396例为研究对象,收集患者的随访记录。根据发病年龄分为老年组(≥60岁)和对照组(<60岁),回顾性比较分析两组患者的一般情况、疾病发作情况及选择治疗方案的异同。 结果 老年组IBD患者37例,占9.3%;其中溃疡性结肠炎(UC)25例,克罗恩病(CD)12例。老年组和对照组比较,男女比、吸烟、IBD家族史和结肠癌家族史的比例分别为1∶0.9和1∶1.3、32.4%和21.7%、0.0%和6.1%、0.0%和1.9%,差异均无统计学意义( P值分别为0.597、0.139、0.247、0.840)。两组患者在症状频发或处于症状持续者和重症患者的比例差异无统计学意义。老年组与对照组炎症受累范围比较,UC广泛型(E3)分别为16.0%(4/25)和41.2%(117/289)( χ ²＝6.123, P＝0.013),CD结肠型(L2)分别为58.3%(7/12)和21.3%(16/75)( χ ²＝6.447, P＝0.011);差异均有统计学意义。两组患者治疗方案选择比较,5-氨基水杨酸(5-ASA)/柳氮磺胺吡啶(SASP)维持治疗1年以上者,老年组为22例(86.5%),对照组为274例(96.9%)( χ ²＝6.382, P＝0.011);诱导治疗阶段全身使用激素,老年组5例(19.4%),对照组122例(43.2%)( χ ²＝7.617, P＝0.006);免疫抑制剂、生物制剂和手术治疗两组比较,差异无统计学意义。 结论 老年发病的IBD患者并非少见,其炎症受累范围比青中年发病者表现为更局限。老年发病IBD患者的治疗方案选择更加谨慎,尤其表现为5-ASA/SASP长期维持治疗和激素的全身使用。     

益生菌对炎症性肠病诱导缓解、维持治疗和贮袋炎作用系统评价

目的 评价益生菌对炎症性肠病缓解、维持治疗和贮袋炎的作用。方法 检索MEDLINE，EMBASE，the Cochrane Con—trolled Trials Register，OVID，BIOSIS和中国生物科技数据库，两位作者独立选取和炎症性肠病缓解率、复发率以及副作用相关的，对比益生菌治疗和非益生菌治疗的随机对照试验，使用Rev Man4．2．10软件统计分析，同时做亚组分析和敏感性分析。结果21项随机对照试验中共有1515例患者符合入选标准：4项研究评估了缓解率，14项研究评估了复发率，3项研究同时评估了缓解率和复发率。通过荟萃分析，炎症性肠病的总体缓解率相对危险度（relativerisk，RR）为1．05（95％CI=0．84—1．31），克罗恩病的缓解率RR0．85（95％CI=0．64—1．13），溃疡性结肠炎的缓解率RR1．18（95％CI=0．87—1．58）；贮袋炎临床复发率RR0．24（95％CI=0．12—0．48），克罗恩病临床复发率RR1．11（95％CI=0．69—1．80）；内镜复发率的RR1．08（95％CI=0．67—1．74）；益生菌与安慰剂比较，炎症性肠病的复发率RR0．51（95％CI=0．29—0．92）；益生菌与5-氨基水杨酸比较，溃疡性结肠炎的复发率RR0．96（95％CI=0．76—1．19）。结论 溃疡性结肠炎患者使用益生菌作为缓解治疗具有和5-氨基水杨酸相同的效果并优于安慰剂，但在炎症性肠病的诱导缓解中无额外益处。     

肝素治疗右旋葡聚糖硫酸钠诱导小鼠结肠炎的疗效观察

目的　评价肝素预防及治疗右旋葡聚糖硫酸钠 (DSS)诱导小鼠结肠炎的有效性。方法　32只小鼠中 ,16只正常小鼠分成两组 ,饮用DSS 7d的同时预防组皮下注射肝素 ,对照组皮下注射生理盐水 ;另 16只饮用DSS 7d后诱导结肠炎的小鼠分成两组 ,治疗组皮下注射肝素 ,对照组皮下注射生理盐水。用疾病活动指数、组织学评分、TNF α的表达和马休斯猩红兰 (MSB)检测微血栓评价疗效。结果　肝素在预防组降低微血栓的形成 ,对照组 8只中 4只微血栓阳性 ,预防组均阴性 (P =0 .0 38)。治疗组组织学评分、TNF α的表达明显降低 ,治疗组与对照组的直肠、横结肠组织学评分和TNF α的表达分别为 1.33和 1.85 (P <0 .0 5 ) ,0 .92和 1.6 8(P <0 .0 5 ) ,(5 .5± 3.5 ) %和 (10 .8± 4.2 ) % (P <0 .0 5 )。结论　肝素可抑制DSS诱导结肠炎小鼠血栓形成和结肠炎症 ,实验结果提示肝素用于溃疡性结肠炎治疗也可能有效