抗生素诱导的菌群紊乱对幼鼠结肠黏膜屏障及免疫反应的影响

摘要

Objective To investigate the effect of antibiotic-induced microbiota dysbiosis on colonic barrier and immune response in juvenile mice.Methods Twenty Balb/c mice were randomly divided into experimental group and control group.Broad spectrum antibiotic mixture were administered by means of gavage to the experimental group every 12 hours for 14 days to induce intestinal microbiota dysbiosis.The mice of control group were given an equal amount of physiological saline.On the 15th day,all mice were intraperitoneally injected with lipopolysaccharide(LPS) to induce intestinal inflammatory reaction.The structure of intestinal flora was analyzed by way of 16SrRNA sequencing,and the morphology of colonic mucosa was observed by means of HE staining.The infiltration of colonic mucosa was observed through toluidine blue staining and immunohistochemistry.The levels of inflammatory cytokines in colon tissue were measured by real-time PCR and intestinal permeability-related parameters were measured by enzyme-linked immunosorbent assay.Results The intestinal microbial composition of the experimental group was significantly changed,and the expression levels of inflammatory cytokines interleukin (IL)-1β,IL-6,IL-8,IL-10,tumor necrosis factor (TNF)-α mRNA in the antibiotic group (0.765 ± 0.062,0.082 ± 0.040,0.442 ± 0.059,0.469 ±0.079,0.736 ± 0.063) were all lower than those in the control group (1.738 ± 0.243,1.090 ± 0.104,1.151 ±0.136,1.066 ± 0.102,1.539 ± 0.218),and the differences were statistically significant (all P ＜ 0.05).The expressions of intestinal barrier related gene of mice ZO-1 and Occludin decreased (0.639 ± 0.071 vs.1.347 ± 0.224,0.770 ±0.067 vs.1.487 ± 0.148) but the level of fecal albumin increased [(6.419 ± 0.552) mg/L vs.(6.079 ± 0.011) mg/L] after antibiotic exposure,and the differences were statistically significant (all P ＜ 0.05).Conclusions Antibiotic exposure leads to changes of enteric microbiota,which adversely affects local mucosal immunity and intestinal barrier function of colon in juvenile mice.%目的 探讨抗生素诱导的菌群紊乱对幼年小鼠结肠黏膜屏障及免疫反应的影响.方法 20只Balb/c小鼠随机分为实验组和对照组,实验组通过灌胃给予抗生素混合物12 h/次,连续14 d,诱导肠道菌群紊乱,对照组予等量9 g/L盐水.第15天经腹腔注射脂多糖(LPS)诱导炎性反应,通过16SrRNA测序分析肠道菌群结构,HE染色观察结肠黏膜形态结构,甲苯胺蓝染色和免疫组织化学观察结肠黏膜免疫细胞浸润,实时荧光定量PCR(RT-PCR)检测炎性因子及紧密连接蛋白的表达情况,酶联免疫吸附试验检测小鼠粪便清蛋白水平.结果 抗生素暴露后小鼠肠道微生物构成明显改变,炎性因子白细胞介素(IL)-1β、IL-6、IL-8、IL-10、肿瘤坏死因子(TNF)-α mRNA表达水平分别为0.765±0.062、0.082±0.040、0.442±0.059、0.469±0.079、0.736±0.063,均明显低于对照组(1.738±0.243、1.090±0.104、1.151 ±0.136、1.066 ±0.102、1.539±0.218),差异均有统计学意义(均P ＜0.05).抗生素暴露后小鼠肠屏障相关基因ZO-1和Occludin mRNA表达水平分别为0.639 ±0.071、0.770±0.067,均明显低于对照组(1.347±0.224、1.487 ±0.148),而粪便清蛋白水平高于对照组[(6.419 ±0.552) mg/L比(6.079 ±0.011) mg/L],差异均有统计学意义(均P＜0.05).结论 抗生素暴露导致肠道微生物群构成改变,对幼年小鼠结肠局部免疫及肠屏障功能的建立产生不利影响.