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目的研究益生菌植物乳杆菌(LP)对炎症状态下肠屏障的修复机理。方法以白介素-10基因敲除(IL-10-/-)小鼠为炎性肠病模型,采用简单随机法(随机表)将空白对照(WT)小鼠和IL-10-/-小鼠分成4组:WT组、WT+LP组、IL-10-/-组和IL-10-/-+LP组,每组10只。分别灌饲Ringer液或LP溶液,持续4周。每周记录小鼠肠炎的临床表现,实验结束后收集小鼠结肠组织,进行病理评分和形态观察,ELISA法检测结肠组织促炎细胞因子肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)浓度,利用Ussing Chamber技术对小鼠结肠通透性及电生理变化进行分析,并用Western blot和免疫荧光分析法对结肠上皮细胞紧密连接蛋白进行定量和定位研究。结果与WT组相比,IL-10-/-组小鼠腹泻、直肠脱垂和体重丧失较明显(P<0.01);TNF-α和IFN-γ浓度明显升高(P<0.01);病理表现为大量炎性细胞浸润,偶见透壁性溃疡和隐窝脓肿形成;超微电镜提示紧密连接结构破坏;紧密连接蛋白(ZO-1、occludin、claudin-1)表达明显降低(P<0.01)且伴有异常分布;结肠上皮细胞甘露醇通透性升高(P<0.001),跨膜电阻值降低(P<0.001)。LP干预4周后,与IL-10-/-组相比,LP能显著改善肠道炎症的临床和病理表现,阻止紧密连接超微结构破坏,促进紧密连接蛋白的表达(P<0.01),降低结肠上皮细胞甘露醇通透性(P<0.001),升高跨膜电阻值(P<0.001),降低了TNF-α和IFN-γ的浓度(P<0.01),减轻了肠道炎症。结论 LP能通过促进肠上皮细胞间紧密连接蛋白的表达修复受损肠屏障功能,从而减轻肠道炎症。
Objective To study the repair mechanism of probiotics probiotic Lactobacillus (LP) on the intestinal barrier under inflammatory conditions. Methods The interleukin-10 gene knockout (IL-10 - / -) mice were used as the model of inflammatory bowel disease. Blank control (WT) mice and IL-10 - / - mice Divided into 4 groups: WT group, WT + LP group, IL-10 - / - group and IL-10 - / - + LP group, Ringer solution were separately poured or LP solution for 4 weeks. The clinical manifestations of mouse enteritis were recorded weekly. The colon tissues of mice were collected at the end of the experiment and pathological score and morphology were observed. The levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured. The colonic permeability and electrophysiological changes of mice were analyzed by Ussing Chamber technique. Western blot and immunofluorescence assay were used to quantify and locate the colonic epithelial tight junction protein. Results Compared with WT group, diarrhea, rectal prolapse and weight loss in IL-10 - / - mice group were significantly increased (P <0.01), TNF-α and IFN-γ concentrations were significantly increased The expression of ZO-1, occludin and claudin-1 were significantly decreased (P <0.01), and the expression of ZO-1, ) With abnormal distribution; the epithelial cell of mannitol increased permeability (P <0.001) and the transmembrane resistance decreased (P <0.001). Compared with IL-10 - / - group, LP could significantly improve the clinical and pathological manifestations of intestinal inflammation, block the ultrastructure destruction of tight junction and promote the expression of tight junction protein (P <0.01) (P <0.001), increased the transmembrane resistance (P <0.001), decreased the concentrations of TNF-α and IFN-γ (P <0.01) and relieved intestinal inflammation. Conclusion LP can repair the impaired gut barrier function by promoting the expression of tight junction protein between intestinal epithelial cells, thereby reducing intestinal inflammation.