TLRs在溃疡性结肠炎小鼠结肠中的表达研究

目的 观察溃疡性结肠炎（UC）小鼠模型结肠组织中TLR2、TLR4和TLR9的表达情况,为UC发病机制的研究提供新思路.方法 将6～8周龄健康雄性BALB/c小鼠随机分为两组：正常对照组（n=10）和UC模型组（n=10）,正常对照组小鼠蒸馏水自由饮用7d.UC模型组小鼠5％ DSS溶液自由饮用7d造模.7d后处死小鼠,采用实时荧光定量PCR方法检测各组小鼠结肠黏膜组织中TLR2、TLR4和TLR9 mRNA的表达情况.结果 正常对照组结肠组织中无TLR2、TLR4和TLR9 mRNA的表达,而UC组结肠组织中TLR2、TLR4和TLR9 mRNA表达明显,两组比较差异有统计学意义（P＜0.05）.结论 TLR2、TLR4、TLR9可能参与了UC的发病过程.     

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.     

Effects of adacolumn selective leukocytapheresis on plasma cytokines during active disease in patients with active ulcerative colitis

AIM: To investigate the relationship between ulcerative colitis (UC) clinical activity index (CAI) and circulating levels of IL-1ra, IL-10, IL-6 and IL-18. METHODS: Blood levels of IL-lra, IL-10, IL-6 and IL-18 were measured in 31 patients with active UC, the mean CAI was 11.1, ranging from 5-25; and 12 healthy individuals as controls. Patients were given granulocyte and monocyte adsorptive apheresis (GMA) with Adacol-umn. Leucocytes which bear the FcyR and complement receptors were adsorbed to the column leucocytapher-esis carriers. Each patient could receive up to 11 GMA sessions over 8 wk. RESULTS: We found strong correlations between CAI and IL-10 (r = 0.827, P < 0.001), IL-6 (r = 0.785, P < 0.001) and IL-18 (r = 0.791, P < 0.001). IL-lra was not correlated with CAI. Following GMA therapy, 24 of the 31 patients achieved remission and the levels of all 4 cytokines fell to the levels in healthy controls. Further, blood levels of IL-lra and IL-10 increased at the column outflow and inflow at 60 min suggesting release from leucocytes that adhered to the carriers. CONCLUSION: Elevated blood levels of IL-6 and IL-18 together with peripheral blood granulocytes and mono-cytes/macrophages in patients with active UC show acti-vative behaviour and increased survival time can be pro-inflammatory and the targets of GMA therapy.     

Effects of baicalin in CD4 + CD29 + T cell subsets of ulcerative colitis patients

AIM: To evaluate the role of baicalin in ulcerative colitis (UC) with regard to the CD4⁺CD29⁺ T helper cell, its surface markers and serum inflammatory cytokines.METHODS: Flow cytometry was used to detect the percentage of CD4⁺CD29⁺ cells in patients with UC. Real time polymerase chain reaction was used to detect expression of GATA-3, forkhead box P3, T-box expressed in T cells (T-bet), and retinoic acid-related orphan nuclear hormone receptor C (RORC). Western blotting was used to analyze expression of nuclear factor-κB (NF-κB) p65, phosphorylation of NF-κB (p-NF-κB) p65, STAT4, p-STAT4, STAT6 and p-STAT6. The concentrations of interferon-γ (IFN-γ), interleukin (IL)-4, IL-5, IL-6, IL-10 and TGF-β in serum were determined by ELISA assay.RESULTS: The percentages of CD4⁺CD29⁺ T cells were lower in treatment with 40 and 20 μmol/L baicalin than in the treatment of no baicalin. Treatment with 40 or 20 μmol/L baicalin significantly upregulated expression of IL-4, TGF-β1 and IL-10, increased p-STAT6/STAT6 ratio, but downregulated expression of IFN-γ, IL-5, IL-6, RORC, Foxp3 and T-bet, and decreased ratios of T-bet/GATA-3, p-STAT4/STAT4 and p-NF-κB/NF-κB compared to the treatment of no baicalin.CONCLUSION: The results indicate that baicalin regulates immune balance and relieves the ulcerative colitis-induced inflammation reaction by promoting proliferation of CD4⁺CD29⁺ cells and modulating immunosuppressive pathways.     

TLR3与TLR9在溃疡性结肠炎中表达状态及临床意义的研究

目的观察TLR3和TLR9在溃疡性结肠炎(UC)患者病变组织和正常大肠组织中的表达情况,探讨其在UC发病机制中的作用。方法收集UC病例及正常对照结肠镜活检标本各30例。采用免疫组化和实时荧光定量PCR技术,检测UC患者及正常对照组肠黏膜中TLR3、TLR9的表达情况。结果 UC病变组织、正常结肠组织中均有TLR3 mRNA及TLR9 mRNA的表达,但UC组织中TLR9 mRNA表达显著高于正常结肠组织,而TLR3 mRNA与正常结肠组织相比差异无统计学意义。在免疫组化染色图片上,发现TLR3、TLR9主要在细胞的胞浆中表达,在UC病变组织中TLR9阳性表达率明显高于正常结肠组织(P0.05)。结论TLR9在UC患者中表达高度上调,推测它可能参与了UC的发病过程。     

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.     

Therapeutic effect of a hydroxynaphthoquinone fraction on dextran sulfate sodium-induced ulcerative colitis

AIM: To evaluate the therapeutic effect of hydroxynaphthoquinone mixture (HM) on dextran sulfate sodium (DSS)-induced colitis and explore the underlying mechanisms.METHODS: BALB/c mice received 3.5% DSS for 6 d to induce ulcerative colitis. Groups of mice were orally administered HM 3.5, 7 and 14 mg/kg and mesalazine 200 mg/kg per day for 7 d. During the experiment, clinical signs and body weight, stool consistency and visible fecal blood were monitored and recorded daily. A disease activity index score was calculated for each animal. At the conclusion of the experiment, the colonic histopathological lesions were evaluated. Myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) levels were determined. Protein expression levels of TNF-α, nuclear factor-κB (NF-κB) p65, inhibitor of κB (IκB) and phosphorylation of IκB (p-IκB) were analyzed by Western blot analysis.RESULTS: Administration of 3.5% DSS for 6 d successfully induced acute colitis associated with soft stool, diarrhea, rectal bleeding, and colon shortening, as well as a loss of body weight. Administration of HM effectively attenuated the severity of colonic mucosa injury. For histopathological analysis, HM treatment improved histological alterations and lowered pathological scores compared with the DSS only group. This manifested as a reduction in the extent of colon injury and inflammatory cell infiltration, as well as the degree of mucosal destruction. In addition, HM at doses of 7 and 14 mg/kg significantly decreased MPO activity in colonic tissue (0.98 ± 0.22 U/g vs 1.32 ± 0.24 U/g, 0.89 ± 0.37 U/g vs 1.32 ± 0.24 U/g tissue, P < 0.05) and serum TNF-α levels (68.78 ± 7.34 ng/L vs 88.98 ± 17.79 ng/L, 64.13 ± 14.13 ng/L vs 88.98 ± 17.79 ng/L, P < 0.05). Furthermore, HM down-regulated the expression of TNF-α, NF-κB p65 and p-IκBα in colonic tissue while up-regulating IκBα protein expression. These results suggest that the significant anti-inflammatory effect of HM may be attributable to its inhibition of TNF-α production and NF-κB activation.CONCLUSION: HM had a favorable therapeutic effect on DSS-induced ulcerative colitis, supporting its further development and clinical application in inflammatory bowel disease.     

Fecal microbiota in pouchitis and ulcerative colitis

AIM To investigate the changes in microbiota in feces of patients with ulcerative colitis(UC) and pouchitis using genomic technology.METHODS Fecal samples were obtained from UC patients with or without an ileal pouch-anal anastomosis(IPAA) procedure, as well as healthy controls. The touchdown polymerase chain reaction technique was used to amplify the whole V3 region of the 16 S r RNA gene, which was transcribed from DNA extracted from fecal samples. Denaturing gradient gel electrophoresis was used to separate the amplicons. The band profiles and similarity indices were analyzed digitally. The predominant microbiota in different groups was confirmed by sequencing the 16 S rR NA gene. RESULTS Microbial biodiversity in the healthy controls was significantly higher compared with the UC groups(P 0.001) and IPAA groups(P 0.001). Compared with healthy controls, the UC patients in remission and those in the mildly active stage, the predominant species in patients with moderately and severely active UC changed obviously. In addition, the proportion of the dominant microbiota, which was negatively correlated with the disease activity of UC(r =-6.591, P 0.01),was decreased in pouchitis patients. The numbers of two types of bacteria, Faecalibacterium prausnitzii and Eubacterium rectale, were reduced in UC. Patients with pouchitis had an altered microbiota composition compared with UC patients. The microbiota from pouchitis patients was less diverse than that from severely active UC patients. Sequencing results showed that similar microbiota, such as Clostridium perfringens, were shared in both UC and pouchitis.CONCLUSION Less diverse fecal microbiota was present in patients with UC and pouchitis. Increased C. perfringens in feces suggest its role in the exacerbation of UC and pouchitis.     

Anti-inflammatory activity of probiotic Bifidobacterium： Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

AIM： To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk （BFM） which is effective against active ulcerative colitis （UC） and exacerbations of UC, and to explore the immunoregulatory mechanisms. METHODS： Peripheral blood mononuclear cells （PBMNC） from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult （BbrY） or Bifidobacterium bifidum strain Yakult （BbiY） to estimate the amount of IL-10 or IL-8 secreted. RESULTS： Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium （CM） and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-α, whereas no such effect was observed with heat- killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IκB-ζ mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM.CONCLUSION： Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.     

NF-κB在溃疡性结肠炎激素抵抗中的意义

目的:探讨NF-κB与溃疡性结肠炎(UC)发病关系及其在激素抵抗中的意义．方法:选择活动期中度以上UC患者35例,均予强的松治疗(20-40 mg/d)4 wk．根据治疗反应分为激素敏感组和激素耐药组,均作为实验组,检测激素治疗前、治疗后结肠病变黏膜组织中的NF-κB表达水平．设立正常对照组 16例．结果:在激素治疗前,激素敏感组、激素抵抗组NF-κB p65表达水平分别与正常对照组比较,差异有显著性(5．9±2．7,6．1±2．9 vs 1.1± 0．8,P<0．01)．在激素治疗前,激素敏感组和激素抵抗组之间比较,差异无显著性(P>0．05)．在激素治疗后,激素敏感组和激素抵抗组之间比较,差异有显著性(1．3±0．8 vs 5．4± 2．4,P<0．01)．在激素治疗前后,激素敏感组自身比较,差异有显著性(5．9±2．7 vs 1．3±0．8, P<0．01)．而激素抵抗组在前后结果的比较中, 差异无显著性(P>0．05)．结论:NF-κB与UC关系密切,在UC的发病机制中起重要作用．NF-κB在激素治疗前后的变化有助于判断UC患者是否对激素抵抗,可以作为进一步预测激素抵抗的一个较早期的诊断指标．     

Toll-like receptor 9 gene mutations and polymorphisms in Japanese ulcerative colitis patients

Abnormal innate immune responses toward luminal bacteria play an important role in the pathogenesis of inflammatory bowel disease.It has been demonstrated that bacteria having CpG DNA ameliorate experimental colitis in mice,and Toll-like receptor 9 (TLR9) signaling mediates the anti-inflammatory effects in mouse colonic inflammation.A gene variation in NOD2/CARD15 has been reported in Crohn’s disease (CD) patients in Western countries,but this variation has not been identified in Japanese CD patients.Therefore,we hypothesized that TLR9 is a key factor in the development of ulcerative colitis (UC),and we investigated gene mutations and polymorphisms of TLR9 in Japanese UC patients.Three single nucleotide polymorphisms (SNPs) in TLR9 were identified in healthy controls,and were assessed in 48 UC patients and 47 healthy controls.Control subjects were matched for age,sex and date of blood sampling from among a subgroup of participants.We found that TLR9-1486CC,1174GG and 2848AA increase the risk of UC [odds ratio (OR) 2.64,95% confidence interval (95% CI):1.73-6.53,P=0.042],and TLR9-1486TT,1174AA and 2848GG decrease the risk of UC (OR 0.30,95% CI:0.10-0.94,P=0.039),although there were no correlations between SNPs and disease phenotype or TLR9 mRNA expression.These findings suggest that TLR9 polymorphisms are associated with increased susceptibility to UC.     

Clinical efficacy of the Toll-like receptor 9 agonist cobitolimod using patient-reported-outcomes defined clinical endpoints in patients with ulcerative colitis

BackgroundThe Toll-like-receptor 9 (TLR-9) agonist cobitolimod (DIMS0150, Kappaproct^®) is a promising therapeutic option for ulcerative colitis (UC) patients.AimsThe objectives of this post-hoc analysis using the COLLECT study data was to investigate the clinical effects of cobitolimod using patient-reported-outcomes (PRO) defined endpoints.MethodsDual topical administration of cobitolimod was studied in a randomised, multicentre clinical trial named COLLECT in moderate-to-severe UC patients. Symptomatic remission (SR) was studied in 104 patients based on their e-diary records and was defined as absence of blood in stool and a mean daily stool frequency (SF) < 4.ResultsSR was achieved at week 4 in 17.1% of cobitolimod vs. 5.9% of placebo treated patients (p = 0.13), at week 8 in 35.7% vs. 17.6% (p = 0.07), and at week 12 in 38.6% vs. 17.6% (p = 0.04) of the patients, respectively.SR rates with cobitolimod and placebo in anti-TNFα experienced patients were smaller but with a broadly similar relative effect-size to anti-TNFα naïve patients. Clinical efficacy was higher in patients with moderate compared to severe disease.ConclusionsApplication of the Toll-like-receptor 9 (TLR-9) agonist cobitolimod is able to induce remission as assessed by PRO measures in UC patients with moderate-to-severe activity as well as in anti-TNFα experienced and naïve patients supporting the overall efficacy of the substance.     

Toll样受体4的信号转导与溃疡性结肠炎的研究进展

溃疡性结肠炎(ulcerative colitis, UC)的发病机制尚不明确,目前研究表明,Toll样受体4(Toll-like receptor 4,TLR4)与UC的发病密切相关,TLR4通过髓样分化因子88 (myeloid differentiation factor 88, MyD88)依赖性和非依赖性等途径激活下游信号分子,引发肠黏膜释放TNF-α、IL-1、IL-6等炎症介质,最终导致UC的发生,同时对此信号转导通路的阻断研究为临床治疗UC带来了新的契机.     

促肾上腺皮质激素释放因子受体2在溃疡性结肠炎中的表达

目的观察促肾上腺皮质激素释放因子受体2（CRFR2）在溃疡性结肠炎（UC）中的表达情况,及其与细胞间黏附分子-1（ICAM-1）、核因子-KB（NF-JcB）p65和白细胞介素-6（IL-6）的关系,探讨其在UC中的作用。方法免疫组织化学法检测UC活动组30例、缓解组30例和30例正常对照组的结肠黏膜中cRFR2、ICAM-1、NF-KBp65的表达情况,ELISA方法检测各组血清中IL-6水平。结果CRFR2在UC活动组肠黏膜组织中的表达明显高于缓解组和对照组（P〈0．05）,而在UC缓解组和对照组之间的表达差异无统计学意义（P〉0．05）。CRFR2在肠黏膜组织中的表达与NF-xBp65、ICAM-1的表达及血清中IL-6水平具有明显相关性（P〈0．05）。结论活动期UC患者的CRFR2的表达明显升高,提示其在UC发病中可能起促炎作用。     

Clinical importance of serum procalcitonin in ulcerative colitis patients

We have read with interest the recently published article entitled “Clinical significance of serum procalcitonin in patients with ulcerative colitis” by Koido et al. They aimed to investigate the association of procalcitonin with ulcerative colitis (UC) activity. They concluded that elevated procalcitonin levels were significantly correlated with UC activity. We would like to thank the authors for their comprehensive contribution.     

Clinical significance of serum procalcitonin in patients with ulcerative colitis

AIM:To investigate the association of procalcitonin(PCT)with ulcerative colitis(UC)activity.METHODS:Serum PCT levels,C-reactive protein(CRP)levels,the erythrocyte sedimentation rate,and the white blood cell count were analyzed in 18 patients with UC and 11 healthy volunteers.Serum PCT levels were analyzed by an electrochemiluminescence immunoassay.Severity assessments were based on Truelove and Witts’severity index.Correlation of serum PCT and CRP levels with UC activity was examined.Moreover,we assessed serum PCT and CRP levels in patients with a Mayo endoscopic subscore.RESULTS:Serum PCT levels in severe UC patients(n=7)(0.096±0.034 ng/mL)were significantly higher than in mild-to-moderate UC patients(n=11)(0.033±0.012 ng/mL)and healthy volunteers(n=11)(0.035±0.005 ng/mL)(P=0.0005 and P<0.0001,respectively).In addition,there was no difference in serum PCT levels between mild-to-moderate UC patients and healthy volunteers.Interestingly,patients with a Mayo endoscopic subscore of 3 points displayed significantly increased levels of serum PCT(0.075±0.043 ng/mL)compared with patients with a subscore of 2 points(0.03±0.011 ng/mL)(P=0.0302).Moreover,CRP levels in patients with severe UC or a Mayo endoscopic subscore of 3 points were not significantly higher than in patients with mild-to-moderate UC or a Mayo endoscopic subscore of 3 points.CONCLUSION:Serum PCT levels were significantly correlated with UC activity.