急性肝内胆汁淤积大鼠小肠上皮紧密连接蛋白ZO-1和Occludin表达的变化

目的 研究急性肝内胆汁淤积大鼠小肠上皮细胞紧密连接蛋白ZO-1和Occludin表达的变化及其发病机制.方法　雄性21日龄SD大鼠40只随机分为空白对照组(10只)和模型组(30只).模型组大鼠采用α-异硫氰酸萘酯50 mg·kg-1一次性灌胃,建立急性肝内胆汁淤积动物模型.空白对照组只灌服等量蓖麻油.于灌胃后24 h、48 h、72 h采用免疫组织化学和Western blot法检测其末端回肠黏膜紧密连接蛋白ZO-1和Occludin的分布和表达,并利用检测分析系统对Western blot图像结果进行定量分析.结果　ZO-1蛋白和Occludin蛋白主要沿大鼠小肠黏膜上皮细胞膜的顶端呈线状分布.模型组大鼠24 h时ZO-1蛋白和Occludin蛋白的阳性染色较空白对照组减少,48 h减少最为明显,72 h阳性染色有所恢复.Western blot结果与免疫组织化学结果一致,模型组24 h已开始下降(ZO-1:0.129 4±0.048 1,Occludin:0.195 0±0.044 1),48 h最低(ZO-1:0.039 5±0.009 5,Occludin:0.013 7±0.009 2),72 h开始恢复(ZO-1:0.202 4±0.049 8,Occludin:0.149 4±0.035 5),模型组各时间点ZO-1与Dccludin与空白对照组(ZO-1:0.288 7±0.023 7,Occludin:0.426 6±0.067 0)比较差异均有统计学意义(Pa＜0.01).结论　急性肝内胆汁淤积大鼠小肠黏膜上皮存在紧密连接蛋白分布异常及数量改变,影响肠黏膜上皮屏障的完整性,破坏小肠黏膜屏障.

Changes of Expressions of Intestinal Mucosal Tight Junction Proteins ZO-1 and Occludin in Rats with Acute Intrahepatic Cholestasis

Objective To study the changes of expressions of intestinal mucosal tight junction proteins ZO-1 and Occludin in rats with acute intrahepatic cholestasis,and explore its mechanisms.Methods Forty SD rats aged 21 days were randomly divided into control group(n=10) and model group(n=30).A single dose(50 mg.kg-1) of α-naphthylisothiocyanate was administered by gavage to each experimental rats to induce acute intrahepatic cholestasis in model group.Rats in control group only administered with caster oil by gavage.At 24 hours,48 hours and 72 hours after gavage,immunohistochemistry and Western blot techniques were used to examine the distribution and expression of tight junction proteins(ZO-1,Occludin),and the results of Western blot were quantitatively analyzed by image analytical system.Results ZO-1 and Occludin were localized along the apical region of the lateral plasma membrane representing the region of tight junctions in surface and crypt epithelial cells.Compared with control group,the positive stainings of ZO-1 and Occludin in model group at 24 hours after gavage were decreased,the decrease was most obviously at 48 hours,and partly recovered at 72 hours.Western blot demonstrated consistent and significant reduction with immunohistochemistry.The expressions of ZO-1 and Occludin in model group were 0.129 4±0.048 1 and 0.195 0±0.044 1 at 24 hours,0.039 5±0.009 5 and 0.013 7±0.009 2 at 48 hours,0.202 4±0.049 8 and 0.149 4±0.035 5 at 72 hours,which were significantly lower than those in control group(ZO-1:0.288 7±0.023 7,Occludin:0.426 6±0.067 0)(Pa<0.01).Conclusions Acute intrahepatic cholestasis induces the abnormal distribution of tight junction proteins and the alteration of their quantity,which affects the intestinal barrier integrity,resulting in impaired barrier function.