| Objective : Meningitis caused by Enterococcus faecalis has caused great public health safety problems,but the mechanism of its crossing the blood-brain barrier is still unclear.In this study,from the perspective of host-pathogen interaction,small RNA sequencing technology was used,and a variety of bioinformatics analysis methods were used to screen and excavate regulatory genes and key pathways related to the permeability change of blood-brain barrier,so as to provide reliable theoretical basis for revealing the mechanism of Enterococcus faecalis meningitis.Methods : The brain injury model of mice infected with Enterococcus faecalis was established with E.faecalis preserved in the laboratory as the experimental strain and clean mice as the experimental animals.Through the isolation and identification of target strains and pathological observation and serum PCT and CRP concentration detection to determine whether mice have meningitis symptoms;to observe the changes of blood brain barrier permeability by detecting the changes of Evans blue in brain tissue at different time points;select three time periods and control group mice brain tissue for small RNA sequencing,and bioinformatics analysis;differential miRNAs related to blood-brain barrier were screened and target gene prediction and target relationship verification were performed;preliminary validation of miRNA target genes in the blood brain barrier function;through the above experiments,the role of miRNA in the destruction of blood-brain barrier by Enterococcus faecalis was explored.Results :Test 1.Establishment of mouse model of artificial infection with Enterococcus faecalisThe results showed that PCT in serum of mice had significant difference at 24 h and 60 h(P< 0.05),and had extremely significant difference at 48 h(P< 0.01).CRP content in serum of mice had significant difference at 12 h and 60 h(P< 0.05),and had extremely significant difference at 24 h and 48 h(P < 0.01).Pathological sections showed brain tissue damage in mice.EB detection showed that the permeability of blood-brain barrier in mice changed,which proved that the model of brain injury in mice infected with Enterococcus faecalis was successfully constructed.Test 2.Construction of miRNA library in brain tissue of blood-brain barrier injury model in miceThe results show that the sequencing results after sequencing data analysis are of high quality and the obtained data are reliable.Successfully screened differentially expressed miRNAs;g O and KEGG enrichment analysis showed that differential miRNAs were mainly related to cell metabolism,cytoskeleton regulation,apoptosis,inflammation and other pathways,and miR-34c-5p and miR-21a-5p related to blood-brain barrier were screened.Test 3.Validation of target relationship between mmu-miR-34c-5p and mmu-miR-21a-5pThe results showed that CCL1 had obvious targeting relationship with mmu-miR-21a-5p,and STK38 L had no obvious targeting relationship with mmu-miR-34c-5p.Test 4.Effect of CCL1 on phosphorylation of key proteins in MAPK pathway in rat brain microvascular endothelial cellsThe results showed that after CCL1 stimulation of cerebral microvascular endothelial cells,the phosphorylation level of JNK increased,with significant difference at 60 min(P < 0.05)and extremely significant difference at 120 min(P< 0.01).The phosphorylation level of p38 increased,showing significant difference at 30 min(P < 0.05)and extremely significant difference at 120 min(P < 0.01).Conclusion: miR-21a-5p is differentially expressed in the brain tissue of Enterococcus faecalis mouse model,and has a significant targeting relationship with CCL1.CCL1 can directly stimulate the phosphorylation of JNK and p38 proteins in brain microvascular endothelial cells,so it is speculated that miR-21a-5p 21a-5p may directly or indirectly regulate the MAPK pathway and thus affect the permeability of the blood-brain barrier. |
PDF Full Text Request