Transcriptomics Analysis Of Myofascial Trigger Points And Mechanism Of EphB In Trigger Points

BackgroundChronic pain affects approximately one-third of the population and is a medical and social problem that needs to be resolved urgently in the clinic.Myofascial pain syndrome(MPS)is one of the most common chronic painful diseases,which is characterized by the existence of one or more myofascial trigger points(MTrPs).The essence of MTrPs is abnormal contractile sarcomeres and defined as a hyperirritable tender spot in a taut band(TB)that can be palpated in skeletal muscle.The continuous contractile activity of muscle in MTrPs leads to local tissue ischemia,hypoxia and neurophysiological changes of nociceptors,which is an important cause of recurrent chronic pain.Therefore,to clarify its pathophysiological mechanism has important clinical significance.At present,researchers have conducted extensive studies on the clinical characteristics of MTrPs,but the specific pathophysiological mechanism of MTrPs is still unclear.In this research,we first used high-throughput techniques,such as RNA Sequencing,protein chip to analyze the MTrPs tissue in MPS patients.Furthermore,through the MTrPs animal model,verify the expression and molecular function of targets related to MTrPs and clarify the pathophysiological mechanism of MTrPs.The purpose of this study is to provide a theoretical basis for finding new targets for the treatment of MPSPart ? Transcriptomics analysis of myofascial trigger pointsObjective:Screening of differentially expressed genes in the MTrPs region of trapezius muscle in MPS patients,so as to find candidate genes and explore their biological functions and mechanisms in the MTrPs.Methods:1.Participants.This study was approved by the Research Ethics Committee of Qilu Hospital of Shandong University.MPS patients were recruited and signed a written informed consent form at the outpatient clinic.According to the international consensus on the diagnostic criteria of MTrPs,the criteria of the patients included in this study are as follows:(1)TBs or nodules in the trapezius muscle of MPS patient;(2)a hyperirritable tender spot in the TB;(3)reproduction of the patient's symptoms;and(4)chronic pain lasting at least 1 month.The exclusion criteria for both groups were conditions that might affect pain sensitivity,such as neuropathic pain,chronic widespread muscle pain,previous trauma in the neck/shoulder area,systemic inflammatory diseases,metabolic disease,high blood pressure,malignancy,pregnancy and a previously failed biopsy.2.Muscle biopsy.Muscle samples were obtained from the TB of the upper trapezius muscle,including the MTrPs.A disposable SuperCoreTM Biopsy instrument was used.First,using the MTrPs palpation protocol,the MTrPs was identified using the index,middle and ring fingers of the right hand.The painful points on the TB were used to define the location of the MTrPs on the superior trapezius muscle.Once the examiner confirmed the position of the MTrPs under the pad of the middle finger,a surgical pen was used to mark a point on the surface of the skin to identify the puncture site of the biopsy needle.A 0.5%lidocaine solution in a 1.5 ml volume was used for subcutaneous injection of anesthetic to minimize pain at the puncture site of the skin.No anesthetic was injected into the muscle.When the biopsy needle reaches the MTrPs,muscle pain can be triggered.The TB was snapped with the thumb and index finger to prevent pneumothorax.The biopsy needle was inserted into the muscle,and,while the local twitch response or pain sensation was triggered,the needle tip was gradually advanced 3 mm(the biopsy instrument had a groove 3 mm from the needle tip),aligning the MTrPs with the groove to allow for biopsy collection.To reduce the sampling error,the biopsy procedures used for all the subjects were performed by an experienced clinician who is a practicing physician with experience in the evaluation and treatment of musculoskeletal disorders and an instructor of these techniques.The biopsied tissues were immediately snap-frozen in liquid nitrogen-cooled isopentane.Then,stored at-80?.3.Genomic library construction and sequencing.Total RNA was isolated using RNeasy mini kit.After the total RNA was extracted,Agilent Bioanalyzer 2100 was used to detect RNA quality.Paired-end libraries were synthesized by using the TruSeqTM RNA Sample Preparation Kit following TruSeq TM RNA Sample Preparation Guide.Purified libraries were quantified by Qubit(?)2.0 Fluorometer and then further validated by Agilent 2100 bioanalyzer to confirm the insert size and calculate the mole concentration.Cluster was generated by cBot with the library diluted to 10pM and then were sequenced on the Illumina NovaSeq 6000.After processing the original data,the RPKM value was used to measure the gene expression.4.Differential expression analysis.After the FPKM values of genes were obtained,the differences of gene expression between two groups were analyzed by edgeR software package.The screening criteria for differential mRNA and lncRNA were:P value<0.05and FC2.5.GO and KEGG enrichment analysis.Based on the GO and KEGG databases,the enrichment analysis is carried out to find out the main biological functions and related signal pathways of the differential genes.Use P value<0.05 as the threshold to screen the enrichment analysis results of differential expression genes.Results:1.Compared with the control group,the results of different expressed mRNAs(DE mRNAs)showed that 2662mRNAs in the MTrPs group was significantly up-regulated,and 4092mRNAs was significantly down-regulated.Compared with the control group,the results of different expressed Lnc RNAs(DE LncRNAs)showed that the expression of 767 LncRNAs in MTrPs group was up-regulated,and the expression of 2356 LncRNAs was down-regulated.2.The results showed that the differential genes were mainly concentrated in various metabolism processes,chemokine activity,growth factor activity,positive regulation of muscle contraction and regulation of protein tyrosine kinase activity in DE mRNAs GO enrichment analysis.In KEGG pathway analysis,the main enrichment results of differential genes focused on autophagy,growth factor signaling pathway,inflammation-related pathway,vascular smooth muscle contraction,glycolysis/gluconeogenesis,apoptosis and metabolic pathway.In the GO enrichment analysis of DE LncRNA target genes,the genes were mainly enriched in Ras GTPase binding,ATP binding,kinase activity,glycolytic process and various metabolic processes,transmembrane receptor protein tyrosine kinase signaling pathway,platelet-derived growth factor receptor signaling pathway,etc.KEGG pathway analysis showed that the DE LncRNA target genes were mainly concentrated in inflammation-related signal pathway,glycerolipid and tyrosine metabolism,glycolysis/gluconeogenesis,Jak-STAT signal pathway,PI3K-Akt signal pathway,chemokine signal pathway,vascular smooth muscle contraction pathway,etc.Conclusion:1.The expression profiles of mRNA and LncRNA in MTrPs tissues changed significantly.2.The results concerning transcriptomics analysis of MTrPs provide valuable insights into the mechanism of MTrPs.Bioinformatics analysis found that a variety of pathways and factors maybe play an important role in the pathophysiological mechanism of MTrPs.Part ? The expression and mechanism of tyrosine kinases in the myofascial trigger pointsObjective:1.To detect the morphological changes between the MTrPs region in the trapeziusmuscle of MPS patients and normal control tissues.2.Using the phosphorylated protein chip to detect the difference expression of thephosphorylation of protein tyrosine kinase(TKs)between MTrPs tissues and normal control tissues.Furthermore,verify the differentially expressed proteins and explore their possible mechanism in MTrPs.Methods:1.HE staining and Masson trichrome staining.The muscle specimens were fixed with formalin.The specimens were subsequently embedded in paraffin wax and sectioned into 3 mm sections,which were mounted on slides and stained with hematoxylin-eosin(H&E)and Masson's trichrome stains.The sections were dehydrated in a graded ethanol series(70%-100%)and xylene,then cover-slipped.2.RayBiotech protein chip detection.After the sample collection,antibody arrays of phosphorylated RTK(p-RTK)were performed using the RayBio Human Phospho Array Kit.In brief,100?l of 1× blocking buffer was added to each well and gently shake it 30 minutes at room temperature.All of the blocking buffer was decanted from each well,then 100 ?l of each sample was added to the appropriate wells.Incubate the array with the sample overnight at 4?.The samples were decanted from each well and washed with wash buffer at room temperature.Then,100 ?l of 1× biotin-conjugated anti-phosphotyrosine antibody was added to each well.The arrays were incubated at room temperature 2 hours with gentle shaking.After the subarrays were washed,100 ?l of 1× fluorescent dye-conjugated streptavidin was added to each well.The arrays were washed and completely dried.The signals were imaged using a laser scanner.3.Western blot was used to verify the differential protein expression.The total proteins from the muscle tissues were separated by 10%SDS-PAGE and transferred to a PVDF membrane,which was blocked with 5%non-fat milk and incubated with primary antibodies at 4? overnight.The second antibody was incubated at room temperature and the transferred blots were developed with a chemiluminescent reagent.4.Immunohistochemistry.Paraffin-embedded sections were heated at 68? for 2 hours,separated in xylene,and rehydrated in graded ethanol at room temperature.The sections were microwaved for 15 min in EDTA buffer.After being rinsed twice in phosphate-buffered saline(PBS),the slices were placed in a wet chamber and incubated in 3%hydrogen peroxide for 10 min.After being washed with PBS three times,the sections were incubated with 10%normal goat serum at 37? for 30 min,then diluted with the antibodies at 4? overnight.After being washed with PBS three times,the slices were incubated with biotin-labelled goat anti-rabbit serum at 37? for 30 min,then treated with a horseradish peroxidase-labelled streptavidin complex at 37? for 30 min.The reaction products were observed with a diaminobenzidine hydrochloride substrate kit.Finally,after weak haematoxylin staining,the sections were dehydrated and covered and observed the expression and localization of differential proteins.Results:1.Following staining,the muscle fibres were observed under a microscope.In the control group,the fibres were uniform in size,polygonal in shape and regular in arrangement in cross-sectional and longitudinal spaces.However,the muscle fibres in the MTrPs displayed annular or elliptical muscle fibres of different sizes in cross-sectional spaces.Continuous expansion of the pyramidal muscle fibres was observed in the longitudinal sections.The muscle fibre space of the transverse section and longitudinal section increased.Abnormal contraction of sarcomeres at MTrPs site was not accompanied by obvious inflammatory cell infiltration and fibrosis.2.The difference of phosphorylation expression of tyrosine kinase family in MTrPs and control tissues was detected.Among the 71 tyrosine kinases,15 proteins had increased phosphorylation levels in MTrPs tissue relative to the levels in control tissue,and two proteins showed decreased levels in MTrPs tissue.The expression of p-EphB 1,p-EphB2 and p-EphB3.The expression of p-EphB1(P<0.05),p-EphB2(P<0.05)and p-EphB3(P<0.001)was higher in MTrPs than in normal tissue.3.Western blot analysis confirmed that compared with the control group,the expression of the p-EphB protein in MTrPs was significantly up-regulated(P<0.01).Immunohistochemical analysis showed an increase in p-EphB levels on the muscle cell membrane of contractile sarcomeres in MTrPs.These results suggest that the expression of p-EphB is up-regulated in the muscle tissue of MTrPs.4.Correlation analysis showed that in MPS patients,the expression of p-EphB1(r=0.723,n=11,P<0.05),p-EphB2(r=0.610,n=11,P<0.05),p-EphB3(r=0.670,n=11,P<0.05),p-PDGFRa(r=0.711,n=11,P<0.05)? p-TRKB(r=0.640,n=11,P<0.05)in the MTrPs site significantly correlated with the numerical rating scale(NRS)of MTrPs in MPS patients.Conclusion:1.Abnormal contractile sarcomeres was found in the MTrPs area of trapezius muscle in patients with MPS,which was consistent with the previous MTrPs hypothesis.2.The expression of p-EphB in TKs family increased in abnormal contractile sarcomeres of MTrPs and was significantly correlated with numerical rating scale of MTrPs in MPS patients.EphB may be involved in the pathophysiological process of MTrPs,which needs to be further explored.Part ? The peripheral EphrinB1/EphB1 signal pathway is involved in mediating myofascial trigger points hyperalgesiaObjective:1.EphB1 ligand agonist EphinB1-Fc was injected into the gastrocnemius muscle of rats to analyze the role of EphrinB1/EphB1 signal pathway in myalgia peripheral sensitization.2.The animal model of MTrPs was established to analyze the expression and mechanism of EphrinB1/EphB1 signal in MTrPs.Methods:1.Experimental animals.The Animal Care and Use Committee of Shandong University approved the experiments.Six-week-old male Sprague-Dawley(SD)rats weighing 200-250g were used in this study.The rats were maintained under a 12-hour light/12-hour dark cycle,room temperature of 24? and 20%-30%relative humidity.Three animals were included per cage,and the animals had free access to food and water2.Local injection.We performed intramuscular(i.m.)injections of EphrinB1-Fc to examine the direct pro-algesic effect of EphrinB1.Different concentrations of EphrinB-Fr(0.02,0.2,and 2 ?g/?l,30 ?l,i.m.,n=4)were injected into the left gastrocnemius muscle of rats.The same volume of PBS served as the control.We examined whether EphrinB1 played its role via its receptor EphB1.Under isoflurane anaesthesia,EphB1-Fc(2 ?g/?l,30 ?l,i.m.,n=6)was injected into the left gastrocnemius muscle prior to the injection of EphrinB1 in the same spot.The same amount of PBS served as the control.The pressure threshold was measured before injection and 0.5 h,1 h,2 h,4 h,8 h and 24 h after injection.3.Construction of MTrPs animal model.6-week-old male SD rats were selected.After one week of environmental adaptation,An active MTrP rat model was established by bluntly striking the left gastrocnemius muscle(GM)of SD rats followed by the performance of eccentric-based exercises for 8 weeks.The rats recovered for 4 weeks.Briefly,the marked left gastrocnemius muscles of the MTrP group were hit with a hand-made stick device that dropped from a height of 20 cm and had a kinetic energy of 2.352 J.The next day,the rats were placed on a treadmill(Sans,Nanjing,China)for 1.5 h with a downward angle of-16°and a speed of 16 m/min.The weight,limb movement,TB changes and behavior of the rats were monitored.Active MTrPs were identified according to the 2017 International Consensus on Diagnostic Standards for Trigger Points of MPS:(1)a palpated TB;(2)mechanical sensitivity on TBs showing a rapid decrease;and(3)ability to elicit local twitch responses(LTRs)by needling.4.Pharmacological experiment.After verifying the success of the rat model,the rats were randomly divided into three groups:MTrPs+PBS(n=6),MTrPs+EphB-Fr(n=6),Control+PBS(n=6).The EphB inhibitor EphB-Fr(2 ?g/?l,i.m.,30?l×3points)was injected into the gastrocnemius muscle of rats,and the Control+PBS group and MTrPs+PBS group were injected with the same volume of PBS as control.After the intramuscular administration of EphB1-Fr,the mechanical withdrawal thresholds were measured at 0.5 h,1 h,2 h,4 h,and 8h using a Randal-Selitto apparatus.5.Western blot:The tissue was collected and the total protein was extracted.The total proteins were separated by 10%SDS-PAGE and transferred to a PVDF membrane,which was blocked with 5%non-fat milk and incubated with primary antibodies at 4?overnight.The second antibody was developed chemically after incubation at room temperature and the transferred blots were developed with a chemiluminescent reagent?Results:1.Muscle hyperalgesia was induced via the intramuscular injection of various doses of EphrinB1-Fc(0.02 ?g/?l,0.2 ?g/?l,2 ?g/?l and PBS,30 ul)into the left gastrocnemius muscle of SD rats.Thirty minutes after injection,the nociceptive threshold decreased in a dose-dependent manner,and the pain response lasted for at least 4 hours.Twenty-four hours after injection,the nociceptive threshold returned to baseline levels.EphB1-Fc is a specific inhibitor of EphB 1,and the intramuscular injection of EphB1-Fc(2?g/?l,30ul)partially blocked the EphrinB1-Fc-induced nociceptive threshold decrease.These results suggest that the EphrinB1-Fc-induced pain response was partially caused by the activation of EphB 1 receptors in muscles.2.The MTrPs model.Two weeks after the first strike,the TBs were palpated subcutaneously in the middle gastrocnemius and were palpated weekly intervals from week 5 to 12 in the MPS group(n=10).Starting from the third week,the mechanical withdrawal thresholds measured using the Randall-Selitto apparatus in the TB sites of the MPS group were significantly lower than the control group,and this difference lasted for 12 weeks.No changes in the palpation or mechanical threshold were found in the control group.Microscopic analysis of haematoxylin and eosin staining revealed that the muscle fibres in the control group were uniform in size,polygonal,and regularly arranged in the cross-sectional space.Large,round muscle cells were observed in the cross-sections of the MTrPs,and obvious inflammatory cell infiltration was also detected.3.Compared with the control group,the expression of p-EphB in MTrPs model rats was significantly increased.The intramuscular administration of EphB1-Fr resulted in significant recovery of mechanical hyperalgesia in the MTrPs group,as detected using a Randall-Selitto apparatus,and this recovery continued for almost 4 hours.The intramuscular administration of PBS as a control did not induce any significant changes in the MTrPs sites in the MTrPs+PBS group(n=6)or Control+PBS group(n=6)animals.In the MTrPs+EphB-Fr group,Eph-Fr(2.5 mg/ml)not only inhibited the expression of p-EphB,but also decreased the expression of p-NR2B.Conclusion:EphrinB1 can induce muscle hyperalgesia through its high affinity receptor EphB1.At the same time,EphB1 plays an important role in the modulation of peripheral nociceptive information related to MTrPs,which may be one of the significant targets for MPS therapy.