乳酸菌对IL-10^(-/-)小鼠肠黏膜屏障损伤修复的机制研究

Mechanism of lactic bacteria on intestinal mucosal barrier injury repair in interleukin-10 knockout mice

目的 :评价乳酸菌对IL-10基因敲除(IL-10^(-/-))小鼠肠黏膜损伤的修复作用,初步阐明其作用机制。方法:以IL-10^(-/-)小鼠为模型,益生菌为植物乳杆菌(LP),随机分成对照组(WT组)、IL-10^(-/-)组、IL-10^(-/-)+LP组,分别灌饲Ringer液(WT组和IL-10^(-/-)组)和LP菌液(IL-10^(-/-)+LP组)4周。实验过程中记录各组小鼠的体重、腹泻及直肠脱垂情况,实验结束后收集各组小鼠结肠组织标本,用于炎症程度的病理评分、促炎细胞因子(TNF-α、IFN-γ)的ELISA检测和内质网应激相关蛋白Grp78表达的Western blot和RT-PCR检测,并利用Ussing chamber技术评估各组小鼠结肠黏膜的电生理及对大分子物质(Inulin-FITC)的通透性影响。结果:与IL-10^(-/-)组相比,IL-10^(-/-)+LP组小鼠腹泻和直肠脱垂的发生率显著降低(40%vs 80%,P<0.05;10%vs 30%,P<0.05),体重增加[(21.6±1.5)g vs(19.3±2.4)g,P<0.05],结肠组织炎症评分降低(4.5±1.1 vs 6.0±1.4,P<0.05),促炎细胞因子TNF-α、IFN-γ表达下调(P<0.05),结肠黏膜上皮对Inulin-FITC的通透性降低(P<0.05),跨膜电阻值增加(P<0.05),Grp78表达降低(P<0.05)。结论:乳酸菌能明显改善IL-10^(-/-)小鼠肠黏膜屏障功能,减轻肠道炎症,其作用机制可能与内质网应激反应的下调有关。

Objective： To evaluate the role of Lactobacillus plantarum （LP） in the treatment of colitis in interteukin-10 knockout（IL-10-/-） mice and to explore its possible mechanisms. Methods： IL-10-/- mice were used as the model of inflammatory bowel disease. Lactobacillus plantarum （LP） was used as probiotics. Wildtype（WT） mice and IL-10-/- mice were randomly assigned to WT group, IL-10-/- group and IL-10-/-＋LP group. The WT and IL-10-/- mice were gaved with 0.5 mL Ringer so- lutions, IL-10-/-＋LP groups were gaved with LP 0.5 mL, took LP 1 x 109 cfu every day, continued for 4 weeks. Colitis was assessed by histological score and clinical manifestation was observed. The gut paracellular permeability was measured by Ussing chamber. The concentrations of TNF- α and interferon y （IFN- γ） were detected by the ELISA method. The expression of Grp78 was detected by Western blot and RT-PCR method. Results： After four-weeks intervention of LP, compared with the IL-10^-/- group, the incidence of diarrhea and rectal prolapsed, and weight loss significantly reduced [（40% vsSO%, P〈0.05; 10% vs 30%, P〈0.05; （21.6± 1.5）g vs （19.3±2.4）g, P〈0.05], the inflammatory score of colonic tissue reduced （4.5± 1.1 vs 6.0 ±1.4, P〈0.05）, the concentrations of TNF-α and IFN-γ significantly decreased （P〈0.05）, the permeability of Inulin-FITC significantly decreased（P〈0. 05）, and transepithelial resistance（TER） significantly improved（P〈0.05）, and the expression of Grp78 significantly reduced （P〈0.05） in the IL-10^-/-＋LP group. Conclusion： Treatment with LP can effec- tively ameliorate colonic epithelial barrier dysfunction and intestinal inflammation in IL-10-/- mice, and these may be associated with the down-regulation of endoplasmic reticulum stress.