Long Non-coding RNA Mediates Caffeine-reduced Electroacupuncture Analgesia

Objective:To explore the effects of caffeine administration in mouse after EA in adjuvant-induced arthritis.This study was aimed to explore the difference expression of lncRNAs and mRNA among the AA model mouse+electroacupuncture accompany with caffeine or not by lncRNAseq technology.We will predict the lncRNAs' potential co-expressed target genes,biofunctional clusters and signal transduction pathways by using bioinformatics method.This study will support the foundation theory for investigation of mechanism of electro-acupuncture analgesia effect antagonism via lncRNAs,guide clinical acupuncture treatment and provide a basis for individualized treatment plan based on caffeine intake.Method:1.In the first part,a total of 24 mouse were used,divided into blank group,model group,model-needle-decaffeinecoffee group and model-needle-caffeine group randomly.Inflammation pain model was established by injecting CFA,taking electroacupuncture as stimulating technique,selecting “Zusanli” at left-hind limb as pricking position.The thermal withdrawal latency was detected before model buildidng,after model building and 30 min after each acupuncture treatment.2.Collection of hypothalamus samples of model+EA+decaffeinated coffee and model+EA+coffee,extract the total RNA.3.Exploration the lncRNA and mRNA expression profile via lncRNA microarray in model+EA+decaffeinated coffee and model+EA+coffee mouse.Screen the outstanding difference expression of lncRNAs and mRNA(difference ratio?2,p?0.05).4.Bio-informatics analysis including potential target genes prediction,DAVID database enriched terms and signal pathways.Construction of networks of TF-lncRNA-target genes.Prediction of differentially expressed lncRNA target genes via the Pearson correlation coefficient.Association analysis for construction of lncRNA-mRNA co-expression network.Analysis of TF-lncRNA-target genes three elementary relationship by Cytoscape.Results:1.After each treatment,the thermal withdrawal latency of mouse in model-needle-caffeine coffee group is much higher than the model group(P<0.01);there is a significant difference between the thermal withdrawal latency after model and the thermal withdrawal latency after treatments finished(P>0.05).After treatment,the thermal withdrawal latency of mouse in model-needle-caffeine has no change.There is no significant difference between the model-needle-caffeine coffee and model-needle-caffeine group when treatments finished(P>0.05).2.Using lncRNA-seq method,differently expressed lncRNAs were detected as follows(differently ratio?2,p?0.05): comparison between model+EA+decaffeinated coffee and model+EA+coffee mouse,total of 43 different lncRNAs were detected,including 25 up-regulated and 18 down-regulated;total of 68 different mRNAs were detected,including 31 up-regulated and 37 down-regulated;3.Comparison of model+EA+decaffeinated coffee and model+EA+coffee mouse: Choose the most significantly differentially expressed known lncRNA(Gm20388,Numb,Ndufa5,Ppp1r1 b,Rps26,Ndufa4,Atp5j2,Atp5 e,Rps6)to predict their target genes via MEM database:MAPK14?CDC42?Penk?Gng7?Rragc?Drd1a?Gpr6?Gnal.4.Perform the gene ontology(GO)analysis and Pathway analysis via DAVID database.Found that the GTPase neurotrphin signaling pathway,MAPK signaling pathway,chemokine signaling pathway so on participate in caffeine-reduced acupuncture analgesia.5.Comparison of model+EA+decaffeinated coffee and model+EA+coffee mouse: Choose the most significantly differentially expressed novel lncRNA(NONMMUG017325.1 and 54709)to predict their target genes and bio-function via Pearson correlation coefficient: Gabrg1?Ikzf4?Pdap1.6.Confirm the regulatory mechanism of 8 lncRNAs(Ttr,Fam117 b,Arhgap39,Ndufa5,Rps26,Hba-a2,Atp5j2,Ndufc1,Atp5e)on the chromosomes via CIS,and there are 4 lncRNA with target genes via Pearson correlation coefficient: Zc3h3?Ppp4r1l-ps?Gm14393?Pdap1?Ikzf4.7.Perform the gene ontology(GO)analysis and Pathway analysis via DAVID database.Found that complement pathway,co-stimulatory signal during T-cell activation,ERK1/ERK2 MAPK signaling pathway and so on participate in caffeine-reduced acupuncture analgesia.8.The transcription factors of LncRNA Fam117b?NONMMUG017315.1?Rpp1r1b1?PS26?54709: AP-2?T-Ag?AP-1?TFIID?MLTF?PEBP2?SIF?Sp1?GCF?APRT?PuF?GATA-1?CTF?Ikzf4 via trans.9.Build three elementarily regulatory networks: Ikzf4(TF)-RPS26,54709(lncRNA)-Gabrg1(mRNA)on the basis of mRNA co-expression via trans analysis.Conclusion:1.Coffee can reduce the analgesic effect of Electro-acupuncture when intaking coffee with caffeine during electro-acupuncture treatment;2.lncRNA Gm20388,Numb,Ndufa5,Ppp1r1 b,RPS26,Ndufa4,ATP5j2,ATP5 e,RPS6,NONMMUG017325.1,54709 participate in caffeine-reduced electroacupuncture analgesia;3.lncRNAs mediate caffeine-reduced electroacupuncture analgesia via neurotrphin signaling pathway;4.Three elementarily mutual regulatory networks such as TF:Ikzf4-lncRNA: RPS26-Gabrg1;TF:Ikzf4-lncRNA: 54709-Gabrg1 participate in caffeine-reduced electroacupuncture analgesia.