益生菌对胆汁淤积性黄疸大鼠小肠上皮细胞紧密连接蛋白的影响

目的探讨双歧三联活菌(培菲康)对胆汁淤积性大鼠小肠上皮细胞紧密连接蛋白ZO-1(zonula oc-cludens-1)和Occludin表达的调控机制。方法雄性3周龄SD大鼠随机分为对照组、模型组和培菲康组,模型组和培菲康组均给予α-异硫氰酸萘酯(ANIT)50 mg/kg一次性灌胃,建立急性肝内胆汁淤积动物模型,培菲康组于造模前4 d开始给予培菲康4.2×107个活菌数/(kg.d)灌胃大鼠。分别于造模后24、48和72 h三个时间点处死大鼠,取末端回肠黏膜组织,采用免疫组化和Western blots免疫印迹法检测紧密连接蛋白ZO-1、闭锁蛋白(Occludin)的分布和表达,并利用图像分析系统对Western blots图像结果进行定量分析。结果ZO-1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和Occludin的阳性染色较对照组减少,48 h减少最为明显,72 h阳性染色有所恢复,而培菲康组大鼠各时间点ZO-1和Occludin的阳性染色和模型组相比均明显增多。Western blots结果与免疫组织化学结果相一致,模型组24 h已经开始下降(ZO-1 0.1294±0.0481)、(Oc-cludin 0.1950±0.0441),48 h达到最低(ZO-1 0.0395±0.0095)、(Occludin 0.0137±0.0092),72 h开始恢复(ZO-10.2024±0.0498)、(Occludin 0.1494±0.0355),各时间点与对照组(ZO-1 0.2887±0.0237)、(Occludin 0.4266±0.0670)相比差异有统计学意义(P0.01);而培菲康组各时间点蛋白表达分别为24 h(ZO-1 0.2110±0.0367)、(Occludin 0.3056±0.0572),48 h(ZO-1 0.1173±0.0423)、(Occludin 0.0521±0.0123),72 h(ZO-1 0.2601±0.0191)、(Occludin 0.2050±0.0721),与模型组相应时间点数据相比差异有统计学意义(P0.05)。结论双歧三联活菌能够影响胆汁淤积性大鼠小肠黏膜上皮紧密连接蛋白的分布和表达,可以恢复肠黏膜上皮屏障的完整性。

Effects of probiotics on the intestinal epithelium tight junction proteins in rats with experimental acute intrahepatic cholestasis

Objective To study the effects of live combination bifidobacterium,lactobacillus and enterococcus（BIFICO） on intestinal mucosal epithelium tight junction protein ZO-1 and Occludin in rats with cholestasis.Method Male SD rats of 3 weeks old were randomly divided into control group,model group and BIFICO group.Before making the animal model,BIFICO4.2×10^7 viable bacterium counts/（kg·d）] were administered to the rats in BIFICO group for 4 days. The model and control groups were fed by normal saline.At the 5th day after administration,after fasting for 12 h,the model group and BIFICO group were intragastrically administered ANIT（50 mg/kg） for modeling.At 24 h,48 h and 72 h after modeling,every 10 rats in each group were executed for taking terminal ileum tissue.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 analysed by image analytical system.Result 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.The positive stainings of ZO-1 and Occludin of model group at 24 h after modeling,were decreased than those of control group;the decrease was most obvious at 48 h,and partly recovered at 72 h.Western blot demonstrated significant reduction consistent with immunohistochemistry.The protein expressions in model group were（ZO-1 0.1294±0.0481）,（Occludin 0.1950±0.0441）at 24 h,（ZO-1 0.0395±0.0095）,（Occludin 0.0137±0.0092）at 48 h,（ZO-1 0.2024±0.0498） and（Occludin 0.1494±0.0355）at 72 h respectively,which were significantly lower than those in control group（ZO-1 0.2887±0.0237）,（Occludin 0.4266±0.0670）（P〈0.01）;The protein expressions in BIFICO group were（ZO-1 0.2110±0.0367）,（Occludin 0.3056±0.0572）at 24 h,（ZO-1 0.1173±0.0423）,（Occludin 0.0521±0.0123）at 48 h,（ZO-1 0.2601±0.0191）,（Occludin 0.2050±0.0721）at 72 h,which have statistical difference compared to those in model group at both time point（P〈0.05）.Conclusion Probiotics supplement can be effective in acute cholestasis by improving the intestinal barrier function.