Expression And Mechanism Of TNF-α/NF-κB/MEF2C In Myofascial Pain Trigger Points

Research BackgroundMyofascial pain syndrome(Myofascial pain syndrome,MPS)is a chronic pain disorder related to inflammation,which is mainly characterized by the existence of myofascial trigger point(Myofascial trigger points,MTrPs).According to the clinical characteristics of MTrPs,it can be divided into active and latent MTrPs.In our previous study,we found that myocyte enhancer 2C(Myocyte enhancerfactor 2C,MEF2C)was differentially expressed between healthy subjects and diagnosed MTrPs subjects by RNA sequencing(RNA-seq),and nuclear factor kappa-B(Nuclear factor kappa-B,NFkappa B)signal pathway was found after KEGG enrichment analysis of differential genes.Previous studies have shown that MEF2C is a key factor in skeletal muscle development and is related to the occurrence of a variety of skeletal muscle diseases.However,there are no studies to explore whether MEF2C is involved in the pathophysiological process of MTrPs.Therefore,the purpose of this study is to investigate whether there is differential expression of MEF2C in MTrPs,and whether TNF-α regulates the expression of MEF2C through NF-κB signal pathway,and then affects the expression of muscle hypertrophy-related molecules in MTrPs.PurposeTo explore whether there is a difference in the expression of MEF2C,and whether NF-κB signal pathway can regulate the expression of MEF2C and the effect of dexmedetomidine on MEF2C.Methods1.Collect MTrPs clinical samples of MPS patients and control muscle tissues,observe the morphological changes by HE staining,and detect the differential expression of MEF2C by immunofluorescence staining.2.In this study,6-week-old male and female Sprague Dawley(SD)rats were subjected to 8-week beating,eccentric exercise and 4-week recovery to establish a rat widely used in the field of myofascial pain syndrome research.MTrPs model to study the upstream and downstream regulation mechanism of MEF2C.3.Recombinant rat tumor necrosis factor alpha(TNF-α)was injected into the gastrocnemius muscle of healthy rats to observe the changes of NF-κB signaling pathway and MEF2C expression.4.The animal model of MTrPs was injected with the specific NF-κB signaling pathway inhibitor Maslinic acid(MA)to observe whether the expression of MEF2C was inhibited.5.In healthy rats,the NF-κB signaling pathway was inhibited before TNF-α stimulation to see whether inflammatory stimulation could also affect MEF2C expression after NF-κB signaling pathway blockade.6.The rat model of MTrPs was intervened by local intramuscular injection and tail vein injection of dexmedetomidine(Dex)to see whether its anti-inflammatory effect could inhibit the expression of MEF2C.7.Detect the change of mechanical pain threshold of rats after applying various interventions through animal behavior experiments.8.HE staining was used to observe the morphological changes of rat skeletal muscle after modeling.Immunohistochemical detection of MEF2C expression in rat MTrPs.qRT-PCR detected changes in the transcriptional level of MEF2C.Western blot was used to detect the protein expression level of NF-κB signaling pathway and MEF2C.9.Statistical analysis:The research data are presented as mean ± standard deviation(Mean± SD),and statistical analysis is performed using Graphpad Prism 8.0 and SPSS 25.The difference between the two groups was analyzed by t test.Data not conforming to normal distribution were tested by Mann-Whitney U.One-way ANOVA or two-way ANOVA was used to determine the main effects and interactions of the two factors,followed by Tukey’s multiple comparison test to identify differences between groups when necessary.Mechanical pain threshold data measured over time were analyzed using repeated measures ANOVA.P<0.05 was considered statistically significant.Results1.MEF2C is up-regulated in human and rat MTrPs tissues(*P<0.05),and participates in the pathophysiological process of MTrPs;2.Injecting TNF-α into rat gastrocnemius muscle can induce the activation of NF-κB signaling pathway,enhance the expression of MEF2C,and cause Mechanical hyperalgesia(*P<0.05);3.Inhibition of NF-κB signaling pathway can inhibit the expression of MEF2C and reverse the pain behavior caused by MTrPs(*P<0.05);4.Peripheral injection of dexmedetomidine can inhibit the up-regulated phosphorylated NF-κBp65,The expression of MEF2C and its downstream muscle hypertrophy markers(*P<0.05);5.The anti-inflammatory effect of peripheral injection of dexmedetomidine is mainly manifested in the local microenvironment of MTrPs,rather than the systemic anti-inflammatory effect through the central system(*P<0.05).Conclusion1.The increased expression of MEF2C in MTrPs may mediate myofascial pain in rats.2.TNF-α promotes the expression of MEF2C by activating NF-κB signal pathway.3.MTrPs local intramuscular injection of dexmedetomidine suppresses MEF2C expression through TNF-α/NF-κB and relieves myofascial pain in rats.