双歧杆菌G9-1保护轮状病毒感染新生大鼠肠道稳态的机制

Mechanisms of Bifidobacterium G9-1 in protecting intestinal homeostasis of neonatal rats with rotavirus infection

目的研究双歧杆菌G9-1保护轮状病毒(RV)感染新生大鼠肠道通透性及肠道稳态的机制。方法将27只新生SD大鼠随机以1∶1∶1比例分为对照组(配方奶灌胃)、RV感染组(配方奶+0.6 ml RV培养液灌胃)和治疗组(配方奶+0.6 ml RV培养液灌胃,次日以双歧杆菌G9-1溶液灌胃),苏木精伊红(HE)染色评估三组新生大鼠肠道组织病理变化,异硫氰酸荧光素-葡聚糖(FITC-Dextran)示踪法检测肠道通透性,酶联免疫吸附法检测血清D-乳酸(DLA)、二胺氧化酶(DAO)水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测肠组织紧密连接相关蛋白-1(ZO-1)、紧密连接蛋白闭锁蛋白(Occludin)、闭合蛋白-1(Claudin-1)mRNA表达,蛋白免疫印迹电泳(Western blot)检测ZO-1、Occludin、Claudin-1蛋白表达,并分析其肠道菌群变化。结果治疗组新生大鼠肠组织病理学评分、FITC-Dextran含量、血清DLA、DAO水平低于RV感染组(P<0.05),治疗组肠组织紧密连接蛋白ZO-1、Occludin、Claudin-1 mRNA和蛋白表达高于RV感染组(P<0.05);治疗组双歧杆菌数量高于RV感染组(P<0.05),乳酸杆菌、大肠埃希菌数量与RV感染组比较,无统计学差异。结论双歧杆菌G9-1可有效降低RV感染新生大鼠肠道通透性,修复肠道屏障功能,改变肠道菌群结构,维持肠道稳态平衡。

OBJECTIVE To study the mechanisms of Bifidobacterium G9-1 in protecting the intestinal permeability and intestinal homeostasis of neonatal rats with rotavirus(RV) infection. METHODS Totally 27 neonatal SD rates were randomly and evenly divided into the control group(formula milk gavage), the RV infection group(formula milk gavage, 0.6 ml RV culture solution gavage) and the treatment group(formula milk gavage, 0.6 ml RV culture solution gavage, Bifidobacterium G9-1 solution gavage on the next day). The pathological changes of intestinal tissues of the three groups of neonatal rats were evaluated by HE staining, the intestinal permeability was detected by means of fluorescein isothiocyanate-dextran(FITC-Dextran) tracer method, the levels of serum D-lactic acid(DLA) and diamine oxidase(DAO) were detected by using enzyme-linked immunosorbent assay, the expression levels of zonula occluden-1(ZO-1), tight junction protein occludin(Occludin) and occludin-1(Claudin-1) mRNA were detected with the use of real-time fluorescent quantitative polymerase chain reaction(real-time PCR), the expression levels of ZO-1, occludin and claudin-1 proteins were detected by means of Western blot, and the changes of intestinal flora were observed. RESULTS The pathological score of intestinal tissues and expression levels of FITC-Dextran, serum DLA and DAO of the treatment group were significantly lower than those of the RV infection group(P<0.05). The expression levels of ZO-1, occludin and claudin-1 mRNA and proteins in intestinal tissues were significantly higher in the treatment group than in the RV infection group(P<0.05). The Bifidobacterium counts of the treatment group were significantly more than those of the RV infection group(P<0.05);there were no significant differences in the Lactobacillus counts and Escherichia coli counts between the treatment group and the RV infection group. CONCLUSION Bifidobacterium G9-1 can effectively reduce the intestinal permeability of the neonatal rats with RV infection, repair the intestinal barrier function, change the structure of intestinal flora and maintainthe intestinal homeostasis.