| Purpose In this study,rheumatoid arthritis(RA)rabbit animal models were induced with egg albumin,which was given buccal needle treatment.The quantitative proteomics and bioinformatics technology were applied to analyze the protein expression level in cerebrospinal fluid,so as to explore the analgesic molecular mechanism of buccal needle treatment for RA in terms of proteomics and cell signaling pathways,and to provide a theoretical basis for clinical application of buccal needle treatment of RA.Methods By pain threshold screening,40 healthy white rabbits with big ears were selected,with 10 randomly selected for control group,and for the rest,rabbits model of RA induced by egg albumin was established,which were randomly divided into 3 groups,namely model group,the buccal needle group and the body acupuncture group.Buccal needle group and body acupuncture group were treated with buccal and body acupuncture respectively,then cerebrospinal fluid and synovial joints were collected to investigate the changes of behavioristics,the pain threshold and the joint synovial pathological histology by behavioristics observation,the pain threshold measurement,the joint synovial pathological histology inspection,quantitative proteomics technology,bioinformatics technology and Western blotting verification of RA related differential protein,so as to establish cerebrospinal fluid protein expression profile and screen out differential protein,and explain the main biochemical metabolic pathway and signal transduction pathway with participation of differential protein.Result 1.Effect Analysis of buccal acupuncture and body acupuncture After the treatment of buccal acupuncture and body acupuncture in rabbits,compared with model group,the rabbits’ mental state improved,diet increased,body weight increased gradually,knee swelling relieved,joint activity enhanced,pain beginning to recover,and the pain threshold difference was significant(P<0.05).While the instant analgesic effect of buccal acupuncture is better than that of body acupuncture(P<0.05)and imaging indicated that swelling of soft tissue around the knee was relieved,damage degree of joint space narrowing and bone was improved,pathological histology observation showed that smooth membrane tissue neovascularization was reduced,the number of inflammatory cells reduced significantly,and synovial variable became thin.2.Identification of differentially expressed proteins in cerebrospinal fluid by mass spectrometry Differentially expressed proteins were identified in cerebrospinal fluid of rabbits in each RA groups using i TRAQ technology.By mass spectrometry analysis,153 differentially expressed proteins were screened from cerebrospinal fluid of rabbits in model group and normal group,of which 78 proteins were up-regulated and 75 proteins were down-regulated.734 differentially expressed proteins were screened from cerebrospinal fluid of rabbits in buccal acupuncture group and model group,of which 329 proteins were up-regulated and 405 proteins were down-regulated.152 differentially expressed proteins were screened from cerebrospinal fluid of rabbits in body acupuncture group and model group,of which 98 were up-regulated and 54 were down-regulated.695 differentially expressed proteins were screened from cerebrospinal fluid of rabbits in buccal acupuncture group and body acupuncture group,of which 283 were up-regulated and 412 were down-regulated.3.Bioinformatics analysis 3.1Differential protein analyzed by GO In terms of the cellular components,biological process and molecular function,analysis of differentially expressed proteins showed that differentially expressed proteins from cerebrospinal fluid of rabbits in model group and normal group involved in biological processes were categorized into 272,90 were classified as cellular components,158 were classified as molecular function;differentially expressed proteins involved in biological process were categorized into 1197 in buccal needle group and the model group,259 were classified as cell location,535 were classified as molecular function;differentially expressed protein from cerebrospinal fluid involved in biological process were categorized into 269 in body acupuncture group and the model group,83 were classified as cellular components,160 were classified as molecular function.Differentially expressed protein from cerebrospinal fluid involved in biological process were categorized into 332 in buccal acupuncture group and acupuncture group,112 were classified as cellular components,190 were classified as molecular function.The RA related biological process of these identified proteins are mainly involved in material transport and energy metabolic process.The cellular components are mainly involved in the cytoplasm,mitochondria,endoplasmic reticulum.Molecular functions are mainly related to energy metabolism,antigen binding,actin binding,inflammatory reaction,chemokine and so on.Based on above analysis,these identified differentially expressed proteins are involved in many types.Except a small number of proteins with independent function,most of proteins have synergistic effect or other interactions in playing their roles,consistent with complexity of lesions in rheumatoid arthritis,signifying a multi-protein participating complex process in the development of rheumatoid arthritis.3.2 Differential protein analyzed by KEGG Through the classical pathway analysis of differentially expressed proteins in cerebrospinal fluid of all rabbit groups,it showed that the differentially expressed proteins in cerebrospinal fluid was involved in 65 classical pathways for model group and the normal group;differential proteins in buccal acupuncture group and model group involved in 199 classical pathways;differential expressed proteins in body acupuncture group and model group involved in 79 classic pathways;differential expressed proteins in buccal acupuncture group and body acupuncture involved in 193 classical pathways.These classical pathways associated with RA mainly include AMPK signaling pathway,cytokine receptor pathway,Jak-STAT signaling pathway,chemokine signaling pathway and so on.Therefore,these identified differentially expressed proteins associated with RA have many signaling pathways,indicating that RA disease is caused by the complex process involved in many signal transduction pathways.Buccal acupuncture can play the analgesic effect by regulating the changes of multiple signal transduction pathways of differential proteins in cerebrospinal fluid of rabbits,indicating that buccal acupuncture have multi-targeted treatment mechanism.4.Verification of differential proteins Some differential protein,such as CD14,TNF-α and CCL3 related to RA in different groups were analyzed by bioinformatics analysis.The results were verified and analyzed by Western blotting,showing that the 3 protein expressions were up-regulated in the model group and the difference was statistically significant(P< 0.05);in the buccal acupuncture and body acupuncture group,the 3 protein expressions were down-regulated,more significantly in body acupuncture group.This result was consistent with that of proteomic analysis.Conclusion 1.The buccal acupuncture therapy can improve general behavioral manifestations,imaging finding,pathological morphology presentation and pain threshold and the instant analgesic effect of buccal acupuncture is better than that of body acupuncture(P<0.05).2.The quantitative proteomics technique combined with bioinformatics analysis showed that the pathogenesis of RA may be the change of multiple abnormal protein expression and signal transduction pathways.3.Buccal acupuncture therapy play an important role in the energy metabolic process of RA by regulating the glycolytic pathway,triose phosphate isomerase,enolase in cerebrospinal fluid of rabbits.4.Buccal acupuncture therapy take the analgesic effect for RA by intervening the AMPK signaling pathway,cytokine receptor pathway,Jak-STAT signaling pathway,chemokine signaling pathway of the inflammation related protein expression in cerebrospinal fluid of rabbits. |
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