Quantitative Proteomics Analysis Of Myofascial Trigger Points And The Changes After Dry Needling And Static Stretching Treatment

ObjectiveNumerous studies have demonstrated the high incidence of myofascial trigger points in various populations.Currently,studies on the mechanisms of MTr Ps and dry needling have mainly focused on histopathology and electromyography(EMG),which basically stay in the verification to support the integrated trigger point hypothesis proposed by Simons and Travell.Furthermore,the exact change in overall proteins in the active MTr P regions compared with healthy tissues is still unknown,as whether there are one or more particular proteins with key roles in dry needling or stretching induced inactivation of MTr Ps.This study aimed to discover the potential pathogenesis of myofascial pain and the therapeutic mechanism underlying dry needling and stretching treatment at the molecular level based on proteomics technology,using an in vivo rat model of MTr Ps,in order to lay a foundation for the specific and pharmacodynamic target therapy to provide a new theoretical guidance for the prevention,diagnosis and treatment of MTr Ps.MethodsA total of 72 male Sprague Dawley rats(220–250 g)were randomly divided into six groups(n=12 in each group): a control group(CG),a model group(MG),a dry needling of MTr Ps group(DG),a sham dry needling of MTr Ps group(NDG),a stretching group(SG)and a dry needling combined with stretching group(SDG).The rats in the CG group were fed as usual without any intervention.In the MG,DG,NDG,SG and SDG groups,the model of active MTr Ps was established by blunt striking on the left gastrocnemius muscle and eccentric based exercise for a period of 8 weeks,followed by a 4-week recovery period.To identify the active MTr P,palpation and EMG device with bipolar electrodes was performed.After the MTr Ps model had been successfully generated,the corresponding dry needling and/or static stretching treatment was adopted,respectively,in the DG,NDG,SG and SDG groups once a week,for 4 weeks.The nociceptive mechanical threshold was tested before and after treatment every week.After the treatment was terminated,the left GM tissues were quickly removed and the protein samples were prepared.A quadrupole Q-Exactive mass spectrometer coupled to an Easy n LC was used to perform TMT(6-plex)based LC-MS/MS analysis,in order to quantify and evaluate the overall protein.A bioinformatics analysis including Hierarchical clustering,Gene ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,and Protein–protein interaction(PPI)network analysis groups were performed to characterize the proteins.To validate the results of TMT,six candidate proteins were verified using Parallel reaction monitoring(PRM)and Western blot analysis.Results1.The nociception of the left hind paw was significantly decreased at 0-week treatment(post-modeling/before treatment)treatment compared to baseline(pre-modeling)in the MG\DG\NDG\SG\SDG groups(P< 0.05).In the last week(4-week treatment),the nociception value was significantly increased in DG\SG\SDG compared to 0-week treatment;value was significantly higher in the DG and SDG than in SG(P < 0.05).2.A total of 180(up-regulated =75;down-regulated =105)between the CG and MG groups,158(up-regulated =86;down-regulated =72)between the DG and MG groups,93(up-regulated =47;down-regulated =60)between the NDG and MG groups,47(up-regulated =30;down-regulated =17)between the SG and MG groups,107(up-regulated =62;down-regulated =31)between the SDG and MG groups were differentially expressed.The results showed that the selected proteins were well distinguished and accurately screened out.3.GO enrichment analysis showed that compared MG to CG or DG,biological process of system process proteins,muscle system process proteins,muscle contraction proteins,molecular function of cytoskeletal protein binding,action binding,structural constituent of muscle,cellular component of supramolecular fiber,supramolecular polymer,supramolecular complex were significantly changed;compared MG to SDG,biological process of glycogen catabolic process,single-organism carbohydrate catabolic process,carbohydrate catabolic process,molecular function of glycogen phosphorylase activity,integrin binding,tropomyosin binding,cellular component of myofibril,contractile fiber,supramolecular fiber were significantly changed.4.KEGG enrichment analysis showed that compared MG to CG or DG,the glycolysis/gluconeogenesis pathway,glucagon signaling pathway,HIF-1 signaling pathway,and cardiac muscle contraction pathway were significantly changed;compared MG to SDG,glucagon signaling pathway,tight junction pathway,and sulfur metabolism pathway were significantly changed.5.The lead proteins(which are differentially expressed between the CG and MG groups)with a high degree of connectivity in the PPI network are Pyruvate kinase PKM,Fructose-bisphosphate aldolase A,Fructose-bisphosphate aldolase,Troponin I type 2,and Actin alpha.The lead proteins(which are differentially expressed between the SDG and MG groups)with a high degree of connectivity in the PPI network are Actinin alpha 3 and Type 2X myosin heavy chain.6.The results of PRM and Western blot showed that compare to CG,Pyruvate kinase muscle isozyme(PKM),Muscle isoform of glycogen phosphorylase(PYGM)significantly decreased and Myozenin 2(MYOZ 2)significantly increased in MG;Compare to MG,PKM,PYGM significantly increased and MYOZ 2 significantly decreased in DG;Actinin alpha 3(Actn ?3),Casq1 and Pvalb ? significantly increased in SDG.ConclusionsThis is the first proteomics study that has investigated the pathogenesis of myofascial trigger point and the mechanisms underlying the treatment effects of dry needling and static stretching treatment in an in vivo rat model of MTr Ps,which might promote our understanding of the molecular mechanisms underlying chronic myofascial pain.The development of MTr Ps were found to be concerned with a total of 180 proteins and 26 pathways.The mechanisms of dry needling treatment were found to be concerned with a total of 158 proteins and 23 pathways.The glycolysis/gluconeogenesis and tight junction pathways played dominant roles in the pathogenesis of MTr Ps and in dry needling combined with static stretching treatment,respectively.The PRM and Western Blot results showed that the six candidate proteins(PKM,PYGM,MYOZ 2,Actn ?3,Casq1 and Pvalb ?)showed similar trends as in the TMT data,which supported the credibility and reliability of the proteomics data.the proteomics data may provide valuable clues to better understand the pathogenesis associated with myofascial pain in order to improve diagnosis and treatment.Further functional verification of the potential signaling pathways and the enriched proteins is warranted.