强化谷氨酰胺及合生元的肠内营养对结肠炎新生小鼠Treg/Th17免疫平衡的影响

Effect of glutamine and synbiotic enteral nutrition on Treg/Th17 immune balance in colitis newborn mice

目的:基于程序性死亡分子-1(PD-1)及其主要配体(PD-L1)信号通路探讨强化谷氨酰胺及合生元的肠内营养对实验性结肠炎小鼠Treg/Th17免疫平衡的影响。方法:动物实验分为正常组(Nor)、模型组(Mod)、对照组(Con)和实验组(Exp),Nor组小鼠每天自由饮用纯净水,Mod组、Con组、Exp组小鼠每天自由饮用含5%DSS的纯净水,连续2周,构建实验性结肠炎小鼠模型。Nor组、Mod组小鼠始终以基础饲料连续喂养;Con组小鼠以基础饲料添加1 mg/kg的PD-L1 Fc融合蛋白喂养,Exp组小鼠以基础饲料添加100 ml/kg百普素、0.5 g/kg的谷氨酰胺和0.5 g/kg的合生元,标准饲养房饲养14 d。试剂盒检测血清中TGF-β1、IL-17含量。免疫组化检测结肠组织中转录因子维甲酸相关核孤儿受体γt(RORγt)和叉状头/翅膀状螺旋转录因子3(Foxp3)表达。流式细胞术检测血清中Treg、Th17细胞变化。Western blot检测结肠组织中PD-1、PD-L1蛋白表达。结果:与Nor组相比,Mod组小鼠血清中TGF-β1含量、Treg细胞、结肠组织中Foxp3阳性细胞比例均明显降低,血清中IL-17含量、Th17细胞比例、结肠组织中RORγt阳性细胞比例、结肠组织中PD-1、PD-L1蛋白表达均明显升高(P<0.05),与Mod组相比,Con组和Exp组小鼠血清中TGF-β1含量、Treg细胞比例、结肠组织中Foxp3阳性细胞比例均明显升高,血清中IL-17含量、Th17细胞比例、结肠组织中的RORγt阳性细胞比例、结肠组织中PD-1、PD-L1蛋白表达均明显降低(P<0.05)。结论:强化谷氨酰胺以及合生元的肠内营养支持可通过抑制PD-1/PD-L1信号调控实验性结肠炎小鼠Treg/Th17的免疫平衡,发挥其对肠黏膜的保护作用。

Objective:To study the effect of glutamine and synbiotic enteral nutrition on Treg/Th17 immune balance in experimental colitis mice based on programmed death-1(PD-1)and its ligand(PD-L1)signaling pathway.Methods:Animal experiments were divided into normal group(Nor),model group(Mod),control group(Con),experimental group(Exp).Nor group mice were free to drink purified water daily,Mod group,Con group and Exp group were free to drink purified water containing 5%DSS daily for 2 consecutive weeks,to build a mouse model of experimental colitis.Mice in Nor group and Mod group were continuously fed with basic feed,mice in Con group were fed with 1 mg/kg of PD-L1 Fc fusion protein with basic feed,and mice in Exp group were fed with 100 ml/kg of buprin,0.5 g/kg glutamine and 0.5 g/kg synbiotic with basic feed,reared for 14 d in the standard house.The kit was used to detect content of TGF-β1 and IL-17 in serum.Immunohistochemistry was used to detect expressions of transcription factor retinoic acid-related nuclear orphan receptorγt(RORγt)and fork-like/wing-like spiral transcription factor 3(Foxp3)in colon tissue.Flow cytometry was used to detect changes of Treg and Th17 cells in serum.Western blot was used to detect expressions of PD-1 and PD-L1 protein in colon tissue.Results:Compared with Nor group,content of TGF-β1 in serum of Mod group mice,Treg,proportion of Foxp3 positive cells in colon tissue were significantly reduced,content of IL-17 in serum,Th17 cells percentage,proportion of RORγt positive cells in colon tissue,expressions of PD-1 and PD-L1 protein in colon tissue were significantly increased(P<0.05).Compared with Mod group,content of TGF-β1 in serum of Con and Exp group mice,Tregs percentage,proportion of Foxp3 positive cells in colon tissue were significantly increased,content of IL-17 in serum,Th17 cells percentage,proportion of RORγt positive cells in colon tissue,expressions of PD-1 and PD-L1 protein in colon tissue were significantly reduced(P<0.05).Conclusion:Enhancing enteral nutritional support of glutamine and synbiotics can regulate the immune balance of experimental colitis Treg/Th17 by inhibiting PD-1/PD-L1 signaling,and exert its protective effect on intestinal mucosa.