基于16S rRNA基因测序研究拉克替醇对小鼠肠道菌群的影响

Effects of lactitol on the intestinal microbiota in mice by 16S rRNA sequencing

目的探讨益生菌、不同浓度拉克替醇及两者合用对小鼠肠道菌群的影响。方法将50只C57BL/6J小鼠分为空白对照组、益生菌组、标准浓度拉克替醇组、高浓度拉克替醇组、益生菌联合拉克替醇组，每组10只。分别予不干预、1×109 CFU/d益生菌、6.6 g·kg－1·d－1拉克替醇、10.0 g·kg－1·d－1拉克替醇、5×108 CFU/d益生菌联合3.3 g·kg－1·d－1拉克替醇灌胃2周。分别收集灌胃前、灌胃1周、灌胃2周后的粪便标本，以及灌胃2周后的肠黏膜组织，送检16S rRNA基因测序。采用α多样性分析、主成分分析和分类学分析菌群变化。结果灌胃前和灌胃1周后，α多样性分析显示5组小鼠粪便菌群差异均无统计学意义（P＝0.552、0.062）；α多样性分析与主成分分析显示，标准浓度拉克替醇和高浓度拉克替醇组肠黏膜菌群差异均无统计学意义（P＝0.270、0.085）。灌胃1周和2周后，标准浓度和高浓度拉克替醇组分别与空白对照组、益生菌组比较，粪便标本Akkermansia菌属均增加（灌胃1周0.114 3比0.003 9、0.013 1，0.071 3比0.003 9、0.013 1，P〈0.01、P＝0.001和P＝0.001、0.005；灌胃2周0.094 0比0.030 5、0.018 9，0.142 4比0.030 5、0.018 9，P＝0.044、0.016和0.001、〈0.01）；益生菌联合拉克替醇组分别与标准浓度和高浓度拉克替醇组比较，粪便标本拟杆菌属均增加（灌胃1周0.115 9比0.037 5、0.041 6，P＝0.013、0.015；灌胃2周0.058 0比0.023 2、0.014 4，P＝0.047、0.009）。益生菌联合拉克替醇组较早（灌胃1周后）出现毛螺旋菌科相应菌属的增加，灌胃2周后，与空白对照组、标准浓度拉克替醇组和高浓度拉克替醇组相比，益生菌组毛螺旋菌科相应菌属增加（P均〈0.05）。标准浓度拉克替醇组与益生菌组比较，肠黏膜Akkermansia菌属增加（0.018 0比0.001 8，P＝0.012），高浓度拉克替醇组分别与空白对照组、益生菌组相比，肠黏膜Akkermansia菌属亦增加（0.037 0比0.010 0、0.001 8，P＝0.002、〈0.01）。益生菌联合拉克替醇组与空白对照组、标准浓度拉克替醇组、高浓度拉克替醇组相比，以及益生菌组与高浓度拉克替醇组相比，肠黏膜脱铁杆菌科Mucispirillum属均增加（0.040 0比0.014 8、0.013 7、0.009 9，0.019 6比0.009 9；P＝0.041、0.040、0.018、0.011）。结论补充益生菌、拉克替醇或两者联用对小鼠肠道菌群均有明显调节作用，外源性益生菌不易在肠道定植。拉克替醇可明显促进粪便和肠道黏膜中Akkermansia菌属增殖。

ObjectiveTo investigate the effects of probiotics, lactitol of different concentrations, and their combination on intestinal microbiota in mice. MethodsFifty C57BL/6J mice were divided into blank control group, probiotics group, lactitol of standard concentration group, lactitol of high concentration group, and combination of probiotics and lactitol group, 10 mice in each group, with no intervention, gavaged with 1×109 colony-forming units（CFU）/d probiotics, with lactitol of standard concentration （6.6 g·kg-1·d-1）, with lactitol of high concentration （10.0 g·kg-1·d-1）, with probiotics （5×108 CFU/d） and lactitol （3.3 g·kg-1·d-1） for two weeks, respectively. The feces before gavage and one week and two weeks after gavage were collected. And intestinal mucosa samples were also collected at two weeks after gavage for 16S rRNA sequencing. Alpha diversity analysis, principal component analysis （PCA） and taxonomy were used for analysis of the changes of microbiota. ResultsThe results of alpha diversity analysis showed there was no statistically significant difference in feces between before gavage and at one week after gavage （P=0.552, 0.062）. The results of alpha diversity analysis and PCA indicated that there was no statistically significant difference in intestinal microbiota between lactitol of standard concentration group and lactitol of high concentration group （P=0.270 and 0.085）. One week and two weeks after gavage, compared lactitol of standard concentration group and lactitol of high concentration group with blank control group and probiotics group, Akkermansia in feces both increased （one week after gavage, 0.114 3 vs. 0.003 9 and 0.013 1, 0.071 3 vs. 0.003 9 and 0.013 1; P〈0.01, P=0.001 and P=0.001, 0.005; two weeks after gavage, which was 0.094 0 vs. 0.030 5 and 0.018 9, 0.142 4 vs. 0.030 5 and 0.018 9; P=0.044, 0.016 and 0.001, 〈0.01）. Compared combination of probiotics and lactitol group with lactitol of standard concentration group and high concentration group, Bacteroides in feces increased （one week after gavage, 0.115 9 vs. 0.037 5 and 0.041 6, P=0.013 and 0.015; two weeks after gavage, 0.058 0 vs. 0.023 2 and 0.014 4, P=0.047 and 0.009）. The increase of Lachnospiraceae appeared earlier in combination of probiotics and lactitol group （at one week after gavage）. Two weeks after gavage, compared with that of blank control group, lactitol of standard concentration group and high concentration group, Lachnospiraceae in feces of probiotics group increased （all P〈0.05）. Compared with that of probiotics group, Akkermansia of mucosa in lactitol of standard concentration group increased （0.018 0 vs. 0.001 8, P=0.012）. Akkermansia of mucosa in lactitol of high concentration group also increased compared with that of blank control group and probiotics group （0.037 0 vs. 0.010 0 and 0.001 8, P=0.002, 〈0.01）. Comparing combination of probiotics and lactitol group with blank control group, lactitol of standard concentration group and lactitol of high concentration group, and comparing probiotics group with lactitol of high concentration group, Mucispirillum in mucosa all increased （0.040 0 vs. 0.014 8, 0.013 7 and 0.009 9, 0.019 6 vs. 0.009 9; P=0.041, 0.040, 0.018 and 0.011）. ConclusionsSupplementary probiotics, lactitol and combination of them all have obvious regulative role in mucosal flora of mice. Exogenous probiotics can not easily colonized in the intestine. Lactitol can obviously promote the proliferation of Akkermansia in feces and intestine.