Effect Of Electroacupuncture On SAMP8 Mice Based On Single-cell Transcriptome Sequencing Technology

Objective:In the early stage of this study,the water maze experiment was used to observe the effect of Electroacupuncture on the cognitive ability of Alzheimer’s disease(AD)rat model.In the later stage,based on single cell sequencing,explore the molecular mechanism of acupuncture improving SAMP8 rat model,and provide more scientific basis for acupuncture treatment of AD from the molecular level.Methods:seven-month-old Senescence-accelerated mouse-prone 8(SAMP8)mice and Senescence-accelerated mouse resistant 1(SAMR1)mice were randomly divided into model group,electroacupuncture group and control group.Electroacupuncture at Baihui Point and Yintang point and rapid pricking at Renzhong point.The treatment cycle is 8 weeks,with 5 consecutive days of intervention and 2 days of rest every week.24 hours after the last electroacupuncture intervention,the cognitive ability of each group was evaluated by water maze test.In addition,two mice in the model group and electroacupuncture group were randomly selected,and the brains of the mice were subjected to single-cell sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG)were used for functional analysis.Methods:Seven-month-old SAMP8 and SAMR1 mice were randomly divided into model group,electroacupuncture group and control group.Electroacupuncture at Baihui Point and Yintang point and rapid pricking at Renzhong point.The treatment cycle is8 weeks,with 5 consecutive days of intervention and 2 days of rest every week.24 hours after the last electroacupuncture intervention,the cognitive ability of each group was evaluated by water maze test.In addition,two mice in the model group and electroacupuncture group were randomly selected,and the brains of the mice were subjected to single-cell sequencing.Gene Ontology(go)and Kyoto Encyclopedia of genes and genomes(KEGG)were used for functional analysis.There are 69 statistically significant pathways,and the top 30 KEGG pathways with high expression are mostly related to neuronal development,inflammatory response and pathways related to cancer and metabolic diseases.Results:1.Results of water maze test: compared with the control group,the escape latency of mice in the model group was significantly prolonged(P < 0.001);The number of crossing the platform decreased significantly,and the residence time in the target quadrant decreased(P < 0.001).Compared with the model group,the escape latency of mice in electroacupuncture group was significantly shorter(P < 0.001);The residence time in the target quadrant and the number of crossing the platform increased significantly(P < 0.05).2.Cell type identification: 49704 cells were finally obtained by single cell sequencing of smap8 mouse brain tissue,including 23919 in model group and 25785 in electroacupuncture group.Through t-sne dimensionality reduction clustering,it has been the marker of cell types.The clusters are called 13 main clusters,namely:astrocyte,perivascular macrophase,oligodendrocyte,endothelium,microglia,neuro,B cell,pericyte,vascular smooth muscle cell,neuroblast,neurophil and vascular leptomeningal cell.3.Screening of differentially expressed genes: compared with the model group,2781 genes were identified.Differential gene analysis showed 90 genes,44up-regulated genes and 46 down-regulated genes.4.Analysis of differential gene enrichment: the first three items with high degree of go enrichment are astrocyte development,cell response to cadmium ion and extracellular region.The top 5 items with high enrichment of KEGG are TNF signaling pathway,IL-17 signaling pathway,nod like receptor signaling pathway,ribosomal signaling pathway and Salmonella infection.The common genes include:CCL2,cebpb,CXCL1,CXCL10,cxcl2,FOS,S100A8,S100A9,TNFAIP3,IL1 B,Jun,pfn1,rpl27,rpl39,rpl9,rps23,rps24,rps27 and UBA52.5.In the brain immune environment of AD model rats,CCL3 and CCL5 chemokines play an important role in regulating the immune environment.6.Analysis of astrocyte subtype: go and KEGG enrichment analysis of top300 marker gene in astrocyte type5 cell cluster shows that electroacupuncture intervention in SAMP8 mice has a direct or indirect relationship with genes regulating cell structure,protein binding,sphingolipid,inflammation and other related signal pathways.It is predicted that these genes may be the targets of electroacupuncture intervention in SAMP8 mice.Top10 marker genes of type5: gbp5,s1pr1,Rnd1,anxa8,ICAM1,CXCL14,PTX3,RNASE1,stx11,Foxg17.The differentially regulated genes pfn1,msrb1,S100A8,S100A9,rps23 and ATF3 after acupuncture may be of great significance to reveal the mechanism of acupuncture in the treatment of AD.Conclusion:1.Electroacupuncture can reduce the cognitive impairment of SAMP8 mice in AD model,delay the progress of AD and have neuroprotective effect.2.Acupuncture can significantly change the number of astrocytes,perivascular macrophages,oligodendrocytes,T cells and neurons.3.This study depicts the single-cell map of SAMP8 mouse brain tissue by single-cell transcriptome sequencing.The heterogeneity after acupuncture was proved,and 13 kinds of cells in the brain tissue of SAMP8 mice were systematically analyzed.Three kinds of immune cells and their subtypes in mouse brain tissue.4.After acupuncture,astrocytes,perivascular macrophages,oligodendrocytes,T cells and neurons have significant inter group differences,which proves that these cells may be closely involved in the process of electroacupuncture in the treatment of AD.5.In the brain immune environment of AD model rats,CCL3 and CCL5 chemokines play an important role in regulating the immune environment.6.The top10 marker genes gbp5,s1pr1,Rnd1,anxa8,ICAM1,CXCL14,PTX3,RNASE1,Stx11 and Foxg1 obtained from astrocyte subtypes can be used as genes for subsequent verification.7.The differentially regulated genes pfn1,msrb1,S100A8,S100A9,rps23 and ATF3 after acupuncture may be of great significance to reveal the mechanism of acupuncture in the treatment of AD.