| Objectives:Porphyromonas gingivalis,(P.gingivalis)and Fusobacterium nucleatum(F.nucleatum)are common periodontal pathogens,and more and more studies have found that periodontitis is associated with Neurodegenerative diseases(NDDs)such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).but the specific molecular mechanism is not well understood.Therefore,this study explores the mechanism in-volved in periodontal pathogenic infection affecting neurodegenerative diseases from the transcriptome perspective.There may be commonalities and differences between different concentrations and types of periodontal pathogens in signaling pathways and key genes affecting neurodegenerative diseases.The purpose of this study includes the following four items:(1)to compare high concentrations of P.gingivalis with low con-centrations of P.gingivalis and F.nucleatum bacteremia Differentially expressed genes in infected rat hippocampus;(2)compare the biological processes,molecular functions,and signaling pathways involved in these genes;(3)screening of key genes in each group;(4)preliminary verification of key genes.Methods:1.A rat bacteremia model was constructed.Twelve 6-8 week-old male Spra-gue Dawley(SD)rats were randomly divided into 4 groups of 3 rats in each group.P.gingivalis High dose group(PgH),P.gingivalis Low dose group(PgL),F.nucleatum Low dose group(Fn).1.0x10 ~8CFU/200μL and 1.0x10~3CFU/200μL of P.gingivalis and F.nucleatum were injected through the rat tail vein,respectively The control group was injected with the same volume of PBS,injected 4 times a week for 8 weeks.2.After 8 weeks,rat hippocampal tissue was taken,and the isolated hippocampal tissue was transcriptome sequenced,and then compared with the control group to screen out DEGs.3.Gene ontology(GO)function annotation of DEGs,Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis and Reactome enrichment analysis,using GSEA software to all unscreened Genes were enriched and the results were vis-ualized using R-related packages.4.Use the STRING website to analyze the DEGs for protein-protein interaction(PPI)and import the results into the Cytoscape software,and filter out Hub gene according to the score from high to low The gene,and then the Hub gene,the three groups of genes in the common pathway are associated with the Alzheimer’s disease-related gene database Alz Base and Parkinson’s disease-related gene database G ene4PD was intersected to obtain key genes of clinical significance.5.Verification of the key genes screened out by qRT-PCR.Results:1.Compared with the control group,there were 271 differential genes in the PgL group,including 137 significantly upregulated genes and 134 significantly down-regulated genes.There were 128 differential genes in the PgH group,including 64 sig-nificantly upregulated genes and 64 significantly downregulated genes.There were a total of 282 differential genes in the Fn group,including 155 significantly upregulated genes and 127 significantly downregulated genes.2.GO enrichment analysis results show:(1)The biological processes of significant enrichment in the DEGs of the PgL group were behavior,cell regulation of secretion,response to decreased oxygen content,adult behavior,social behavior,etc.;The significantly enriched cellular components were do-pamine synthesis and external power arm;The significantly enriched molecular func-tions were G protein-coupled peptide receptor activity and neuropeptide hormone ac-tivity.(2)The biological processes significantly enriched in DEGs in the PgH group were hormone level regulation,epithelial cell differentiation,anion transport,and pos-itive regulation of ion transport;The significantly enriched cellular components are the cell tip moiety,and the significantly enriched molecular function is ion channel activity.(3)The biological processes significantly enriched in the DEGs of the Fn group were epithelial cell differentiation,system process regulation,hormone-stimulated cellular response,metal ion transport,hormone level regulation,etc.;The significantly enriched cellular components were cilia,apical plasma membrane,etc.The significantly en-riched molecular function is organic acid binding.3.The results of KEGG enrichment analysis show that:(1)The pathways significantly enriched in the PgL group were neuroactive ligand-re-ceptor interaction pathway,calcium ion signaling pathway,cAMP signaling pathway,neurotrophic factor signaling pathway,etc.(2)The pathways significantly enriched in the PgH group were neuroactive ligand-receptor interaction signaling pathways and cell adhesion molecules.(3)The pathways significantly enriched and enriched in the Fn group were neuroactive ligand-receptor interaction signaling pathway,estrogen signaling pathway,renin secretion,etc.4.The results of Reactome enrichment analysis show:(1)The pathways significantly enriched in the PgL group were peptide-ligand binding receptor,GPCR(G protein-coupled receptor)ligand binding,GPCR signaling,GPCR downstream signaling,etc.(2)The significantly enriched pathway in the PgH group is the immune system.(3)The Fn group was significantly enriched and enriched,and the pathways were GPCR ligand binding.5.The results of GSEA analysis show that:(1)The PgL group was significantly enriched with GABA synthesis-release-reuptake-degradation pathway,serotonin neurotransmitter release cycle,dopamine neurotrans-mitter release cycle,and norepinephrine neurotransmitter release cycle.(2)The PgH group was significantly enriched with glycolytic gluconeogenesis and glucose metabo-lism pathways.(3)The Fn group significantly enriched the assembly of collagen fibers and other polymers,collagen chain trimers,and cross-connection pathways of collagen fibers.6.Screening and verification results of key genes:(1)In the PgL group,9 key genes were found:Avp,Sstr,Nr4a2,Oxt,Cldn4,Drd1,Lhb,Nmb,Nts(2)In the PgH group,4 key genes were found:Atf3,Prl,Ttr and Kl.(3)In the Fn group,4 key genes were found:Avp,Sstr,Dpp4,Prlr,LhbAmong them,the key genes of PgL group(Avp,Sstr,Nr4a2,Oxt,Cldn4)and the key genes of PgH group(Atf3,Prl,Ttr,Kl),the key genes of Fn group(Avp and Sstr)had significant changes in q PT-PCR experiments and were consistent with transcrip-tome sequencing results.Conclusion:P.gingivalis and F.nucleatum bacteremia significantly affected the expression of signaling pathways and related molecules associated with neurodegenerative diseases in hippocampus of rats.P.gingivalis bacteremia is concentration-dependent on the regulation of these processes. |
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