ABSTRACT
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.
ABSTRACT
Objective:To investigate the changes of inflammatory factors, serum amylase and lipase and pancreatic damage at different time points after establishment of cecal ligation and perforation in a rat model of sepsis, which provides a stable and reliable animal model for the study of pancreatic injury following sepsis in children.Methods:Sixty male SD rats were randomly divided into 6 h group, 12 h group, 24 h group and 48 h group in control group, sham group and cecal ligation and puncture (CLP) group.Rat model of sepsis was established by cecal ligation and perforation in CLP group, the sham group was treated with sham operation, and rats in control group were not treated.The levels of interleukin (IL)-1β, IL-10, amylase and lipase in each group were detected, and pancreatic tissue was taken for HE staining.Results:In this model, the 48 h survival rate of rats in the CLP group was 70%.With the prolongation of postoperative time in the CLP group, pancreatic tissue congestion, cell edema, and inflammatory cell infiltration gradually worsened.The pathological changes of pancreatic tissue in the Sham group were mild.The expression levels of IL-1β in CLP group and sham group increased first and then decreased, and reached the peak at 12 h. The expression levels of IL-1β in CLP group were significantly higher than those in control group and sham group at all time points( P<0.05). The expression level of IL-10 showed a gradually increasing trend, reaching a peak at 48 h. The expression level of IL-10 in CLP group was significantly higher than that in control group and sham group at all time points( P<0.05), which in sham group was significantly higher than that in control group( P<0.05). Serum amylase and lipase in CLP group showed an upward and then downward trend, reaching the peak at 24 h. The serum amylase levels at 24 h and 48 h were significantly higher than those in control group and sham group( P<0.05). Serum amylase levels in sham group were significantly different from those in control group at 12 h, 24 h and 48 h( P<0.05). Serum lipase level at 12 h, 24 h and 48 h was significantly higher than that in control group and sham group( P<0.05). There was no significant difference in serum lipase between sham group and control group( P>0.05). Conclusion:After CLP modeling, IL-1β, IL-10 elevation and pancreatic pathological changes appeared at the early stage, and the pathological changes gradually increased with time.Significant differences in serum amylase and lipase began to appear at 24 h and 12 h respectively.