实验性结肠炎大鼠内毒素-Toll样受体4-核因子κB信号通路的变化及益生菌的作用

Changes of lipopolysaccharide-Toll-like receptor 4 and nuclear factor-kappaB signal pathway of experimental colitis rats and effects of probiotics

目的探讨和分析实验性结肠炎大鼠血浆内毒素水平的变化及结肠Toll样受体4(TLR4)和核因子κB(NF-κB)表达及意义,并分析益生菌的作用。方法30只雄性W istar大鼠均分为正常对照组(NC组)、模型对照组(UC组)和益生菌治疗组(PC组);UC和PC组建立2,4,6-三硝基苯磺酸(TNBS)实验性结肠炎大鼠模型,PC组大鼠给予双歧三联活菌悬液(2.2×109CFU/只)治疗,1次/d,共4周。光镜下观察肠黏膜炎性反应并评分;鲎试验检测各组大鼠血浆内毒素水平;W estern印迹法和实时荧光定量PCR法检测大鼠结肠TLR4和NF-κB p65蛋白及mRNA的表达,分析其变化及益生菌的作用。结果PC组炎性反应评分较UC组明显降低(P<0.05),但高于NC组(P<0.01);PC组血浆内毒素明显低于UC组(P<0.05),但高于NC组(P<0.01);UC组TLR4和NF-κB p65的蛋白及mRNA表达明显高于PC组和NC组(P<0.05、0.01),PC组高于NC组(P<0.01)。结论内毒素-TLR4-NF-κB信号通路参与了大鼠实验性结肠炎的发生和发展,减少内毒素的产生、降低大鼠结肠TLR4和NF-κB表达可能是益生菌减轻大鼠结肠炎症的机制之一。

Objective To investigate changes of the plasma endotoxin levels,protein and mRNA expressions of Toll-like receptor 4（TLR4） and nuclear factor kappaB（NF-κB） on colons of rats with trinitrobenzene sulfonic acid（TNBS）-induced colitis,and to evaluate effects of probiotics.Methods Thirty male Wistar rats were randomly divided into normal control group（NC group）,model control group（UC group） and probiotics-treated group（PC group）.The experimental colitis was induced by TNBS/ethanol enema.Rats in PC group were fed with Bifico [live probiotics of combined bifidobacterium（Bif）,lactobacillus（Lac） and enterococcus] by 2.2×10^9 CFU/d for 4 weeks.Inflammatory scores were observed.Plasma endotoxin levels were measured with Limulus test.Protein and mRNA expressions of TLR4 and NF-κB p65 were measured by Western blot and real-time quantitative polymerase chain reaction（RT-PCR）,respectively.Results The inflammatory score obviously decreased in PC group compared with UC group（P〈0.05）,but it was higher than that in NC group（P〈0.01）.There was significant difference of plasma endotoxin levels among three groups（P〈0.05）.Protein and mRNA（copies） expressions of TLR4 and NF-κB p65 in UC group were higher than those in PC and NC groups（P〈0.05 and P〈0.01）,which in PC group were higher than those in NC group（P〈0.01）.Conclusion Endotoxin-TLR4-NF-κB signal pathway may play a role in development of experimental colitis of rats.Reducing the plasma endotoxin levels and decreasing expressions of TLR4 and NF-κB on colons of rats may be one of mechanisms of probiotics to ameliorate inflammation of rats with TNBS-induced colitis.