Study On The Analgesic Activation Mechanism Of Three Methods And Three Acupoints In Rats With Neuropathic Pain Mode

BackgroundThe clinical analgesia of tuina is characterized by rapid action,safety and reliability.And the previous studies have proved part of the analgesic mechanism,but they are all based on multiple times of tuina interventions,ignoring when and how tuina analgesia is initiated,which is not conducive to the clarification of tuina analgesia clinical application scope and international application and promotion.ObjectiveThis study was carried out to evaluate the immediate analgesic effect of tuina,clarify the initiation time of tuina analgesia,explore the initiation and cascade mechanisms of tuina analgesia,further reveal the action mechanism of tuina analgesia,improve the theoretical basis of tuina analgesia,and provide scientific evidence to explain the analgesic efficacy and time in the clinical application of tuina therapy.MethodsReplicate the minor chronic constriction injury(minor CCI)model to simulate clinical peripheral nerve injury causing neuropathic pain(NP),using the Tuina Manipulation Simulator to simulate "Three-Manipulation"(finger pressing,plucking,and kneading manipulation),acting on "Three-Acupoint"(BL 37,BL 57,and GB 34),for the "Three-Manipulation and Three-Acupoint" treatment,giving the model rats qualitative,timed and quantitative interventions,and the study was investigated through 3 sets of experiments as follows:Experiment 1 Study on the immediate analgesic effect and initiation of the "ThreeManipulation and Three-Acupoint" treatment:To evaluate the immediate analgesic effect of the "Three-Manipulation and Three-Acupoint" treatment through nociceptive behavioral and morphological studies,and to initially screen the initiation time point of tuina analgesia.Nociceptive changes after 1,2,3,5 and 7 times of tuina interventions in rats were observed by mechanical withdrawal threshold(MWT),thermal withdrawal latency(TWL),and accumulated pain score(APS).The ultrastructure of the spinal dorsal horn(SDH)oligodendrocytes and organelles were observed by transmission electron microscopy.Experiment 2 Study on the initiation time and mechanism of the "Three-Manipulation and Three-Acupoint" treatment analgesia:To clarify the initiation time point and explore the initiation mechanism of tuina analgesia through systems biology studies.On the basis of determining the time range for the initiation of tuina analgesia,detect and analyze SDH protein changes in L4-6 segments after 1 time of tuina intervention by data independent collection proteomics.And SDH gene changes in L4-6 segments after 1,2 and 3 times of tuina interventions by eukaryotic genome sequencing,and multiple bioinformatic analyses,such as Venn analysis,time series analysis,promoter and transcription factor prediction.Experiment 3 Study on the initiation and cascade mechanism of the "ThreeManipulation and Three-Acupoint" treatment analgesia:To reveal the initiation and cascade mechanisms of tuina analgesia through molecular biology studies.Detect expression changes of Ndl,Crabp1,Rapgef5,Sema7a proteins after 1 time of tuina intervention and Cptlb,Mtnd3,Angptl4,Plin4,Pla2g4b,Cyp2j4,Mapk12,Gabre,Bdkrb2,Ndufal,Vegfa,Egf,Flt4,Cxcr4,Efna4 genes after 1,2 and 3 times of tuina interventions in the SDH of the L4-6 segment.Results1 The immediate analgesic effect of tuina was significant1.1 The results of nociceptive behavioral tests:7 d after modeling,the nociceptive behavioral performance of the tuina group was the same as that of the model group(P>0.05,P>0.05,P>0.05),and the results of mechanical pain,thermal pain,and tactile pain of the rats were significantly different compared with those of the model group after giving tuina intervention 1,2,3,5,and 7 times(P<0.01,P<0.01,P<0.01).And mechanical pain was close to the level of the negative control group after 3 times of tuina intervention(P>0.05),thermal pain was close to the level of the negative control group after 2 times of tuina intervention(P>0.05).1.2 The results of electron microscopic observation:The ultramorphological structures were relatively normal in the SDH of the sham group.The ultramorphological structures were mildly damaged in the oligodendrocytes and organelles in the SDH of the model group,which was consistent with the nociceptive behavior of the rats.The differences in ultramorphological structures were smaller in the SDH of the tuina group compared with the model group.The present results suggest that tuina analgesia was effective immediately after the 1 time of tuina intervention,and no morphological changes in SDH were observed;the initiation time point of tuina analgesia was after 1,2,or 3 times of tuina interventions.2 The initiation of tuina analgesia was presented in a dynamic,cascade wayThe following results were obtained by RNA sequencing techniques combined with various bioinformatic analysis methods that detected the SDH differentially expressed gene(DEG)after 1,2,and 3 tuina interventions:2.1 Basic results:A total of 725 genes were affected after 1 time of tuina intervention,which was the most of all groups.And the number of affected DEG gradually decreased with the increase in the tuina intervention;after 1,2 and 3 times of tuina intervention,the DEG showed 3 types of time series trends of up-regulation,up-regulation and then down-regulation,and down-regulation of expression(P<0.05,P<0.05,P<0.05);a total of 1269 promoters and 52 transcripts were affected after 1 time tuina intervention,which was the most of all groups.2.2 The result of bioinformatics analysis after 1,2 and 3 times of tuina intervention:The analgesic mechanism after 1 time of tuina intervention was related to the signaling pathways of AMPK,IL-17,tumor necrosis factor,arachidonic acid metabolism,and neurotrophic factors;the analgesic mechanism after 2 times of tuina intervention was related to the signaling pathways of JAK-STAT,chemokines,cytokine-cytokine receptor interaction,and retrograde endocannabinoid;the analgesic mechanism after 3 times of tuina intervention was related to the signaling pathways of neurotrophic factors,MAPK,GnRH,inflammatory mediator regulation of TRP channels,apoptosis,and Toll-like receptors.2.3 The results of initiation time series bioinformatic analysis of tuina analgesia:At the genetic level,the cascade reaction of tuina analgesic was initiated after 2 times of tuina intervention.It was related to the signaling pathways of apoptosis,IL-17,MAPK.2.4 The results of initiation promoter and transcription factor bioinformatics analysis of tuina analgesia:At the genetic level,the initiation of tuina analgesia can occur after 1 time of tuina intervention,which was related to signaling pathways of thermogenesis and PPAR through related promoters;after 2 times of tuina intervention,which initiate signaling pathways of retrograde endocannabinoid,regulation of TRP channels by inflammatory mediators,and MAPK;after 3 times of tuina intervention,which initiate signaling pathways of apoptosis,Toll and Imd,and JAK-STAT;related transcription factors may initiate signaling pathways of IL-17,MAPK,apoptosis,and PPAR.The present results suggest that the initiation of tuina analgesia was after 1 time of tuina intervention.The following results were obtained by proteomic techniques combined with various bioinformatic analysis methods that detected the differentially expressed protein(DEP)of SDH after 1 time of tuina intervention:2.5 The results of proteomic bioinformatics analysis after 1 time of tuina intervention:A total of 106 proteins were affected after 1 time of tuina intervention,and combined with analysis of the protein interaction network,the analgesic mechanism after 1 time of tuina intervention was related to signaling pathways of MAPK,axon guidance,thermogenesis,lipid metabolism,retrograde endocannabinoid,and regulation of TRP channels by inflammatory mediators.2.6 The results of the genomic and proteomic combined analysis:The targets associated with the initiation of tuina analgesia include proteins(Nd1,Crabp1,Rapgef5,Sema7a),ligands(Egf,Vegfa,Efna4,Cxcr4,Mt-nd3,Ndufal),receptors or kinases(Cxcr4,Flt4,Bdkrb2,Egf),transcription factors(Pla2g4b,Mapk12),and target genes(Mt-nd3,Cptlb,Plin4,Ndufal,Gabre,Angptl4,Cyp2j4).The present results suggest that the initiation of tuina analgesia is dynamic and may be cascade initiated.At the genetic level,the initiation of tuina analgesia was after 1 time of tuina intervention,and the cascade reaction of tuina analgesia was after 2 times of tuina intervention.3 Tuina exerts analgesia by modulation of lipids,initiated endocannabinoid system and TRP channels and cascade activated multiple signaling pathways including MAPK3.1 Changes in the expression of relevant proteins in SDH after 1 time of tuina intervention:Proteins of Nd1,Crabp1,Rapgef5 and Sema7a were down regulated after 1 time of tuina intervention,but there was no statistical difference compared with the model group(P>0.05,P>0.05,P>0.05,P>0.05).3.2 Changes in the expression of relevant genes in SDH after 1,2 and 3 times of tuina intervention:①The tuina intervention regulated the thermogenesis signaling pathway through down regulation of Cptlb expression(P>0.05,P<0.01,P<0.01);②tuina intervention regulated PPAR signaling pathway through down regulation of Angptl4,Plin4 expression(P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01);③tuina intervention regulated the signaling pathway of inflammatory mediators on TRP channels through down regulation of Pla2g4b,Cyp2j4,Mapk12 expression(P<0.01,P>0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.05);④tuina intervention regulated retrograde endocannabinoid signaling pathway through down regulation of Gabre,Bdkrb2 expression and up regulation of Ndufal expression(P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01);⑤tuina intervention regulated MAPK signaling pathway through down regulation of Vegfa,Flt4 expression(P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01);⑥tuina intervention regulated axon guidance signaling pathway through down regulation of Cxcr4 expression and up regulation of Efna4 expression(P>0.05,P>0.05,P<0.05,P<0.01,P<0.01,P<0.01).The present results suggest that the initiation mechanism of tuina analgesia was related to changes in the expression of genes such as Cptlb in SDH,and tuina exerts analgesia by modulation of lipids(thermogenesis and PPAR signaling pathway),initiated endocannabinoid system and TRP channels and cascade activated multiple signaling pathways including MAPK.Conclusions1 The 1 time of tuina intervention exerted analgesia immediately and effectively relieved mechanical pain,thermal pain,and tactile pain of the minor CCI model rats,and no morphological changes in SDH were observed.2 The process of the initiation of tuina analgesia was dynamic and cascading,with analgesia initiated after 1 time of tuina intervention and cascade reaction initiated after 2 times of tuina intervention.3 Tuina exerted analgesic effects through Cptlb and other genes that modulated lipids to initiate endocannabinoid system and TRP channels and cascade activate multiple signaling pathways including MAPK.