NF-κB信号转导通路在小鼠先天性巨结肠相关性小肠结肠炎发病中的作用

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目的:探讨NF-κB信号转导通路在先天性巨结肠(Hirschsprung's disease,HSCR)相关性小肠结肠炎(Hirschsprung's disease associated enterocolitis,HAEC)发病中的作用。方法:选择21日龄Ednrb基因敲除小鼠(Ednrb-/-小鼠)建立HSCR小鼠模型作为实验组,以相同日龄野生型小鼠作为对照组,每组各6只。收集结肠标本,依据形态将实验组小鼠结肠分为狭窄段及扩张段,对照小鼠肠管相应分为结肠远段、近段。采用HE染色法检查结肠组织的病理改变;采用酶联免疫吸附法定量分析各组结肠组织的乙酰胆碱含量;采用蛋白质印迹法检测结肠组织磷酸化ⅠκBα(p-ⅠκBα)、ⅠκBα、TNF-α蛋白的表达水平;采用实时荧光定量聚合酶链反应(RT-qPCR)检测结肠组织ⅠκBα mRNA的水平。结果:对照组小鼠结肠远段和近段结肠组织炎症损伤的病理评分均为0级,实验组小鼠结肠狭窄段为Ⅱ级或更低,而扩张段为Ⅲ级或更高,结果显示HAEC主要发生在结肠的扩张段。实验组小鼠扩张段结肠组织p-ⅠκBα、ⅠκBα、TNF-α蛋白的相对表达量分别0.208±0.058、0.068±0.013和2.230±0.142,狭窄段结肠组织分别为0.099±0.030、0.046±0.014和0.135±0.011,扩张段较狭窄段结肠组织均显著升高,差异均有统计学意义(n P<0.05);实验组小鼠扩张段结肠组织ⅠκBα mRNA的表达水平明显高于狭窄段,差异有统计学意义(n P<0.05);实验组小鼠扩张段结肠组织乙酰胆碱含量下降,狭窄段升高,两者差异有统计学意义(n P<0.05)。n 结论:HSCR小鼠模型结肠扩张段的炎症程度较狭窄段严重,其机制与NF-κB信号转导通路被激活有关;而狭窄段炎症较轻,与NF-κB信号转导通路被抑制有关,为HAEC的治疗提供新的思路和靶点。“,”Objective:To explore the role of NF-κB signal transduction pathway in the pathogenesis of Hirschsprung's disease associated enterocolitis (HAEC).Methods:Ednrb knockout mice were bred and raised in a specific pathogen free (SPF) laboratory. Experimental group: Twenty-one-day-old EdnrB-/- mice (n=6) and comparable-age wild-type controls (n=6) were selected. Intestinal samples were harvested. Experimental colons were divided into narrow and dilated segments according to morphological changes. Control colons were divided into distal and proximal segments. Colonic hematoxylin-eosin (HE) staining was employed for judging HAEC. Acetylcholine level in colon was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylated IκBα (p-IκBα), IκBα and tumor necrosis factor-alpha (TNF-α) were examined by Western blot and mRNA level of IκBα was detected by real-time qualitative polymerase chain reaction (RT-qPCR).Results:In the control group, the pathological score of colon tissue inflammatory injury in the distal and proximal colon was zero, the colon stenosis was level Ⅱ or lower, while the colon expansion was level Ⅲ or higher, the results showed HAEC occurs mainly in the expansion of the colon. Expansion period of experimental mice colon tissue, relative expression of p-ⅠκBα、ⅠκBα、TNF-α were 0.208±0.058, 0.068±0.013 and 2.230±0.142, narrow section of the colonic tissue were 0.099±0.030, 0.046±0.014 and 0.135±0.011, the expansion period of relatively narrow section of colonic tissue were significantly increased, differences were statistically significant (n P<0.05); Experimental mice expansion period of colon tissue p-ⅠκBα mRNA expression level was significantly higher than the narrow section, the difference was statistically significant (n P<0.05); In the experimental group, the content of acetylcholine in colon tissues in the dilated segment decreased, while that in the stricture segment increased, and the difference was statistically significant (n P<0.05).n Conclusions:During HAEC, inflammation is more severe in dilated segment than that in narrow segment. It may be associated with an activation or inhibition of NF-κB signal transduction pathway.
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