乳杆菌对幼年小鼠肠道菌群紊乱的缓解和恢复作用

Alleviation and recovery effects of Lactobacillus on the antibiotic-induced intestinal dysbiosis in early life stages of mice

目的探索乳杆菌对头孢曲松诱导的小鼠幼年期肠道菌群紊乱的缓解和恢复作用,以及对机体免疫的可能影响。方法48只4周龄Balb/c雄性小鼠按体重随机分为空白对照组、抗生素组、泡菜直投菌组(PC)和鼠李糖乳杆菌组(LGG),每组12只。实验共计4周,第1周对照组、抗生素组、PC组和LGG组分别灌胃生理盐水、头孢曲松(40 mg)、头孢曲松和PC菌悬液(109CFU)、头孢曲松和LGG菌悬液(109 CFU),之后3周对照组和抗生素组不予灌胃,PC组和LGG组继续灌胃菌悬液。每3天测一次体重。每周收集一次粪便,二代测序分析粪便细菌的群落多样性和结构变化。实验第4周处死小鼠,采用实时荧光定量PCR法测定脾脏中白细胞介素-6(interleukin-6,IL-6)、IL-12和肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)的mRNA表达水平。结果经抗生素灌胃的3组小鼠体重均低于空白对照组(P<0.05),PC组在第18 d首先恢复至正常水平,第21 d时抗生素组和LGG组也得到恢复。菌群多样性分析发现,投用抗生素1周后抗生素组simpson指数显著降低(P<0.001),PC组和LGG组未见降低;第4周时PC组observed species指数显著升高(P<0.001),抗生素组和LGG组simpson指数显著升高(P<0.01)。菌群结构分析发现,第1周空白对照组和其他3组群落结构出现差异(P<0.05),PC组和LGG组间未见差异,抗生素组、PC组和LGG组厚壁菌门/拟杆菌门(F/B)比值升高,抗生素组阿克曼菌丰度下降程度大于PC组和LGG组;第4周时4组群落结构互不相同(P<0.05),各组F/B值和阿克曼菌丰度均有所恢复,抗生素组出现瘤胃菌科、拟杆菌科、坦纳菌科等特征性物种,PC组以乳杆菌属为主,LGG组特征性物种为阿克曼菌。第4周时PC组和LGG组脾脏IL-12、TNF-αmRNA表达量均低于抗生素组(P<0.01),LGG组IL-6表达量低于空白对照组(P<0.01)。结论幼年期抗生素暴露可抑制体重增长、降低肠道菌群多样性并抑制功能性菌种的生长,健康机体的菌群结构具有一定的自我恢复能力,但机体仍长期处于炎症状态。乳杆菌可缓解幼年期抗生素暴露引起的菌群紊乱,稳定机体免疫水平,但尚未发现治疗性效果。

OBJECTIVE To investigate the effects of Lactobacillus on the alleviation and recovery of ceftriaxone-induced intestinal dysbiosis in early life stages of mice,and the possible effect on immunity of the host.METHODS A total of 48 fourweek-old Balb/c male mice were randomly divided into four groups,with 12 in each group.The experiment lasted for four weeks.In the first week,mice were given normal saline,ceftriaxone,ceftriaxone plus Lactobacillus rhamnosus GG,ceftriaxone plus direct vat set pickle(PC)by gavage respectively in the control group(ctrl),ceftriaxone group(ceftri),PC group and LGG group.The PC group and LGG group were given PC and LGG respectively for the last three weeks.The feces were collected once a week,and the profile composition of fecal bacteria were analyzed by next-generation sequencing.The mRNA expression levels of interleukin-6(IL-6),IL-12 and tumor necrosis factorα(TNF-α)in the spleen were analysed using quantitative real-time PCR.RESULTS In the first week,the simpson index decreased significantly in the ceftri group(P<0.001),but not in PC and LGG groups.At week four,the observed-species index in the PC group increased significantly(P<0.001)and the simpson index increased in both LGG group and ceftri group(P<0.01).As for the flora structure,in the first week,the ctrl group differed from the other three groups significantly(P<0.05)while no differences were found between the PC group and the LGG group.The firmicutes/bacteroides(F/B)ratio of three ceftriaxone-treated groups increased while the abundance of Akkermansia decreased,particularly in the ceftri group.In the fourth week,the intestinal flora structure were different between four groups(P<0.05)and the F/B ratio and Akkermansia abundance recovered.Ruminococcaceae,Bacteroidaceae and Tannerellacea appeared in the ceftri group,while Lactobacillus was dominant in the PC group,and Akkermansia was the characteristic species of the LGG group.Weight lost was found in ceftriaxone-treated groups(P<0.05)and the PC group recovered first.IL-12 and TNF-αmRNA expression of the spleen were lower in PC and LGG groups compared with the ceftri group(P<0.01).IL-6 mRNA expression level in the LGG group were lower than the ctrl group(P<0.01).CONCLUSION The administration of antibiotics in the early life stage might inhibit weight gain,reduce the diversity of intestinal flora,damage functional bacteria and cause long-term inflammation,even though self-recovery ability exist.Lactobacillus might be helpful to alleviate the damage of antibiotics.But no therapeutic effect has been found.