基于16S rRNA高通量测序技术初步探讨双歧杆菌乳杆菌三联活菌片对系统性红斑狼疮模型小鼠MRL/lpr肠道菌群的影响

Preliminary Study of the Effect of Live Combined Bifidobacterium and Lactobacillus Tablets on Intestinal Microflora in MRL/lpr Mouse Model with Systemic Lupus Erythematosus Based on 16S rRNA High Throughput Sequencing Technology

目的:基于16S rRNA高通量测序技术初步探讨双歧杆菌乳杆菌三联活菌片(金双歧)对系统性红斑狼疮模型小鼠MRL/lpr肠道菌群的影响。方法:将12只6~8周龄雌性SPF级MRL/lpr小鼠随机分为模型对照组和金双歧高、中、低剂量组,每组3只,以3只雌性6~8周龄SPF级BALB/C小鼠作为空白对照组。适应性喂养3 d后,金双歧高、中、低剂量组分别以金双歧溶液234μg、78μg、26μg灌胃,模型对照组及空白对照组每日灌胃等量生理盐水。灌胃1周后,从12只MRL/lpr小鼠和3只BALB/C小鼠的粪便中提取DNA,对核糖体RNA V3-V4区进行扩增,进行高通量测序得到原始数据,通过生物信息学的方法对数据进行质控过滤,对样品Alpha/Beta多样性进行分析以及后续差异分析。结果:Chao1、ACE、Shannon、Simpson指数的Alpha多样性分析显示,5组之间差异无统计学意义(P>0.05)。灌胃后,金双歧高、中、低剂量组Neisseria、Pseudomonas、Haemophilus的相对丰度明显低于模型对照组(P<0.05);金双歧中剂量组Actinobacteriota的相对丰度高于模型对照组、金双歧高剂量组、金双歧低剂量组(P<0.05);与空白对照组比较,金双歧高、中、低剂量组及模型对照组unidentified-Blautia的相对丰度降低(P<0.05)。结论:金双歧高、中、低剂量灌胃后,使Neisseria、Pseudomonas、Haemophilus、Lachnospira等有害菌的丰度下调。金双歧低剂量灌胃后,有益菌Fusobacteriota上调,说明金双歧能影响系统性红斑狼疮模型小鼠MRL/lpr的肠道菌群。

Objective:Based on 16S rRNA high-throughput sequencing technology,to study the effect of Live Combined Bifidobacterium and Lactobacillus Tablets(Golden Bifid)on intestinal microbiota in MRL/lpr mouse model with systemic lupus erythematosus.Methods:Twelve female SPF grade 6-8-week-old MRL/lpr mice were randomly divided into a model control group and high-dose,medium,and low-dose groups of Golden Bifid,with three mice in each group.Three female SPF grade 6-8-week-old BALB/C mice were used as the blank control group.After three days of adaptive feeding,the high-dose,medium,and lowdose groups of Golden Bifid were treated with Golden Bifid Solution(234μg,78μg,26μg)respectively.The model control group and blank control group were given an equal amount of physiological saline by gavage daily.After one week of gastric lavage,DNA was extracted from the feces of 12 MRL/lpr mice and 3 BALB/C mice.The V3-V4 region of ribosomal RNA was amplified and highthroughput sequencing was performed to obtain the raw data.The data was filtered through bioinformatics methods for quality control,and the Alpha/Beta diversity of the samples was analyzed and subsequent differential analysis was performed.Results:The Alpha diversity analysis of Chao1,ACE,Shannon,and Simpson indices showed no statistically significant differences between the five groups(P>0.05).After gastric lavage,the relative abundance of Neisseria,Pseudomonas,and Haemophilus in the high-dose,medium,and low-dose groups of Golden Bifid was significantly lower than that in the model control group(P<0.05).The relative abundance of Actinobacteriota in the medium-dose group of Golden Bifid was higher than that in the model control group,the high-dose and lowdose groups of Golden Bifid(P<0.05).Compared with the blank control group,the high,medium,and low-dose groups of Golden Bifid and the model control group showed a decrease in the relative abundance of unidentified Blautia bacteria(P<0.05).Conclusion:After gastric lavage of the high,medium,and low-dose groups of Golden Bifid,the abundance of harmful bacteria such as Neisseria,Pseudomonas,Haemophilus,and Lachnospira was downregulated.After gastric lavage of the low-dose group of Golden Bifid,the beneficial bacterium Fusobacteriota was upregulated,indicating that Golden Bifid can affect the intestinal microbiota of MRL/lpr mouse model with systemic lupus erythematosus.