基于16S rDNA测序的脓毒症大鼠早期肠道微生态变化分析
[Analysis of the changes in intestinal microecology in the early stage of sepsis rat based on 16S rDNA sequencing].
作者信息
Li Hongyi, Zhai Ruiqing, Liang Huoyan, Zhu Yanhui, Yan Yan, Wang Chengyang, Ding Xianfei, Song Gaofei, Sun Tongwen
机构信息
Department of General Intensive Care Unit, the First Affiliated Hospital of Zhengzhou University, Henan Provincial Key Laboratory of Critical Medicine, Henan Provincial Engineering Research Center for Critical Care Medicine, Zhengzhou Key Laboratory of Sepsis, Zhengzhou 450052, Henan, China.
College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150081, Heilongjiang, China. Corresponding author: Sun Tongwen, Email:
出版信息
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022 Jan;34(1):28-34. doi: 10.3760/cma.j.cn121430-20201215-00754.
OBJECTIVE
To investigate the changes of intestinal microecology in the early stage of sepsis rat model by 16S rDNA sequencing.
METHODS
Sixty male Sprague-Dawley (SD) rats were randomly divided into cecal ligation and puncture (CLP) group and sham operation group (Sham group), with 30 rats in each group. In the CLP group, sepsis rat model was reproduced by CLP method; the rats in the Sham group only underwent laparotomy without CLP. At 24 hours after the operation, the intestinal feces and serum samples of 8 rats in each group were collected. The survival rate of the rest rats was observed until the 7th day. The level of serum tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA). Intestinal feces were sequenced by 16S rDNA sequencing technology. The operational taxonomic unit (OTU) data obtained after sequence comparison and clustering was used for α diversity and β diversity analysis, principal coordinate analysis and linear discriminant analysis effect size analysis (LEfSe) to observe the changes of intestinal microecology in early sepsis rats and excavate the marker flora.
RESULTS
At 24 hours after the reproduction of the model, the rats in the CLP group showed shortness of breath, scattered hair and other manifestations, and the level of serum TNF-α increased significantly as compared with that in the Sham group (ng/L: 43.95±9.05 vs. 11.08±3.27, P < 0.01). On the 7th day after modeling, the cumulative survival rate of the Sham group was 100%, while that of the CLP group was 31.82%. Diversity analysis showed that there was no significant difference in α diversity parameter between the Sham group and the CLP group (number of species: 520.00±52.15 vs. 492.25±86.61, Chao1 richness estimator: 707.25±65.69 vs. 668.93±96.50, Shannon index: 5.74±0.42 vs. 5.79±0.91, Simpson index: 0.93±0.03 vs. 0.94±0.05, all P > 0.05). However, the β diversity analysis showed that the difference between groups was greater than that within groups whether weighted according to OTU or not (abundance weighted matrix: R = 0.23, P = 0.04; abundance unweighted matrix: R = 0.32, P = 0.01). At the phylum level, the abundance of Proteobacteria and Candidatus_sacchari in the CLP group increased significantly as compared with the Sham group [18.100% (15.271%, 26.665%) vs. 6.974% (2.854%, 9.764%), 0.125% (0.027%, 0.159%)% vs. 0.018% (0.008%, 0.021%), both P < 0.05]. At the genus level, the abundance of opportunistic pathogen including Helicobacter, Ruthenium, Streptococcus, Clostridium XVIII in the CLP group was significantly higher than that in the Sham group [5.090% (1.812%, 6.598%) vs. 0.083% (0.034%, 0.198%), 0.244% (0.116%, 0.330%) vs. 0.016% (0.008%, 0.029%), 0.006% (0.003%, 0.010%) vs. 0.001% (0%, 0.003%), 0.094% (0.035%, 0.430%) vs. 0.007% (0.003%, 0.030%), all P < 0.05], and the abundance of probiotics such as Alloprevotella and Romboustia was significantly lower than that in the Sham group [7.345% (3.662%, 11.546%) vs. 22.504% (14.403%, 26.928%), 0.113% (0.047%, 0.196%) vs. 1.229% (0.809%, 2.29%), both P < 0.01]. LEfSe analysis showed that the probiotics belonging to Firmicutes were significantly enriched in the Sham group, and Romboustia was the most significantly enriched species. Opportunistic pathogens such as Helicobacter, Streptococcus and Clostridium XVIII were significantly enriched in the CLP group, Helicobacter_NGSU_ 2015 was the most significantly enriched species.
CONCLUSIONS
In the early stage of sepsis, the intestinal microbiota structure of rats is significantly changed, which mainly shows that the abundance of Alloprevotella and other probiotics is significantly reduced, while that of Helicobacter and other opportunistic pathogens is significantly increased.
目的
通过16S rDNA测序研究脓毒症大鼠模型早期肠道微生态的变化。
方法
将60只雄性Sprague-Dawley（SD）大鼠随机分为盲肠结扎穿刺（CLP）组和假手术组（Sham组），每组30只。CLP组采用CLP法复制脓毒症大鼠模型；Sham组大鼠仅行剖腹术，不行CLP。术后24小时，收集每组8只大鼠的肠道粪便和血清样本。观察其余大鼠至第7天的生存率。采用酶联免疫吸附测定（ELISA）法检测血清肿瘤坏死因子-α（TNF-α）水平。采用16S rDNA测序技术对肠道粪便进行测序。将序列比对和聚类后得到的操作分类单元（OTU）数据用于α多样性和β多样性分析、主坐标分析和线性判别分析效应大小分析（LEfSe），以观察脓毒症早期大鼠肠道微生态的变化并挖掘标志性菌群。
结果
模型复制后24小时，CLP组大鼠出现呼吸急促、毛发稀疏等表现，血清TNF-α水平较Sham组显著升高（ng/L：43.95±9.05 vs. 11.08±3.27，P < 0.01）。建模后第7天，Sham组的累积生存率为100%，而CLP组为31.82%。多样性分析显示，Sham组和CLP组之间的α多样性参数无显著差异（物种数：520.00±52.15 vs. 492.25±86.61，Chao1丰富度估计值：707.25±65.69 vs. 668.93±96.50，香农指数：5.74±0.42 vs. 5.79±0.91，辛普森指数：0.93±0.03 vs. 0.94±0.05，均P > 0.05）。然而，β多样性分析显示，无论是否根据OTU加权，组间差异均大于组内差异（丰度加权矩阵：R = 0.23，P = 0.04；丰度未加权矩阵：R = 0.32，P = 0.01）。在门水平上，CLP组中变形菌门和暂定糖杆菌属的丰度较Sham组显著增加[18.100%（15.271%，26.665%）vs. 6.974%（2.854%，9.764%），0.125%（0.027%，0.159%）% vs. 0.018%（0.008%，0.021%），均P < 0.05]。在属水平上，CLP组中包括幽门螺杆菌、钌菌属、链球菌属、 XVIII梭菌属等机会致病菌的丰度显著高于Sham组[5.090%（1.812%，6. .598%）vs. 0.083%（0.034%，0.198%），0.244%（0.116%，0.330%）vs. 0.016%（0.008%，0.029%），0.006%（0.003%，0.010%）vs. 0.001%（0%，0.003%），0.094%（0.035%，0.430%）vs. 0.007%（0.003%，0.030%），均P < 0.05]，而别普雷沃菌属和罗姆布斯菌属等益生菌的丰度显著低于Sham组[7.345%（3.662%，11.546%）vs. 22.504%（14.403%，26.928%），0.113%（0.047%，0.196%）vs. 1.229%（0.809%，2.29%），均P < 0.01]。LEfSe分析显示，厚壁菌门中的益生菌在Sham组中显著富集，罗姆布斯菌属是最显著富集的物种。幽门螺杆菌、链球菌属和 XVIII梭菌属等机会致病菌在CLP组中显著富集，幽门螺杆菌_NGSU_2015是最显著富集的物种。
结论
在脓毒症早期，大鼠肠道微生物群结构发生显著变化，主要表现为别普雷沃菌属等益生菌的丰度显著降低，而幽门螺杆菌等机会致病菌的丰度显著增加。
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