摘要
炎症性肠病(inflammatory bowel disease，IBD)的病因和发病机制未明，严重危害健康，但缺乏特效治疗方法，因此IBD的机制和治疗的研究成为目前医学研究的热点。研究显示肠道菌丛在IBD的发病中起关键作用，使用益生菌治疗IBD有一定的疗效，但其机制尚不清楚。2，4，6-三硝基苯磺酸(2，4，6-trinitrobenzene sulfonic acid，TNBS)引起的结肠炎为乙醇造成结肠粘膜损伤后暴露的结肠蛋白与TNBS结合后形成自身抗原，导致免疫异常引起炎症，其发病机制和临床病理改变与人类IBD相似，其作为IBD的动物模型被国内外广泛用于IBD的实验研究。本课题通过建立TNBS诱导的大鼠IBD模型，观察和探讨大鼠肠道部分菌群、内毒素水平、结肠TLR2、TLR4和NF-κBp65的表达及肠系膜淋巴结树突状细胞表型和功能的变化及其意义，并分析和探讨双歧三联活菌的作用。
第一部分实验性结肠炎大鼠结肠菌群和血浆内毒素水平的变化及双歧三联活菌的作用
目的
建立TNBS诱导的大鼠结肠炎模型，观察和分析其结肠炎症、菌群和血浆内毒素水平的变化及双歧三联活菌的作用，并与美沙拉嗪的作用进行比较。
方法
雄性Wistar大鼠40只，随机分为正常对照组(NC组)、阳性对照组(UC组)、双歧三联活菌治疗组(Bifico，BC组)和美沙拉嗪对照组(MC组)，每组10只，采用TNBS(100mg／kg)乙醇溶液一次性灌肠法建立大鼠结肠炎模型；双歧三联活菌(2.2×10~9CFU／只)和美沙拉嗪(200mg／只)治疗4周，邻甲联苯胺法检测大便隐血情况。实验结束时处死大鼠留取结肠及心脏采血分离血浆；HE染色观察各组大鼠结肠炎症情况，并盲法行炎症评分，对结肠内的部分细菌进行培养；鲎试验测定大鼠血浆内毒素水平，分析其变化，并观察该益生菌对大鼠结肠炎症、细菌和血浆内毒素水平的影响。
结果
1．一般情况
TNBS灌肠后大鼠活动减少，懒动；食欲和食量下降、厌食，毛发无光泽，并出现腹泻、粘液血便或隐血实验阳性、体重下降等；经Bifico和美沙拉嗪治疗4周后，活动及体重增加，腹泻及便血等症状好转；于第2周时，UC组、BC组和MC组分别死亡2只、1只和1只大鼠，这4只大鼠明显消瘦，腹部膨隆，解剖发现结肠局部增厚、水肿，与周围组织粘连，近端结肠和小肠明显扩张，肠壁变薄。NC组大鼠饮食和活动无明显异常，体重较其他三组增加。
2．结肠粘膜损伤情况
肉眼观察：UC组大鼠结肠与周围组织粘连明显，肠壁水肿、增厚，部分可见粘膜下出血；而BC组和MC组大鼠结肠与周围组织粘连较轻，水肿及肠壁厚度轻于UC组；BC组较MC组重；NC组大鼠结肠肉眼观察无明显异常。光镜下，UC组粘膜结构损伤明显，表现为广泛粘膜糜烂，隐窝炎及脓肿，腺管排列紊乱，固有层充血水肿明显，大量炎性细胞浸润，部分标本见大片组织坏死：BC组和MC组有明显的炎性细胞浸润，部分见隐窝炎，但腺管排列整齐，无明显脓肿及大片的组织坏死，BC组炎症浸润较MC组明显；NC组大鼠结肠有轻至中度炎性细胞浸润，但腺管排列整齐，无组织坏死及隐窝炎。
3．炎症评分
四组炎症评分分别为NC组：4.35±0.88，UC组：10.25±1.36，BC组：7.94±0.85和MC组：6.78±0.71。BC组和MC组大鼠结肠炎症评分明显低于UC组(P＜0.05和P＜0.01)，但高于NC组(P＜0.01)；BC组炎症评分高于MC组(P＜0.05)。
4．细菌培养结果
(1)BC组结肠细菌中乳酸杆菌和双歧杆菌数量明显高于UC组(P＜0.05)且接近NC组(P＞0.05)；肠杆菌和真菌数量明显低于UC组(P＜0.05)，但肠杆菌数量高于NC组(P＜0.01)，真菌数量与NC组相比无明显差异(P＞0.05)。(2)MC组肠杆菌数量明显低于UC组(P＜0.05)，乳酸杆菌和双歧杆菌计数虽然高于UC组，但差异无统计学意义(P＞0.05)；其肠杆菌、双歧杆菌和乳酸杆菌较BC组低(P＜0.05)，真菌高于BC组(P＜0.05) MC组肠杆菌与NC组比较无明显差异(P＞0.05)，真菌高于NC组(P＜0.01)，乳酸杆菌和双歧杆菌低于NC组(P＜0.01)。(3)UC组大鼠结肠中肠杆菌和真菌数量明显高于NC组(P＜0.01)，乳酸杆菌和双歧杆菌数量明显低于NC组(P＜0.01)。(4)四组大鼠粪便培养未见葡萄球菌和产气荚膜杆菌。
5．内毒素水平比较
BC组内毒素水平明显低于UC组(P＜0.05)，但高于NC组和MC组(P＜0.01和P＜0.05)；MC组内毒素水平明显低于UC组(P＜0.01)，但高于NC组(P＜0.01)；UC组内毒素水平明显高于NC组(P＜0.01)。
结论
TNBS诱导的结肠炎大鼠，其结肠粘膜炎症明显，存在结肠菌群紊乱，双歧杆菌和乳酸杆菌显著减少，同时伴血浆内毒素水平明显增高。双歧三联活菌可减轻大鼠的结肠炎症，改善菌群紊乱，降低内毒素水平，但其减轻炎症的效果不如美沙拉嗪。
第二部分实验性结肠炎大鼠肠系膜淋巴结中树突状细胞表型和功能的变化及双歧三联活菌的作用
目的
观察和分析结肠炎大鼠肠系膜淋巴结中树突状细胞(Dendritic Cells，DCs)表型及刺激同种异体T淋巴细胞增殖能力的变化；并探讨双歧三联活菌对结肠炎大鼠肠道DCs成熟与功能的影响。
方法
实验动物、分组、模型的建立及给药见第一部分(无MC组)，动物处死时取出全部肠系膜淋巴结。免疫组化染色法检测肠系膜淋巴结DCs表面特征性标志0X-62和成熟标志CD83的表达；密度梯度离心法和淘洗法分离纯化DCs；流式细胞仪分析DCs表面分子MHC-Ⅱ及共刺激分子CD86的表达；同种混合淋巴细胞反应(MLR)检测DCs刺激同种异体T淋巴细胞增殖的能力。
结果
1．大鼠肠系膜淋巴结中0X-62及CD83的表达分别为：(1)0X-62：Nc组：198.6±87.7，UC组：1422.1±598.2，BC组：805.7±298.8。(2)CD83：NC组：87.6±24.4，UC组：709.2±199.6，BC组：461.7±172.3。BC组大鼠二者的表达明显低于UC组(P＜0.05)，但高于NC组(P＜0.01)，UC组明显高于NC组(P＜0.01)。
2．大鼠肠系膜淋巴结DCs上MHC-Ⅱ和CD86的表达率分别为：(1)MHC-Ⅱ：NC组：(26.23±7.19)％，UC组：(84.55±9.38)％，BC组：(66.49±8.40)％。(2)CD86：NC组：(20.46±7.69)％，UC组：(80.63±8.84)％，BC组：(59.79±10.03)％。BC组二者的表达明显低于UC组，(P＜0.05)，但高于NC组(P＜0.01)，UC组明显高于NC组(P＜0.01)。
3．MLR试验显示BC组大鼠DCs刺激同种异体大鼠T淋巴细胞增殖的能力明显低于UC组，但强于NC组；UC组大鼠DCs刺激同种异体大鼠T淋巴细胞增殖的能力明显强于NC组大鼠。
结论
结肠炎大鼠肠系膜淋巴结中成熟的DCs明显增多，表面分子MHC-Ⅱ和共刺激分子CD86表达明显上调，刺激T淋巴细胞增殖的能力增强，提示该结肠炎大鼠存在DCs活化增多，功能增强。双歧三联活菌可明显减少其肠系膜淋巴结中成熟的DCs，使DCs表面的共刺激分子表达下调，提示该益生菌可减少肠道DCs成熟与活化，降低其刺激T淋巴细胞增殖的能力，间接减少肠系膜淋巴结T淋巴细胞的活化，从而减轻炎症。
第三部分实验性结肠炎大鼠结肠TLR2、TLR4和NF-κBp65的表达及双歧三联活菌的作用
目的
观察和分析Toll样受体2(Toll-like receptor 2，TLR2)、Toll样受体4Toll-like receptor 4，TLR4)及核因子κBp65(Nuclear factor-kappap65，NF-κBp65)蛋白及mRNA在结肠炎大鼠结肠中的表达，并观察双歧三联活菌干预后的变化，探讨双歧三联活菌对该信号通路的影响。
方法
实验动物、分组、模型的建立及给药同第一部分(无MC组)，动物处死时留取全部结肠(包括盲肠)。免疫印记法(Western Blotting)和实时荧光定量PCR分别检测大鼠结肠TLR2、TLR4、NF-κBp65蛋白及mRNA的表达。
结果
1．大鼠结肠TLR2、TLR4、NF-κBp65蛋白表达分别为：(1)NC组：TLR2：10.26±4.24，TLR4：8.16±4.50，NF-κBp65：3.16±1.52。(2)UC组：TLR2：47.20±4.62，TLR4：25.84±4.46，NF-κBp65：42.96±10.32。(3)BC组：TLR2：36.64±3.67，TLR4：19.71±3.28，NF-κBp65：30.74±7.71。分析显示三者在BC组大鼠结肠的表达明显低于UC组(P＜0.05)，但高于NC组(P＜0.01)，UC组明显高于NC组(P＜0.01)。
2．TLR4、TLR2、NF-κBp65mRNA的表达(拷贝)结果为：(1)TLR2 mRNA在NC组、UC组和BC组的表达分别为(0.24±0.14)×10~3、(10.57±2.69)×10~3和(6.64±1.42)×10~3。BC组明显低于UC组(P＜0.05)，高于NC组(P＜0.01)，UC组明显高于NC组(P＜0.01)。(2)TLR4mRNA在NC组、UC组和BC组的表达分别为(0.21±0.11)×10~3，(8.58±2.71)×10~3和(5.29±1.54)×10~3；BC组的表达明显低于UC组(P＜0.05)，高于NC组(P＜0.02)，UC组高于NC组(P＜0.02)。(3)NF-κBp65mRNA在NC组、UC组和BC组的表达分别为(0.17±0.07)×10~3、(9.69±2.71)×10~3和(6.21±1.33)×10~3。BC组的表达明显低于UC组(P＜0.05)，但高于NC组(P＜0.01)，UC组的表达明显高于NC组(P＜0.01)。
结论
TNBS诱导的结肠炎大鼠结肠上模式识别受体TLR2、TLR4和转录因子NF-κBp65表达明显增高，表明与抗原接触机会增多和核转录增加；双歧三联活菌可明显降低上述模式识别受体和NF-κBp65的表达，提示该益生菌可降低肠腔抗原的摄取，减少抗原的递呈及核转录产物的生成。
第四部分实验性结肠炎大鼠粘膜与系统水平细胞因子的变化及双歧三联活菌的作用
目的
观察和分析结肠炎大鼠粘膜和系统水平细胞因子的情况及双歧三联活菌对细胞因子产生的影响。
方法
实验动物、分组、模型的建立及给药见第一部分(无MC组)。实验结束时无菌取脾及小肠派伊尔氏淋巴滤泡(Peyers’patches，PP)，分离脾细胞和PP免疫细胞，与大肠杆菌标准株共同培养72h后，酶免疫分析法(EIA)检测上清液中TNF-α、IFN-γ、IL-12、IL-10和TGF-β水平。
结果
1．大鼠脾细胞培养上清液细胞因子的变化
BC组TNF-α和IFN-γ水平明显低于UC组(P＜0.05和P＜0.02)，但高于NC组(P＜0.01和P＜0.03)，其IL-10水平高于UC组(P＜0.05)，低于NC组(P＜0.02)。UC组TNF—α和IFN-γ水平明显高于NC组(P＜0.01)，而IL-10明显低于NC组(P＜0.01)。IL—12水平在三组大鼠间无明显差异(P＞0.05)；各组TGF-β保持在相似的水平，虽有差异，但均无统计学意义(P＞0.05)。
2．大鼠PP细胞培养上清液细胞因子的变化
BC组TNF—α、IFN-γ和IL-12水平明显低于UC组(P＜0.05)，高于NC组(P＜0.01)，其IL-10水平高于UC组(P＜0.05)但低于NC组(P＜0.02)。UC组TNF—α、IFN-γ和IL-12水平明显高于NC组(P＜0.01)，IL-10明显低于NC组(P＜0.01)。三组大鼠的TGF-β水平相似，各组间差异小，无统计学意义(P＞0.05)。
结论
TNBS诱导的结肠炎大鼠PP细胞和脾细胞对革兰氏阴性大肠杆菌标准株的反应增强，促炎症性细胞因子TNF—α、IFN-γ和IL-12产生增加，抗炎症性细胞因子IL-10产生减少，粘膜水平和系统水平均存在促炎症性细胞因子和抗炎症性细胞因子间的平衡失调。双歧三联活菌可降低粘膜水平和系统水平的促炎症性细胞因子的产生，增加抗炎症性细胞因子的产生，减轻两类细胞因子间的失平衡。
PARTⅠ
Changes of gut flora and plasma endotoxin levels in ratswith TNBS-induced colitis and effects of Bifico
Objective
To investigate the changes of gut flora and plasma endotoxin in ratswith TNBS-induced colits, and to evaluate the effects of Bifico on gutinflammation, flora and plasma endotoxin levels of experimental colitisrats.
Methods
Forty Wistar rats were randomly divided into normal control (NC) group,positive control (UC) group, Bifico-treated (BC) group and mesalazine-treated (MC) group, 10 rats in each group. The rat model of experimentalcolitis (IBD model) was induced by 2, 4, 6-trinitrobenzene sulfonic acid(TNBS)/ethanol enema(100mg/kg). Rats in BC group were fed by gavage withBifico[live probiotics of combined bifidobacterium(Bif),lactobacillus(Lac) and enterococcusl by 2.2×10~9 colony-formingunit(CFU)/rat and rats in MC group were fed by gavage withmesalazine(200mg/rat), once a day for 4 weeks. Inflammatory score wasmeasured, plasma endotoxin levels were tested, and enteric microorganismsin cecum were recruited with standard methods.
Results
1. Inflammatory scores were 4.35±0.88 in NC group, 10.25±1.36 in UCgroup, 7.94±0.85 in BC group and 6.78±0.71 in MC group. Inflammatoryscores in UC group increased significantly compared with those in NC, MCand BC groups (P＜0.01 and P＜0.05). Inflammatory scores in BC and MC groupswere higher than those in NC group(P＜0.01). Inflammatory scores in BCgroup were higher than those of MC group (P＜0.05).
2. There was a significant alteration in the enteric microbial flora:Bif and Lac were decreased significantly(P＜0.01 and P＜0.05) but Bacillus coil and Mycetes were increased in UC group than those in NC andBC groups(P＜0.01 and P＜0.05). Bifico supplement ameliorated thisimbalance. Though the counts of Bacillus coli in BC group were higher thanthose in NC group(P＜0.01), there was no significance of the differencein Bif and Lac counts between Be and NC groups (P＞0.05). Bacillus colicounts in Me groupwere lower than those in UC group (P＜0.05), but therewere no significance in the difference of other three kinds of flora countsbetween in MC group and in UC group (P＞0.05). The counts of Bacillus coli,Bif and Lac in MC group were lower than those in Be group (P＜0.05). Staphylococci and Clostridium porfringens were not detected in fourgroups.
3. Plasma endotoxin levels were (35.20±15.12)pg/ml in NCgroup, (121.25±39.07)pg/ml in UC group, (93.33±21.28) pg/ml in BC groupand (67.55±19.30) pg/ml in Me group. Plasma endotoxin levels in UC groupincreased significantly compared with those in NC, Me and Be groups(P＜0.01 and P＜0.05), which in BC group were higher than those in NC andMC groups (P＜0.01 and P＜0.05). Plasma endotoxin levels in MC group werehigher than those in NC group(P＜0.01).
Conclusions
1. There was imbalance in gut flora and changes of plasma endotoxinlevels in rats with TNBS-induced colitis.
2. Bifico could attenuate inflammation, decrease plasma endotoxin level,and increase Bif and Lac numbers in colons of TNBS-induced colitis rats,its mechanisms of attenuating inflammation may be the reinstitution ofgut flora balance.
PARTⅡ
Changes in phenotype and functions of dendritic cells inmesenteric lymphoid nodes of rats with TNBS-inducedcolitis and effects of Bifico
Objective
To investigate the expressions of 0X-62, CD83, MttC-Ⅱand costimulantory molecules CD86 and function of dendritic cells(DCs) inmesenteric lymphoid nodes. To study effects of Bifico on maturation andactivation of dendritic cells, and to explore its immunoregulatoryfunctions in rats with experimental colitis.
Methods
1. Method of establishment of the rat model of experimental colitis wasas in PARTⅠ(no MC group).
2. All mesenteric lymph nodes of rats in the three groups were removedunder aseptic condition at the time of sacrifice. One was kept forexpressions of 0X-62 and CD83 measured by immunohistochemical staininganalysis. DCs in mesenteric lymph nodes were freshly isolated andpulificated by density gradient centrifugation and panning. Expressionsof MHC-Ⅱand co-stimulantory molecules CD86 on DCs in mesenteric lymphnodes were measured by flow cetometry. The capacity of stimulating T cellswas determined by allogeneic mixed leukocyte reaction (MLR).
Results
1. Expressions of 0X-62 and CD83 on DCs in mesenteric lymph node ofNC group, UC group and BC group were 198.6±87.7, 1422.1±598.2, 805.7±298.8 and 87.6±24.4, 709.2±199.6, 461.7±172.3, respectively.Expressions of 0X-62 and CD83 on DCs in UC group were significantly higherthan those of rats in NC group (P＜0.01),and also higher than those ofrats in BC group (P＜0.05) .Expressions of 0X-62 and CD83 of rats in BCgroup were higher than those in NC group(P＜0.01).
2. Expressions of MHC-Ⅱand CD86 on DCs in mesenteric lymph nodes ofNC group, UC group and BC group were (26.23±7.19)％, (84.55±9.38)%, (66.49±8.40)% and (20.46±7.69)%, (80.63±8.84)%, (59.79±10.03)%, respectively. Expressions ofMltC-Ⅱand CD86 on DCs in UC group weremarkedly higher than those in other two groups (P＜0.01 and P＜0.05), andthose in BC group were higher than those in NC group (P＜0.01).
3. Stimulatory Capacity of allogeneic T cells of DCs in UC group andBC group were higher than that in NC group, which was decreased in BC groupthan that in UC group.
Conclusions
1. DCs in mesenteric lymph nodes of rats with TNBS-induced colitisare increased and in a state of maturation and activation, and theirstimulatory capacity of allogeneic T cells was enhanced indicating moreimmature DCs of rats becoming mature and activated and the enhancementof the function of antigen presenting and activating naive T lymphocytes.
2. Bifico might decrease naive T lymphocytes activation throughpreventing immature DCs of rats becoming mature and activated suggestingBifico may regulate immune function of rats with TNBS-induced colitis.
PARTⅢ
Expressions of TLR2, TLR4 and NF-κBp65 on colons ofrats with TNBS-induced colitis and effects of Bifico
Objective
To investigate the expressions of protein and mRNA of Toll-likereceptor 2(TLR2),Toll-like receptor 4(TLR4) and nuclear factorkappaBp65(NF-κB p65) on colons of rats with TNBS-induced colitis.Tostudy effects of Bifico on expressions of TLR2, TLR4 andNF-κBp65 andto explore its immunoregulatory functions in rats with experimentalcolitis.
Methods
1. Method of establishment of the rat model of experimental colitis wasas in PARTⅠ(no MC group).
2. The whole colon including cecum in the three group rats were removedat the time of sacrifice. Total protein and mRNA of the colon wereextracted. Expressions of protein and mRNA of TLR2, TLR4 andNF-κB p65were measured by Western Blotting and real-time quantitative polymerasechain reaction(PeR),respectively.
Results
1. Protein expressions: Protein expressions of TLR2, TLR4 and NF- κBp65 on colons in NC, UC and BC groups were TLR2:10.26±4.24, 47.20±4.62, 36.64±3.67;TLR4:8.16±4.50, 25.84±4.46, 19.71±3.28, and NF-κBp65:3.16±1.52, 42.96±10.32, 30.74±7.71, respectively. Proteinexpressions of TLR2, TLR4 and NF-κBp65 on colons in NC group were verylow, but markedly increased in UC group(P＜0.01). Protein expressionsof TLR2、TLR4、NF-κBp65 in BC group were lower than those in UC group(P＜0.05), and higher than those in NC group (P＜0.01).
2. mRNA expressions: mRNA expressions(copies) of TLR2、TLR4, NF-κBp65 in NC, UC and BC groups were TLR2: (0.24±0.14)×10~3, (10.57±2.69)×10~3, (6.64±1.42)×10~3;TLR4: (0.21±0.11)×10~3, (8.58±2.71)×10~3, (5.29±1.54)×10~3, and NF-κBp65: (0.17±0.07)×10~3, (9.69±2.71)×10~3, (6.21±1.33)×10~3, respectively, mRNA expressions of TLR2,TLR4, NF-κBp65 in UC group were significantly increased compared withthose in NC group (P＜0.02) and in BC (P＜0.05). mRNA expressions ofTLR2, TLR4 and NF-κBp65 in Be group were higher than those in NC group(P＜0.01).
Conclusions
1. Expressions of TLR2, TLR4 and NF-κBp65 were increasedsignificantlyon colons of rats with TNBS-induced colitis.
2. Bifico might decrease expressions of protein and mRNA of TLR2 andTLR4, and downregulate expression of NF-κBp65 of rats withTNBS-induced colitis.
PARTⅣ
of cytokines both at the mucosal and SystemicalChanges
levels in rats with TNBS-induced colitis and effects of BificoObjective
Imbalance between pro-inflammatory and anti-inflammatory cytokinesplays an important role in the onset and development of IBD. The purposeof the study was to investigate the changes of pro-inflammatory cytokinesincluding tumor necrosis factor alpha(TNF-α), interferon-γ(IFN-γ), interleukin-12(IL-12) and anti—inflammatory interleukin-10(IL-10) andtransforming growth factor beta(TGF-β) at mucosal and systemicallevels. We assessed the impacts of Bifico on cytokine production, bothat the mucosal and systemical levels.
Methods
1. Method of establishment of the rat model of experimental colitiswas as in PARTⅠ(no MC group).
2. The spleens of all rats were removed under aseptic condition at thetime of sacrifice, and the splenocytes were isolated and cocultured withthe standard Bacillus coli strain (1×10~6 cells/ml) for 72 hours at 37℃. Supernatants were harvested and stored at -20℃for cytokineanalysis by using enzyme immunoassay (EIA). Cytokines analyzed were TNF-α, FN-γ, IL-12, IL-10 and TGF-β.
3. The small intestines of the rats were removed and the lymphoidfollicles of the Peyers' patches (PP) carefully removed from theintestinal serosal side with curved scissors. Seven to ten Peyers'patches were obtained per rat, and immunocytes at Peyers' patches wereisolated and cocultured with the standard Bacillus coli strain (1×10~6 cells/ml) for 72 hours at 37℃. Supernatants were collected and storedat -20℃for analysis of TNF-α, FN-γ, IL-12, IL-10 and TGF-β. Cytokineproduction was measured by EIA.
Results
1. Cytokine analysis was performed in splenocyte supernatants by EIAfollowing stimulation in vitro with standard Bacillus coli strain. TNF—αand IFN-γwere increased significantly in UC group than those inNC group(P＜0.01) and in BC(P＜0.05), IL-10 were decreased(P＜0.01 andP＜0.05). TNF—αand IFN-γin BC group were higher and IL-10 was lowercompared with those in NC group(P＜0.03). There was no significance indifference of IL-12 among the three groups(P＞0.05). Moreover,production of TGF-βwas maintained in three groups(P＞0.05).
2. Cytokine analysis was performed in supernatants of Peyers'patches cells by EIA following stimulation in vitro with standard Bacillus coli strain. TNF—α, IFN-γand IL-12 were significantlyincreased in UC group compared with those in NC group and in BC group(P＜0.01 and P＜0.05), but IL-10 was markedly reduced compared withthose in other two groups (P＜0.01 and P＜0.05). TNF—α、IFN-γandIL-12 in BC group were higher than those in NC group (P＜0.01), but IL-10was lower (P＜0.02). The difference of TGF-βin three groups was slightand didn't reach significance (P＞0.05).
Conclusions
1. Imbalance between pro-inflammatory and anti-inflammatory cytokineswas existed in rats with TNBS-induced colitis, both at the mucosal andsystemical levels.
2. Following challenge with Bacillus coli in vitro, there were reductionin Th1 cytokines (TNF-α, IFN-γ, and IL-12) and increase in Th2 cytokine(IL-10) in the Bifico group whereas production of the immunoregulatorycytokine TGF-βwas maintained.
3. The Bifico could improve the imbalance between pro-inflammatorycytokines and anti-inflammatory cytokines at the mucosal and systemicallevels.
引文
1. Hanauer SB. Inflammatory bowel disease:epidemiology, pathogenesis, and therapeutic opportunities[J]. Inflamm Bowel Dis, 2006, 12 (Suppll):S3-S9.
2. Crohn' s & Colitis Foundation of America. Living with Crohn' s Disease [educational brochure]. Available at:Http:www.ccfa.org.Accessed August 19, 2005.
3. Crohn' s & Colitis Foundation of America. Living with Ulcerative Colitis [educational brochure]. Available at: Http:www.ccfa.org. Accessed August 19, 2005.
4. Andres PG, Friedman LS. Epidemiology and the natural course of inflammatory bowel disease[J]. Gastroenterol Clin North AM.1999;28 (2): 255-281.
5. Jiang XL, Cui HF. An analysis of 10218 ulcerative colitis cases in China[J]. World J GastroenteroL 2002;8 (1): 158-161.
6. Ouyang Q, Tandon R, Gob KL, et al. The emergence of inflammatory bowel disease in the Asian Pacific region[J]. Curt Opin Gastroenterol. 2005;21 (4): 408-413.
7. Abreu MT. The pathogenesis of inflammatory bowel disease: translational implications for clinicians[J]. Curt 6astroenterol Rep. 2002 ; 4 (6):481-489.
8. Podolsky DK. Inflammatory bowel disease[J]. N Engl J Med. 2002:347(6): 417-429.
9. Donnenberg MS. Pathogenic strategies of enteric bacteria[J].Nature. 2000 ; 406 (6797): 768-774.
10. Neish AS, Gewirtz AT, Zeng H, et al. prokaryotic regulation of epithelial responses by inhibition of Ⅰ κ B-α ubiquitination[J]. Science. 2000 ;289 (5484): 1560-1563.
11. Schenk M, Bouchon A, Birrer S, et al. Macrophages expressing triggering receptor expressed on myeloid cells-are underrepresented in the human intestine [J]. J Immunol. 2005;174 (1):517-524.
12. Shanahan F. Probiotics and inflammatory bowel disease: is there a scientific rationale [J]? Inflamm Bowel Dis. 2000;6(2):107-115.
13. Fiocchi C. Non-stop progress in inflammatory bowel disease: new players, new understanding, new therapies [J], Curt Opin Gastroenterol. 2006 ;22 (4): 347-348.
14. Colletti T. IBD-recognition, diagnosis, and therapeutics [J]. JAAPA. 2004; 17(5): 16-24.
15. Schuler G, Steinman RM. Dendritic cells as adjuvants for inmmuno-mediated resistance to tumors [J]. J Exp Med. 1997; 186(8):1183-1187.
16. Hart DN. Dendritic cells:unique leukocyte population which control the primary immune response [J]. Blood 1997 ; 90 (9):3245-3287.
17. Akira S, Takeda K, Kaisho T. Toll-like receptors:critical proteins linking innate and acquired immunity [J]. Nat Immunol. 2001;2 (8):675-680.
18. Miles L. Are probiotics beneficial for health [J]? Nutrition Bulletin. 2007;32 (1):2-5.
19. Burke DA, Axon AT. Adhesive escherichia coli in inflammatory bowel disease and infective diarrhea [J]. BMJ. 1988 ; 297 (6641): 102-104.
20. Venturi P, Gionchetti P, Rizell F, et al. Impact on the composition of the faecal flora by a new probiotic preparation: Preliminary data on maintenance treatment of patients with ulcerative colotis [J]. Aliment Pharmacol Ther. 1999; 13 (8): 1103-1108.
21. gamada N, Inoue N, Hisamatsu T, et al. Nonpathogenic Eschericbia coli strain Nissle 1917 prevents murine acute and chronic colitis [J]. Inflamm Bowel Dis. 2005 ; 11 (5): 455-463.
22. Fedorak RN, Madsen KL. Probiotics and the management of inflammatory bowel disease [J]. Inflamm Bowel Dis. 2004; 10(3):286-299.
23. Morris GP, Beck PL, Herridge MS, et al. Hapten-induced model of chronic inflammation and ulceration in the rat colon [J]. Gastroonterology. 1989; 96 (3): 795-803.
24. Llopis M, Antolin M, Guarner F, et al. Mucosal colonization with Lactobacillus casei mitigates barrier injury induced by exposure to trinitronbenzene sulphonic acid [J]. Gut. 2005;54(7):955-959.
25. Togawa J, Nagase H, Tanaka K, et al. Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance [J]. Am J Physiol Gastrointest Liver Physiol. 2002 ;283 (1):G187-G195.
26. McCarthy J, O' Mahony L, O' Callaghan L, et al. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance [J].Gut 2003;52(7):975-980.
27. Diebel LN, Liberati DM, Diglio CA, et al. Synergistic effects of candida and Escherichia cili on gut barrier function [J].J Traum.1999;47(6): 1045-1051.
28. Madsen KL, Doyle JS, Jewell LD, et al. Lactobacillus species prevents colitis in interlerleukin 10 gene-dificient mice [J]. Gastroenterology. 1999; 116(5):1107-1114.
29. Philippe D, Dubuquoy L, Groux H, et al. Anti-inflammatory properties of the mu opioid receptor support its use in the treatment of colon inflammation [J].J Clin Invest. 2003;111(9):1329-1338.
30. Tozawa K, Hanai H, Sugimoto K, et al. Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice [J]. Gastroenterol Hepatol. 2003; 18(5):578-587.
31. Sartor RB. Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics [J]. Gastroenterology. 2004;126(6):1620-1633.
32. Broekaert IJ, Walker WA. Probiotics and chronic disease [J]. J Clin Gastroenterol. 2006; 40(3):270-274.
33. Santosa S, Farnworth E, Jones PJ. Probiotics and potential health claims [J].Nutr Rev. 2006; 64(6):265-274.
34. Ounne C, O' Mahony L, Murphy L, et al. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings [J]. Am J Clin Nutr. 2001;73(2 Suppl):386-392.
35. Murphy L, Dunne C, Kiely B, et al. In vivo assessment of potential probiotic Lactobacillus salivarius strains: evaluation of their establishment, persistence, and localisation in the murine gastrointestinal tract [J].Micro Ecol Health Dis. 1999;11(1):149-157.
36. Ishikawa H, Akedo I, Umesaki Y, et al. Randomized controlled trial of the effect of bifidobacteria-fermented milk on ulcerative colitis [J]. J Am Coll Nutr. 2003; 22(1):56-63.
37. Malchow HA. Crohn's disease and escherichia coli. A new approach in therapy to maintain remission of colonic Crohn's disease [J]? J Clin Gastroenterol.1997; 25(4):653-658.
38. Schultz M, Veltkamp C, Dieleman LA, et al. Lactobacillus plantarum 299V in the treatment and prevention of spontaneous colitis in interleukin-10 dificient mice [J]. Inflamm Bowel Dis. 2002;8(2): 71-80.
39. Bibiloni R, Fedprak RN, Tannock GW, et al. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis [J]. Am J Gastroenterol. 2005; 100(7):1539-1546.
40. Kruis W, Fric P, Pokrotnieks J et al. Maintaining remission of ulcerative colitis with the probiotic Escherichia Coli Nissle 1917 is as effective as with standard messalazine [J]. Gut. 2004; 53 (11): 1617-1623.
41. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines [J]. Cell. 2001; 106(3):255-258.
42. Neutra MR, Frey A, Kraehenbuhl JP, Epithelial M cells: gateways for mucosal infection and immunization [J]. Cell. 1996;86(3): 345-348.
43. Uhlig HH, Powrie F. Dendritic cells and the intestinal bacterial flora: a role for localized mucosal immune responses [J]. J Clin Invest. 2003; 112(5): 648-651.
44. Kelsall BL, Biron CA, Sharma O, et al. Dendritic cells at the host-pathogen interface [J]. Nat Immunol. 2002;3(8):699-702.
45. Steinman RM, Hawiger D, Liu K, et al. Dendritic cell function in vivo during the steadystate: a role in peripheral tolerance [J]. Ann N Y Aead Sci. 2003;987 (1):15-25.
46. Fujii S, Liu K, Smith C, et al. The linkage of innate to adaptive innmuunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CDSO/CD86 costimulation [J]. J Exp Med. 2004; 199(12): 1607-1618.
47. Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells [J]. Cell. 2001; 106(3):263-266.
48. Liu YJ. Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity [J]. Cell. 2001; 106(3):259-262.
49. Jonuleit H, Schmitt E, Schuler G, et al. Induction of interleukin 10 producing, nonproliferating CD4 (+) T cells with regulatory properties by repetitive stimulation with allogeneie immature human dendritic cells [J]. J Exp Med. 2000; 192 (9): 1213-1222.
50. Manfred B. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity [J]? Trends in Immunol. 2002; 23 (9): 445-449.
51. Brenan M, Puklavec M. The MRC 0X—62 antigen: a useful marker in the purification of rat veiled cells with the bio-chemical properties of an integrin [J]. J Exp Med 1992 ; 175 (6): 1457-1465.
52. Brenan M, Rees DJ. Sequence analysis of rat integrin a E1 and a E2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph [J]. Eur J Immuol. 1997 ;27 (11): 3070-3079.
53. 赵谨，王心，树突状细胞．日本医学介绍[J]. 2005;26:286-287
54. Ridge JP, Fuchs EJ. Matzinger P. Neonatal tolerance revisited: Turning on neworn T cells with dendritic cells [J]. Science. 1996 ;271 (5256): 1723-1726.
55. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991;9(2):271-296
56. Turnbull EL, YrlidU, Jenkins CD, Macpherson GG. Intestinal dendritic cell subsets: differential effects of systemic TLR4 stimulation on migratory fate and activation in vivo [J]. J Immunol. 2005; 174(3):1374-1384.
57. Bimczok D, Sowa EN, Faber-Zuschratter H, et al. Site-specific expression of CDllb and SIRPalpha (CD172a) on dendritic cells: implications for their migration patterns in the gut immune system. Eur J Immunol. 2005; 35 (5): 1418-1427.
58. Kraus TA, Brimnes J, Muong C, et al. Induction of mucosal tolerance in Peyer' s patch-deficient, ligated small bowel.loops. J Clin Invest 2005; 115 (8):2234-2243.
59. Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria [J]. Science 2004; 303(5664):1662-1665.
60. Chirdo FG, Millington OR, Beacock-Sharp H, et al. Immunomodulatory dendritic cells in intestinal lamina propria [J]. Eur J Immunol. 2005; 35(6): 1831-1840.
61. Worbs T, Bode U, Yah S, et al. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells [J]. J Exp Med. 2006; 203(3):519-527.
62. Shananan F. Crohn's disease [J]. Lancet. 2002;359(9300):62-69
63. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 2004;118(2):229-241
64. Hoshino, K, Takeuchi O, Kawai T, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162(7):3749-3752.
65. Abreu MT, Vora P, Faure E, et al. Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide [J]. J Immunol.2001;167(3):1609-1616.
66. Hausmann M, Kiessling S, Mestermann S, et al. Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation [J].Gastroenterology. 2002;122(7):1987-2000.
67. Melmed G, Thomas LS, Lee N, et al. Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host-microboal interactions in the gut. J Immunol. 2003; 170(3):1406-1415.
68. Otte JM, Cario E, Podolsky Dk. Mechanisms of cross-hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells [J]. Gastroenterology. 2004;126(4):1054-1070.
69. Hart AL, Al-Hassi HO, Rigby RJ, et al. Characteristics of intestinal dendritic cells in inflammatory bowel diseases [J]. Gastroenterology. 2005; 129(1): 50-65.
70. O'Neill LA. How NOD-ing off leads to Crohn disease [J]. Nat Immunol. 2004; 5 (8): 776-778,
71. NeteaMG, Kullberg BJ, de Jong DJ, et al. NOD2 mediates anti-inflammatory signals induced by TLR2 ligands: implications for Crohn's disease [J]. Eur J Immunol 2004;34(7):2052-2059.
72. Neurath MF, Pettersson S, MeyerzumbuMChenfelde KH, et al. Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice [J]. Nat Med 1996;2(9):998-1004.
73. Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins:evolutionarily conserved mediators of immune responses [J]. Annu Rev Immunol. 1998; 16(1): 225-260.
74. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol. 2004; 5(10): 971-974.
75. Sasaki M, Jordan P, Houghton J, et al. Transfection of IL-10 expression vectors intoendothelial cultures attenuates alpha4beta7-dependent lym phocyte adhesion mediated by MAdCAM-1. BMC Gastroenterol. 2003; 3(2): 3
76. Weninger W, von AndrianUH. Chemokine regulation of naive T cell traffic in health and disease [J]. Semin Immunol. 2003; 15(5): 257-270.
77. D' Haens G, Deventer SV, Uogezand RV, et al. Endoscopic and histological healing with infliximab anti-tumor-necrosis factor antibodies in Crohn's disease: a European multicenter trial[J]. Gastroenterology. 1999; 116(5): 1029-1034.
78. Niess JH, Reinecker HC. Dendritic cells: the commanders-in-chief of mucosal immune defenses [J]. Curt Opin Gastroenterol. 2006; 22(4): 354-360.
79. Fuss IJ, Neurath M, Boirivant M, et al. Disparate CD4+lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IL-5 [J]. J Immunol. 1996; 157(3): 1261-1270.
80. Van Hogezand RA, Verspaget HW. Selective immunomodulation in patients with inflammatory bowel disease-future therapy or reality [J]? Neth J Med. 1996; 48(2): 64-67.
81. Neurath MF, Fuss I, Kelsall BL, et al. Antibodies to interleukin 12 abrogate established experimental colitis in mice [J]. J Exp Med. 1995; 182(5): 1281-1290.
82. Shanahan F. Inflammatory bowel disease: immunodiagnonostics, immunotherapeutics and ecotherapeutics [J]. Gastroenterology. 2001; 120 (3): 622-635
83. Barbara G, xing Z, HogabomCM, et al. Interleukin 10 gene transfer prevents experimental colitis in rats [J]. Gut. 2000; 46(3): 344-349.
84. Xiao CQ, Kao KJ. Suppression of interleukin-12 production through endogenously secreted interleukin-10 in activated dendritic cells: in-volvement of activation of extracellular signal-regulated protein kinase. Scand J Immunol. 2003; 58(1): 23-32.
85. Monteleone G, Mann J, Monteleone I, et al. A failure of transforming growth factor-betal negative regulation maintains sustained NF-kappaB activation in gut inflammation [J].J Biol Chem. 2004;279(6):3925-3932.
86. Kitani A, Fuss IJ, Nakamura K, et al. Treatment of experimental (Trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-betal plasmid: TGF-betal-mediated suppression of T helper cell type 1 response occurs by inter]eukin (IL)-10 induction and IL-12 receptor beta-chain downregulation. J Exp Med. 2000;192(1):41-52.
87. Strobl H, Knapp W. TGF-β regulation of dendritic cells [J].Microbes Infect 1999;1(15):1283-1290.
1. Marin AR, Ruiz S, Parrillas V, et al. Origin and differentiation of dendritic cells [J]. Trends Immunol. 2001;22(12):691-700.
2. Coombes BK, Mahony JB. Dendritic cell discoveries provide new insight into the cellular immunobiology of DNA vaccines [J]. Immmunol Lett. 2001;78(2): 103-111.
3. Shortman K, Liu YJ. Mouse and human dendritic cell subtypes [J]. Nature Rev Immunol. 2002;2 (3): 151-161.
4. Syme R, Gluck S. Generation of dendritic cells: role of cytokines and potential clinical applications [J]. Transfus Apheresis Sci 2001; 24 (2): 117-124.
5. Maldonado-Lopez R, Moser M. Dendritic cell subsets and the regulation of Th1/Th2 responses [J]. Semin Immunol. 2001;13 (5):275-282.
6. Abe M, Kajino K, Akbar SM, et al. Loss of immunogenecity of liver dendritic cells from mouse with chronic hepatitis [J]. Int J Mol Med. 2002 9(1):71-76.
7. Mowat AM. Anatomical basis of tolerance and immunity to intestinal antigens [J]. Nat Rev Immunol.2003; 3 (4):331-341.
8. Sundquist M, Johansson C, Wick MJ. Dendritic cells as inducers of antimicrobial immunity in vivo [J]. APMIS.2003 ; 111 (7-8): 715-724.
9. Castellaneta A, Abe M, Morelli AE, et al. Identification and characterization of intestinal Peyer' s patch interferon-alpha producing (plasmacytoid) dendritic cells []]. Hum Immunol. 2004; 65 (2): 104-113.
10. Uhlig HH, Powrie F. Dendritic cells and the intestinal bacterial flora:a role for localized mucosal immune responses [J].J Clin Invest. 2003; 112 (5):648-651.
11. Sundquist M, Rydstrom A, Wick MJ. Immunity to Salmonella from a dendritic point of view [J]. Cell Microbiol. 2004;6(1):1-11.
12. Foti M, Ricciardi-Castagnoli P. Antigen sampling by mucosal dendritic cells [J]. Trends Mol Med.2005; 11 (9):394-396.
13. O'Hara AM, O'Regan P, Fanning A, et al.Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius [J]. Immunology. 2006 ; 118 (2): 202-215.
14. Zaph C, Troy AE, Taylor BC, et al. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis [J]. Nature. 2007; 446(7135): 552-556.
15. Hase K, Ohno H. Epithelial cells as sentinels in mucosal immune barrier [J]. Nihon Rinsho Meneki Gakkai Kaishi.2006;29(1):16-26.
16. Eberl G. Inducible lymphoid tissues in the adult gut: recapitulation of a fetal developmental pathway [J] ? Nat Rev Immunol. 2005 ; 5 (5): 413-420.
17. Sanders DS. Mucosal integrity and barrier function in the pathogenesis of early lesions in Crohn's disease[J]. J Clin Pathol. 2005;58(6):568- 572.
18. Dubois B, Goubier A, Joubert G, et al. Oral tolerance and regulation of mucosal immunity [J]. Cell Moll Life Soi.2005;62 (12): 1322-1332.
19. Mora JR, Iwata M, Eksteen B, et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells [J]. Science. 2006;314 (5802): 1157-1160
20. Niess JH, Reinecker HC. Lamina propria dendritic cells in the physiology and pathology of the gastrointestinal tract [J]. Curt Opin Gastroenterol. 2005 ;21 (6):687-691.
21. Williams IR. CCR6 and CCL20: partners in intestinal immunity and lymphorganogenesis [J]. Ann N Y Acad Sci. 2006; 1072 (1): 52-61.
22. Annacker O, Coombes JL, Malmstrom V, et 21. Essential role for CD103 in the T cell-mediated regulation of experimental colitis [J].J Exp Med 2005; 202 (8): 1051-1061.
23. Pulendran B, Palucka K, Banchereau J. Sensing pathogens and tuning immune responses [J]. Science. 2001 ;293 (5528):253-256.
24. Mohamadzadeh M, Olson S, Kalina WV, et al. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization [J]. Proc Natl Acad Sci USA. 2005; 102 (8):2880-2885.
25. Kikuchi T, Kobayashi T, Gomi K, et al. Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses [J]. J Immunol. 2004; 172 (3): 1727-1734.
26. Guinet E, Voshida K, Nouri-Shirazi M. Nicotinic environment affects the differentiation and functional maturation of monocytes derived dendriticcells (DEs) [J]. Immunol Lett. 2004;95 (1):45-55.
27. KobetsN, Kennedy K, Garside P. An investigation of the distribution of antigen fed in tolerogenic or immunogenic forms [J]. Immunol Lett. 2003; 88(2): 147-155.
28. Viney JL, Mowat AM, O' Malley JM, et al. Expanding dendritic cells in vivo enhances the Induction of oral. tolerance [J].J Immunol. 1998; 160(12): 5815-5825.
29. Powrie F, Maloy KJ. Immunology. Regulating the regulators [J]. Science. 2003;299 (5609): 1030-1031.
30. Granucci F, Ricciardi-Castagnoli P. Interactions of bacterial pathogens with dendritic cells during invasion of mucosal surfaces [J]. Curr Opin Microbiol. 2003;6 (1):72-76.
31. Stumbles PA, Upham JW, Holt PG. Airway dendritic cells: co-ordinators of immunological homeostasis and immunity in the respiratory tract [J].APMIS. 2003;111 (7):741-755.
32. Lindsay JO, Whelan K, Stagg AJ, et al. Clinical, microbiological, and immunological effects of fructo-oligosaccharide in patients with Crohn' s disease [J]. Gut. 2006;55 (3):348-355.
33. Bilsborough J, Viney JL. In vivo enhancement of dendritic cell function [J]. Ann N Y Acad Sci. 2004; 1029(1):83-87.
34. Mora JR, Bono MR, Manjunath N, et al. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells [J]. Nature. 2003;424(6944): 88-93.
35. Sun J, Van Houten N. CD40 stimulation in vivo does not inhibit CD4~+ T cell tolerance to soluble antigens [J]. Immunol Lett. 2002;84(2):125-132.
36. Niedergang 'F, Kweon MN. New trends in antigen uptake in the gut mucosa [J]. Trends Microbiol. 2005; 13 (10):485-490.
37. McKenzie BS, Corbett AJ, Brady JL, et al. Targeting lymphocyte Peyer's patch adhesion molecule-l: a relay approach to gut immunization [J]. Vaccine. 2005 ; 23 (28): 3668-3678
1. Seitz M. Toll-like receptors: sensors of the innate immune system[J]. Allergy. 2003; 58 (12): 1247-1249.
2. Bell JK, Mullen GED, Leifer CA, et al. Leucine-rich repeats and pathogen recognition in Toll-like receptors[J]. Trends Immunol. 2003;24(10):528-533.
3. Kawai T, Akira S. Pathogen recognition with Toll-like receptors[J]. Curr Opion Immunol. 2005 ; 17 (4): 338-344.
4. Abreu MT, Vora P, Faure E, et al. Decreased expression of Toll-likereceptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide [J]. J Immunol. 2001; 167 (3): 1609-1616.
5. Hausmann M, Kiessling S, Mestermann S, et al. Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation[J], Gastroenterology. 2002 ; 122 (7): 1987-2000.
6. Medvedev AE, Lentschat A, Wahl LM, et al. Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor associated kinase 1 activation in endotoxin-tolerant cells[J], J Immunol. 2002; 169 (9): 5209-5216.
7. Otte JM, Cario E, Podolsky DK, et al. Mechanisms of cross hyporesponsiveness to Toll-like receptor bacterial ligands in intestinal epithelial cells[J]. Gastroenterology. 2004; 126 (4): 1054-1070.
8. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, et al. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis[J]. Cell. 2004; 118 (2):229-241.
9. Cario E, Gerken G, Podolsky DK, et al. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C[J]. Gastroenterology. 2004;127 (1):224-238.
10. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity[J]. Nat Immunol. 2004;5 (10):971-974.
11. Netea MG, Kullberg BJ, de Jong DJ, et al. NOD2 mediates anti-inflammatory signals induced by TLR2 ligands: implications for Crohn' s disease[J]. Eur J Immunol. 2004; 34 (7): 2052-2059.
12. O'Neill LA. How NOD-ing off leads to Crohn disease[J].Nat Immunol. 2004; 5 (8): 776-778.
13. Kobayashi M, Kweon MN, Kuwata H, et al. Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice[J]. J Clin Invest. 2003; 111 (9):1297-1308.
14. Boone DL, Ma A. Connecting the dots from Toll-like receptors to innate immune cells and inflammatory bowel disease[J]. J Clin Invest. 2003;111 (9):1284-1286.
15. Dubuquoy L, Jansson EA, Deeb S, et al. Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis[J]. Gastroenterology. 2003; 124 (5):1265-1276.
16. Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease [J]. Nat Immunol. 2004 ; 5 (1 O): 975-979.
17. Franchimont D, Vermeire S, E1 Housni H, et al. Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn' s disease and ulcerative colitis[J]. Gut. 2004; 53 (7):987-992
18. Torok HP, Glas J, Tonenchi L, et al. Polymorphisms of the lipopolysaccharide-signaling complex in inflammatory bowel disease: association of a mutation in the Toll-like receptor 4 gene with ulcerative colitis[J]. Clin Immunol. 2004; 112 (1):85-91.
19. Glasgow JN, Wood T, Perez-Polo JR. Identification and characterization of nuclear factor kappaB binding sites in the murine bcl-x promoter[J]. Neurochem. 2000;75 (4): 1377-1389.
20. Tak PP, Firestein GS. NF-κB: a key role in inflammatory diseases[J]. Clin Invest. 2001 ; 107(1):7-11.
21. Schmid RM, Adler G. NF-kappaB/Rel/IkB:implications in gastrointestinal diseases [J]. Gastroenterology. 2000; 118 (6): 1208-1228.
22. Ghosh S, May MJ, Kopp EB. NF-κB and Rel proteins:evolutionarily conserved mediators of immune responses[J]. Annu Rev Immunol. 1998; 16(1): 225-260.
23. Zhang G, Ghosh S. Toll-like receptor-mediated NF-kappaB activation: a phylogenetically conserved paradigm in innate immunity[J]. J Clin Invest. 2001 ; 107 (1): 13-19.
24. Wang Z, Castresana MR, Detmer K, et al. An IkappaB-alpha mutant inhibits cytokine gene expression and proliferation in human vascular smooth muscle cells[J]. Surg Res. 2002; 102 (2): 198-206.
25. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-kappaB activity [J]. Annu Rev Immunol. 2000 ; 18 (1): 621-663.
26. Rothwarf DM, Zandi E, Natoli G, et al. IKK-gamma is an essential regulatory subunit of the IkappaB kinase complex[J]. Nature. 1998;395 (6699): 297-300.
27. MacDonald TT, Monteleone G, Pender SL. Recent developmentsin the imm-unology of inflammatory bowel disease [J]. Scand J Immunol. 2000 ; 51 (1): 2-9
28. Stoyanova Ⅱ, Gulubova MV. Mast cells and inflammatory mediators in chronic ulcerative colitis. Acts Histochem. 2002; 104 (2): 185-192.
29. Kirsner JB. Historical origins of current IBD concepts[J]. World J Gastroenterol. 2001 ;7 (2): 175-184.
30. Brown KA, Back SJ, Ruchelli ED, et al. Lamina propria and circulating interleukin-6 in newly diagnosed pediatric inflammatory bowel disease patients[J]. Am J Gastroenterol. 2002;97(10):2603-2608.
31. Sasaki M, Jordan P, Houghton J, et al. Transfection of IL-10 expression vectors intoendothelial cultures attenuates alpha4beta7-dependent lym phocyte adhesion mediated by MAdCAM-1 [J]. BMC Gastroenterol.2003 ; 3 (2): 3.
32. Tamuxa K, Fukuda Y, Sashio H, et al. IL-18 polymorphism is associated with an increased risk of Crohn's disease[J]. Gastroenterology. 2002;37 (Suppl 14):111-116.
33. Ito H, Hirotani T, Yamamoto M, et al. hnti-IL-6 receptor monoclonal antibody inhibits leukocyte recruitment and promotes T-cell apoptosis in a murine model of Crohn' s disease [J]. J Gastroenterol. 2002 ; 37 (Suppl 14): 56-61.
34. Schmidt C, Marth T, Wittig BM, et al. Interleukin-12 antagonists as new therapeutic agents in inflammatory bowel disease[J] Pathobioloy. 2002 ;70 (3): 177-183.
35. Lochner M. Forster I. hnti-interleukin-18 therapy in murine models of inflammatory bowel disease[J].Fathobiology. 2002;70(3):164-169.
36. Baldwin AS Jr. Series introduction the transcrtion factor NF-κB and human disease[J].J Clin Invest.2001;107(1):3-6.
37. Brannigan hE, Watson RW, Beddy D, et al. Increased adhesion molecule expression in serosal fibroblasts isolated from patients with inflamm-atory bowel disease is secondary to inflammation[J].Am Surg.2002;235(4): 507-511.
38. Jobin C, Hellerbrand C, Licato LL, et al. Mediation by NF-kappaB of cytokine induced expression of intercellular adhesion molecule I(ICAM-1) in an intestinal epithelial cell line, a process blocked by proteasome inhibitors[J].Gut. 1998;42(6):779-787.
39. Conner EM, Brand S, Davis IM, et al. Proteasome inhibition attenuates nitric oxide synthase expression. VCAM-1 transcription and the development of chronic colitis[J].pharmacol Exp ther. 1997;282(3):1615-1622.
40. Nakase H, Okazaki K, Tabata Y, et al. An oral drug delivery system targeting immune-regulating cells ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis[J].Pharmacol Exp Ther. 2001;297(3):1122-1128.
41. Brueckmann M, Marx h, Martin WH, et al. Stabilization of monocyte chem-oattractan tprotein-l-mRNh by activated protein C[J]. ThrombHaemost. 2003;89(1):149-160.
42. FiettaAM, iorosini M, Meloni F, et al. Pharmacological analysis of signal transduction pathways required for mycobacterium tuberculosis-induced IL-8 and MCP-1 production in human peripheral monocytes[J]. Cytokine. 2002;199 (5):242-249.
43. Schreiber S, Nikolaus S, Hampe J. Activation of nuclear factor kappaB inflammatory bowel disease[J].Gut. 1998;42(4):477-484.
44. Thiele K, Bierhaus A, Autschbach F, et al. Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha systerm in patients with Crohn's disease[J].Gut. 1999;45(5):693-704.
45. Weber CK, Liptay S, Wirth T, et al. Suppression of NF-kappaB activity by sulfasalazine is mediated by direct inhibition of IkappaB kinases alpha and beta[J]. Gastroeaterology. 2000;11g(5):1209-1218.
46. Segain JP, Raingeard de la Bletiere D, Bourreille A, et al. Butyrate inhibits inflammatory responses through NF-kappaB inhibition:implications for Crohn's disease[J].Gut. 2000;47(3):397-403.
47. Neurath MF, Pettersson S, Meyerzum Buschenfelde KH, et al. Local administration of antisense phosphorothioate oligo-nucleotides to the p65 subunit of NF-kappaB abrogates established experimental colitis in mice[J].Nat Med. 1996;2(9):998-1004.
48. Neurath MF.Pettersson S.Predominant role of NF-kappaBp65 in the pathogenesis of chronic intestinal inflammation. Immunobiology[J]. 1997;198(1):91-98.