| Objective:1.Explore the method of constructing mice models for different prognosis of septic intestinal injury.2.Screen hub genes for different prognosis of septic intestinal injury based on RNA-seq technology and bioinformatics analysis.Methods:1.The 60 mice were randomly divided into 6 groups(10mice/group).Mice in each group were intraperitoneally injected with an equal volume of PBS or lipopolysaccharide(LPS)solution at doses of 0mg/kg,10 mg/kg,15 mg/kg,20 mg/kg,30 mg/kg,and 40 mg/kg.the 7-day survival curve of each group of mice was drawn.According to the 7-day survival of each group of mice,the optimal modeling doses of the survival group and the death group were screened out.2.Two different LPS doses were used to construct the mouse models of the survival group and the death group respectively,and an equal volume of PBS solution was used to construct the control group mouse mode.Observe the activity status of mice in each group.The expression levels of TNF-α,IL-6 and IL-1β in the plasma of mice in each group were detected by ELISA.HE staining was used to observe the morphological changes of the small intestine of mice in each group.3.Differentially expressed genes in small intestinal tissue were screened by RNA-seq technology.GO function analysis and KEGG pathway enrichment analysis were performed.Using the STRING database to complete the protein interaction network analysis.The analysis results were upload into the Cyto Scape software,and the Cyto Hubba plug-in was used to calculate the score according to the Degree value,and the topscoring genes in the entire network were screened out.Quantitative analysis of the screened hub genes by RT-PCR.4.SPSS 20.0 software was used for statistical analysis of data.If the data conformed to a normal distribution,one-way ANOVA was used for comparison among three groups,Turkey test was used for pairwise comparison among three groups.If the data did not conform to a normal distribution,the Kruskal-Wallis test was used for the comparison among three groups,and the Dunn’s test was used for the pairwise comparison among the three groups.The results were presented as mean ± standard deviation(±s),and P<0.05 indicated that the difference was statistically significant.Results:1.With the increase of LPS dose,the mortality rates of the six groups of mice were 0%,10%,30%,60%,80% and 90%,respectively.According to the mortality rate of mice,15 mg/kg(with a mortality of 30%)and 30 mg/kg(with a mortality of 80%)were selected as the LPS doses for constructing different prognosis mouse models.2.ELISA test found that compared with the control group,the expressions of TNF-α,IL-6 and IL-1β in the plasma of the mice in the survival group and the death group were significantly increased.The expression levels of plasma inflammatory factors in the control group,the survival group,and the death group were295.8±30.56,371.6±39.76,516.4±49.91 pg/m L,respectively,for TNF-α,73.99±4.63,84.61±4.61,97.87±5.71 pg/m L,respectively,for IL-1β,55.4±6.76,66.7±7.76,84.50±6.09 pg/m L,respectively,for IL-6.HE staining showed that both the survival group and the death group had different degrees of intestinal mucosal integrity damage and inflammatory cell infiltration,and the death group had more obvious intestinal damage.3.Gene transcription data of intestinal tissues were analyzed and 185 significantly differentially expressed genes were screened,including 79up-regulated genes and 106 down-regulated genes.Among them,Myc,mtNd1,Ndufb7,and Uqcrc2 may be the hub genes that affect the different outcomes of septic intestinal injury.4.RT-PCR results showed that mt-Nd1,Ndufb7,and Uqcrc2 were highly expressed in the death group,while the expression of Myc was decreased in the death group,the difference was statistically significant.Conclusions: 1.Intraperitoneal injection of different concentrations of LPS solution can construct mouse models of septic intestinal injury with different prognosis.2.Myc,mt-Nd1,Ndufb7 and Uqcrc2 play key roles in the prognosis of septic intestinal injury and may be potential therapeutic targets for septic intestinal injury. |
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