Exploring Key Genes For The Outcome Of Sepsis Based On The Transcriptome Sequencing Of Mouse Spleen

Objective: High-throughput sequencing and bioinformatics techniques were combined to screen key differentially expressed genes(DEGs)affecting sepsis prognosis in mouse spleens.Methods: 1.Mice were randomly divided into control group and sepsis model group,and sepsis models with different prognosis were established by intraperitoneal injection of different doses of lipopolysaccharide.According to the 7-day survival experiment,the optimal modeling dose of sepsis survival group and death group was selected.2.Enzyme-linked immunosorbent assays(ELISAs)were carried out by using peripheral plasma from the control group,sepsis survival group and death group respectively,in order to verify the expression level of inflammatory factors(TNF-α,IL-1β,IL-6 and IL-10),and spleens of three groups were taken out for HE staining.3.High-throughput sequencing was conducted on the spleens of the survival group and death group,DEGs were screened out by bioinformatics and displayed in the form of volcano map and heat map.GO and KEGG annotation enrichment analyses were performed on differential genes.A PPI network was constructed to screen out key DEGs,with the help of Mcode and Cytohubba plug-ins in Cytoscape software.4.DEGs were further validated by RT-PCR analysis.Results:1.By observing and statisticizing the behaviors and survival rate of mice that were injected with different doses of LPS,the LPS dosage in the survival group was 15mg/kg(with a mortality of 30%),and the LPS dosage in the death group was 30mg/kg(with a mortality of 80%).The best sampling time was 24 h after modeling.2.ELISA assay results of inflammatory factors:IL-6(46.54 ± 5.185,59.66 ± 11.17,94.78 ± 4.619 pg/ml),TNF-α(282.3 ± 50.97,356.9 ± 33.54,162.7 ± 19.19 pg/ml),IL-1β(67.70 ± 1.82,76.57 ± 3.74,103.2 ±6.77 pg/ml),IL-10(301.1 ± 34.48,216.5 ± 18.30,162.7 ± 19.19 pg/ml).The expression levels of peripheral pro-inflammatory factors(TNF-α,IL-1β,IL-6)in septic mice were significantly higher than those of the control group,while the expression level of the anti-inflammatory factor(IL-10)decreased(P<0.05).The comparison of sepsis model groups showed that the pro-inflammatory factors in the death group were higher than those in the survival group,while the anti-inflammatory factor was lower than that in the survival group(P <0.05).The HE staining indicated that compared with the control group,the white pulp of spleens in the survival group and death group presented different degrees of dilation and fusion,with unclear boundaries,which was more distinct in the death group.3.A total of 2,999 DEGs in the survival group and death group were screened out by bioinformatics,among which 1,185 genes were up-regulated and 1,814 genes were down-regulated.Top 5 DEGs enrichment pathways were screened out,i.e.,“hematopoietic cell lineage”,“primary immunodeficiency”,“African trypanosomiasis”,“leishmaniasis” and“B-cell receptor signaling pathway”.Ifit1,Ifit3 and Mx1 were the three key DEGs that were screened out.4.RT-PCR results revealed that compared with the survival group,the expressions of Ifit1,Ifit3 and Mx1 were down-regulated in the death group,with statistically difference(P<0.05).Conclusion: Through high-throughput sequencing and bioinformatics,three key DEGs,Ifit1,Ifit3,and Mx1,were predicted to be associated with the prognosis of death from sepsis.The above key genes may affect the prognosis of sepsis through immune mechanisms related to viral infection..