Mechanistic Insights Of α7AChR And Cytokine Inducible SH₂ Containing Protein On The Interventions Of Pediatric E.coli Sepsis And Meningitis

BACKGROUND AND AIMSNowadays,neonate and infant mortality rates are the important indicators evaluating the countries’ medical level.Despite the availability of antibiotics over the last several decades,an unmet requirement still exists for new effective therapeutic approaches to treat neonatal infections.Most of these deaths were caused by bacterial sepsis and meningitis（BSM）,which is characteristic with high mortality and adverse prognosis.According to the surveillance and investigation of bacterial drug resistance from CHINET reported that "the most common gram-negative bacterial pathogens causing BSM is Escherichia coli（E.coli）,which is exhibiting an uncontrollable,increasing multi-antibiotics resistance".Hence,the crosstalk between host-pathogen becomes a new strategy and research point for the development of antibiotic replacement therapies.Inflammation-activated chemokines induce permeability increasing in brain microvascular endothelial cells,which is one of the typical pathological features for evaluating BBB integrity.This is also a non-negligible reason for explaining BSM among children.Preliminary studies demonstrated that alpha7 nicotinic acetylcholine receptor（α7nAChR）played a detrimental role in the pathogenesis of BSM and relative blood-brain barrier（BBB）injuries,accompanied with increasing of cytokine-inducible SH₂-containing protein（CISH）at the transcriptome level.Recent studies suggested the activations of Janus protein tyrosine kinase 2（JAK2）and the related pathways were associated with BBB injuries,while JAK2 could be generated aggregation and phosphorylation by α7nAChR.Whether this biological signaling pathway could explain the pathogenesis of BSM is still unknown.Is there any more complicated interactions exist?In this proposal,we will explore the underlying mechanism ofα7nAChR,CISH and their down-stream during pediatric E.coli induced BSM.By involving in vitro（Human brain microvascular endothelial cell infected with E.coli K1）and in vivo（Wild type and α7nAChR knockout mice intragastric administration with E.coli K1）,we dissected both positive regulation（α7nAChR-JAK2-STAT5）and negative regulation（CISH-JAK2-STAT5）in the pathogenesis of E.coli K1 induced BBB injuries and brain microvascular leakage.In addition,two small molecule compounds target to α7nAChR including methyl lycaconitine citrate（MLA）and memantine hydrochloride（MEM）will also be discussed when BSM infection and BBB injuries.METHODSBioinformatics methods:The datasets GSE80496 and GSE72829 were obtained from Gene Expression Omnibus（GEO）.GEO2R,gene set enrichment analysis（GSEA）and weighted gene co-expression network analysis（WGCNA）were applied to screen out the differentially expressed genes（DEGs）among the patients with bacterial sepsis,when compared to the healthy.Gene ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genome（KEGG）enrichment analysis were also performed on the key genes by R software,hubs genes were obtained by using String 11.0 and Cytoscape subsequently.In addition,the dataset GSE25504 was involved to verify the expression profiles of hub genes in whole blood samples among infants.And the relationships between gene expression and samples’ baseline data（such as:gender,gestational age,birth weight,and antibiotic exposure）were also discussed in the study.In vitro:Constructing the human microvascular endothelial cells（HBMECs）infected with E.coli K1 model;CISH expression in HBMECs were overexpressed/silenced by siRNA and plasmid transfection techniques,respectively;the adhesion and invasion abilities of E.coli infect to HBMECs were evaluated by dilution method of plate counting;survival rates of HBMECs after infected with E.coli were detected by cell counting kit-8（CCK-8）method;IL-6,TNF-α and IL-1β levels were detected by ELISA;JAK2,p-JAK2,STAT5b,p-STAT5b,CISH,tight junction protein（Occludin）in HBMECs pre-treated with different drugs were collected and detected by western blotting,while the morphological profiles of specific protein were observed by immunofluorescence technique,etc.In vivo:Constructing the wild type and α7nAChR knock-out mice model infected with E.coli by intragastric administration;mice genotypes were identified by PCR-electrophoresis technique;the activation of α7nAChR was evaluated by α-BTX assay;Bacteria in peripheral blood and cerebrospinal fluid of mice were detected by dilution method of plate counting;morphological section of brain microvascular were stained by hematoxylin-eosin method and observed by optical microscope with 200×,400×amplification;immunohistochemistry was used to labelled the specific protein in mice tissues,etc.RESULTS932 DEGs were screened out in GSE80496 dataset,while 319 DEGs were screened out in GSE72829 dataset.We identified 10 hub genes（MMP9,ITGAM,CSTD,GAPDH,PGLYRP1,FOLR3,OSCAR,TLR5,IL1RN and TIMP1）.GSEA analysis shows key pathways（amino acid carbohydrate ribose metabolism,PPAR signaling pathway,glycan biosynthesis,autophagy regulatory pathway,complement/coagulation factor cascade reaction,nicotine and nicotinamide metabolism,unsaturated fatty acids biosynthesis and Alzheimer’s disease pathway）and biological processes（steroid hormone secretion,adenylate cyclase activation,extracellular matrix degradation and metal ion transport）associated with bacterial sepsis.The expressions of JAK2,p-JAK2,STAT5b,p-STAT5b were up-regulated after E.coli infection,consistently with IL-6,TNF-α increasing and tight junction collapsing significantly.Nicotine（α7nAChR agonist）could aggravate the E.coli-induced BBB injuries,while MLA（α7nAChR antagonist）exhibited the protective effect via regulating JAK2-STAT5 signaling pathway when infection.In addition,CISH protein was proved that functioned as a negative regulatory inhibit the activations of JAK2,p-JAK2,STAT5b,p-STAT5b in HBMECs infected with E.coli.Notably,the expression of CISH protein could be regulated by α7nAChR membrane receptor in HBMECs.Moreover,we determined the protective effects of MLA and MEM（functioned as α7nAChR antagonist）on HBMEC cells when E.coli infection,and suggested that α7nAChR and CISH protein were considered for explaining several small molecule compounds’protective effects on E.coli-induced BBB injuries.CONCLUSIONSBacterial sepsis was associated with the activation of nicotine and nicotinamide metabolism pathways in human peripheral blood.On the stage of BBB injuries induced by E.coli infection,α7nAChR is a key regulator affects the integrity of HBMECs via activating JAK2-STAT5 signaling pathway,while CISH inhibits JAK2-STAT5 activation and exhibits protective effects on E.coli infection.In addition,MLA and MEM were determined to improve BBB injuries regulated by α7nAChR and CISH protein.