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. Conclusion: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.

Objective:To observe the effect of metformin on intestinal metabolites and its protective effect on lung injury in an elderly sepsis rat.Methods:SD rats were fed at the Animal Laboratory Center of Zhengzhou University, fourteen elderly SD rats were randomly divided into three groups: sham surgery (age-Sham, AgS group, n=4), cecal ligation and perforation induced sepsis (age-Cecal ligation and puncture, AgCLP group, n=5), and oral administration of metformin (100 mg/kg) after 1 h of CLP treatment (age-Metformin, AgMET group, n=5). Collected rat feces 24 h after modeling, and analyzed the composition and inter group differences of metabolites in the feces using liquid chromatography tandem mass spectrometry non targeted metabolomics. Collected rat lung tissues and detected the expression levels of inflammation related genes and pathological changes in the tissue. The visualization of metabolic changes between groups were presented using orthogonal partial least squares discriminant analysis, heatmaps, and unsupervised principal component analysis, respectively. MetaboAnalyst 3.0 was used to evaluate the Pathway analysis of metabolites, and this software was based on the KEGG database and the human metabolome database. Results:The expressions of CCL4 ( F=203.00, P<0.001), CXCL1( F=65.69, P<0.001), IL-6 ( F=38.94, P<0.002), TNF-α ( F=14.85, P=0.005) between two groups of rats were significantly different (all P<0.05). However, there was no significant difference in CCL2 expression between AgCLP group and AgMET group. Furthermore, compared with the AgS group, the relative intensities of 17 metabolites such as 7-methylxanthine, N-Arachidonylglycine and Manolide in AgCLP group were significantly increased, whereas the 9 metabolites such as Phenazone, Gly-Phe and Valyproline were significantly decreased, and metformin treatment could reverse these changes of the above metabolites. Correlation analysis showed that the IL-6 and TNF-α levels were positively correlated with the relative strength of 7-Methylxanthine, N-Arachidonylglycine and other metabolites, but negatively correlated with the Phenazone and Gly-Phe. CCL4 and CXCL1 were positively correlated with Manolide, but negatively correlated with Valyproline. Conclusion:The results of this study showed that metformin improved sepsis induced acute lung injury and regulates the host intestinal metabolites, which might provide a potential and effective treatment for elderly sepsis induced acute lung injury.