肠屏障在自身免疫性肝炎发病中的作用

Role of intestinal barrier in the pathogenesis of autoimmune hepatitis

目的观察并分析肠屏障在自身免疫性肝炎(AIH)发病中的作用,为阐释AIH的发病机制和探索基于肠道的治疗策略提供方向。方法纳入2017年1至12月在天津医科大学总医院就诊的14例AIH患者(AIH无肝硬化组6例,AIH肝硬化组8例)和10名健康对照(健康对照组),采用ELISA法检测各组血清D-乳酸、二胺氧化酶(DAO)含量,实时荧光定量PCR检测各组回肠末端组织紧密连接蛋白[闭锁小带蛋白-1(ZO-1)、闭合蛋白(occludin)]、细胞因子[IL-2、干扰素γ、IL-4和IL-10]和TLR4的相对表达量,蛋白质印迹法检测回肠末端组织分泌型免疫球蛋白A(sIgA)的相对表达量。选取30只BALB/c小鼠分为空白组、葡聚糖硫酸钠(DSS)组、刀豆蛋白A(ConA)组、DSS+ConA组、DSS+灌菌+ConA组,每组6只,检测各组小鼠结肠组织ZO-1和闭合蛋白的相对表达量,血清转氨酶水平(ALT、AST)和肝组织学炎症活动度Knodell评分。统计学方法采用独立样本t检验和单因素方差分析。结果AIH肝硬化组和AIH无肝硬化组的血清D-乳酸和DAO水平均高于健康对照组[(1768.2±147.1)μg/L、(436.2±197.0)μg/L比(100.2±10.9)μg/L和(11.5±2.5)U/L、(5.4±0.9)U/mL比(3.5±0.9)U/mL],且AIH肝硬化组血清D-乳酸和DAO水平最高,差异均有统计学意义(t=5.512、36.010、4.088和9.443,F=396.958、46.640,P均<0.01)。AIH肝硬化组的肠黏膜ZO-1、闭合蛋白的相对表达量均低于健康对照组(0.20±0.14比1.67±0.51,0.12±0.09比0.90±0.21),AIH无肝硬化组ZO-1的相对表达量低于健康对照组(0.99±0.37比1.67±0.51),差异均有统计学意义(t=8.641、7.407、2.295,P均<0.05)。AIH肝硬化组回肠末端组织中IL-2、干扰素γ的相对表达量均高于健康对照组(1.11±0.43比0.24±0.16和3.50±1.90比0.32±0.30),肠黏膜sIgA的相对表达量低于健康对照组(0.506±0.024比1.081±0.102),差异均有统计学意义(t=4.679、3.981、5.493,P均<0.05);AIH肝硬化组和AIH无肝硬化组IL-10的相对表达量均低于健康对照组(0.30±0.20、0.42±0.24比0.84±0.23),回肠末端组织TLR4的相对表达量均高于健康对照组(8.74±5.13、6.74±3.65比0.89±0.70),差异均有统计学意义(t=3.095、4.816、3.856、3.685,P均<0.05)。DSS+ConA组肠黏膜ZO-1和闭合蛋白的相对表达量均低于ConA组(0.14±0.08比0.98±0.13和0.09±0.02比0.98±0.16),血清ALT、AST水平和Knodell评分均高于ConA组[(5496.67±618.83)U/L比(3325.00±1030.06)U/L、(8825.00±1165.35)U/L比(5433.33±1691.14)U/L和(18.00±2.00)分比(9.33±3.01)分],差异均有统计学意义(t=13.480、13.520、4.427、4.045、-2.892,P均<0.01)。DSS+灌菌+ConA组肠黏膜ZO-1和闭合蛋白的相对表达量均高于DSS+ConA组(0.46±0.08比0.14±0.08和0.53±0.15比0.09±0.02),血清ALT、AST水平均低于DSS+ConA组[(4343.33±252.16)U/L比(5496.67±618.83)U/L和(6123.33±1086.60)U/L比(8825.00±1165.35)U/L],差异均有统计学意义(t=6.928、7.122、4.228、4.153,P均<0.01)。结论AIH患者的肠道通透性增高、肠屏障被破坏,且肝硬化患者较非肝硬化患者的程度更严重。肠屏障被破环会加重ConA诱导的免疫性肝损伤,而保护和修复肠屏障则可相对减轻ConA诱导的免疫性肝损伤。

Objective To observe and analyze the role of intestinal barrier in the pathognesis of autoimmune hepatitis(AIH),to explain the pathogenesis of AIH and to explore the intestinal based new treatment strategies.Methods A total of 14 AIH patients from January to December 2017 at Tianjin Medical University General Hospital(six patients without liver cirrhosis,and eight patients with liver cirrhosis)and 10 healthy controls were enrolled.The serum levels of D-lactic acid(D-Lac)and diamine oxidase(DAO)were detected by enzyme-linked immunosorbent assay.Real time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of connexin(zonula occluden-1(ZO-1),occludin),cytokines(interleukin(IL)-2,interferon(IFN)-γ,IL-4,IL-10)and Toll-like receptor 4(TLR4)in terminal ileal tissues of each group.The relative expression of secretory immunoglobulin A(sIgA)in the terminal ileum was determined by Western blotting.Thirty BALB/c mice were selected and divided into blank control group,dextran sulfate sodium(DSS)group,concanavalin A(ConA)group,DSS+ConA group,and DSS+bacterium+ConA group,with six mice in each group.The relative expression levels of ZO-1,occludin in mouse colonic tissues,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels,and inflammatory activity degree of liver tissues(Knodell score)of each group were measured.T-test and one-way analysis of variance were performed for statistical analysis.Results The serum D-Lac and DAO levels of AIH with liver cirrhosis group and AIH without liver cirrhosis group were both higher than those of healthy control group((1768.2±147.1)μg/L,(436.2±197.0)μg/L vs.(100.2±10.9)μg/L,and(11.5±2.5)U/L,(5.4±0.9)U/mL vs.(3.5±0.9)U/mL),and the levels of D-Lac and DAO of AIH with liver cirrhosis group were the highest;and the differences were statistically significant(t=5.512,36.010,4.088 and 9.443,F=396.958 and 46.640,all P<0.01).The relative expression levels of ZO-1 and occludin in the terminal ileal mucosa of AIH with liver cirrhosis group were lower than those of healthy control group(0.20±0.14 vs.1.67±0.51,0.12±0.09 vs.0.90±0.21),and the relative expression of ZO-1 in AIH without liver cirrhosis group was lower than that in healthy control group(0.99±0.37 vs.1.67±0.51);and the differences were statistically significant(t=8.641,7.407 and 2.295,all P<0.05).The relative expression levels of IL-2 and IFN-γin terminal ileal tissues of AIH with liver cirrhosis group were higher than those of healthy control group(1.11±0.43 vs.0.24±0.16,and 3.50±1.90 vs.0.32±0.30),however the relative expression of sIgA in terminal ileal tissues was lower than that of healthy control group(0.506±0.024 vs.1.081±0.102);and the differences were statistically significant(t=4.679,3.981 and 5.493,all P<0.05).While the relative expression levels of IL-10 in AIH with liver cirrhosis group and AIH without liver cirrhosis group were lower than that in healthy control group(0.30±0.20,0.42±0.24 vs.0.84±0.23),and the relative expression levels of TLR4 in ileum mucosa of the both groups were higher than that of healthy control group(8.74±5.13,6.74±3.65 vs.0.89±0.70);and the differences were statistically significant(t=3.095,4.816,3.856 and 3.685,all P<0.05).The relative expression levels of ZO-1 and occludin of DSS+ConA group were lower than those of ConA group(0.14±0.08 vs.0.98±0.13,and 0.09±0.02 vs.0.98±0.16),however serum ALT,AST levels and the Knodell score were all higher than those of ConA group((5496.67±618.83)U/L vs.(3325.00±1030.06)U/L,(8825.00±1165.35)U/L vs.(5433.33±1691.14)U/L,and 18.00±2.00 vs.9.33±3.01);and the differences were statistically significant(t=13.480,13.520,4.227,4.045 and-2.892,all P<0.05).The relative expression levels of ZO-1 and occludin in DSS+bacterium+ConA group were higher than those in DSS+ConA group(0.46±0.08 vs.0.14±0.08,and 0.53±0.15 vs.0.09±0.02),while serum ALT and AST levels were lower than those of DSS+ConA group((4343.33±252.16)U/L vs.(5496.67±618.83)U/L,and(6123.33±1086.60)U/L vs.(8825.00±1165.35)U/L);and the differences were statistically significant(t=6.928,7.122,4.228 and 4.153,all P<0.01).Conclusions AIH patients have increased intestinal permeability and impaired intestinal barrier which is more serious in patients with liver cirrhosis than in patients without cirrhosis.The intestinal barrier injury can aggravate ConA-induced immune-mediated liver injury.While the protection and repair of intestinal barrier can alleviate immune-mediated liver injury induced by ConA.