Peripheral And Central Mechanism Of Inflammatory Osteoarthritis Pain Conduction

Research BackgroundOsteoarthritis(OA)is the most common arthroplasty and can cause any joint pain and dysfunction including temporomandibular joint Osteoarthritis(TMJOA),knee Osteoarthritis(KOA),and severe OA pain can cause disqualify life of patients.OA was originally being recognized as a predominant disease of cartilage disease,a previous study of the correlation between many joint space stenosis(imaging assessment of cartilage damage)and clinical pain levels,however these studies eventually produced conflicting results.OA pain can occur independently of cartilage damage.And currently there is no effective way to cure OA pain.Not only do we focus on cartilage injury research,but we must also understand more about the pathogenesis of chronic joint pain to meet the needs of clinicians to improve quality of life.Nociceptive stimuli such as inflammatory stimuli or inflammation itself generated in OA joints,activate the nociceptor terminals(the end of the afferent fibers)which in turn activate primary afferent fibers whose cell bodies in the Ganglion,can further lead to changes in the expression of certain genes in neurons in the ganglion,such as changes in ion channels,increased excitability of neurons,etc.,so that nociceptive afferent fibers turn nerve impulses from nociceptors to the central nervous system(CNS)and provides the brain with information on the location,intensity and duration of the stimulus.This article will systematically explore the mechanisms of the development and maintenance of TMJOA and KOA,two major osteoarthritic pains from the peripheral and central nervous system,including the role of peripheral sensitization and central sensitization in this inflammatory pain overall effect.TMJOA pain severely affects the quality life of patients,its treatment effect is poor,and the specific molecular mechanism of pain is unclear.Previous studies have shown that complete Freund's adjuvant(CFA)intra-mandibular joint injection can significantly increase tumor necrosis factor-?(TNF-?)and its receptor in TMJ tissues.However,it is unclear whether TNF-? is involved in the development of TMJOA pain in the trigeminal pain system.In this experiment,We used CFA-induced mouse TMJOA inflammatory pain model,increased expression of TNF-? in Trigeminal ganglia(TG)and Spinal trigeminal nucleus Caudalis(Sp5C)were found in TMJOA mice.In the first and second part of this thesis,we focused on the related mechanisms of TNF-? involved in the occurrence and development of TMJOA chronic pain.KOA pain severely affects the quality life of patients,the clinical treatment of pain is poor,and the specific molecular mechanism of pain is unclear.The Dorsal Root Ganglion(DRG)is the primary sensory neuron of the KOA pain conduction pathway.NaV1.9 in DRG is one of the voltage-gated sodium channel subunits and plays a key role in nociceptive pain.However,whether NaV1.9 expression increases in DRG neurons in the KOA pain model is still inconclusive.In this experiment,a CFA-induced rat KOA inflammatory pain model was used.Increased protein kinase C(PKC)and NaV1.9 were found in DRG.In the third and fourth part of this paper,we focus on PKC.regulation of NaV1.9 involved in the development of chronic pain in KOA.Research purposesThis study will clarify the behavioral characteristics of chronic inflammatory pain caused by TMJOA and KOA;Reveal the molecular basis of increased expression of related molecules in pain conduction-related neurons induced by CFA-induced TMJOA and KOA;The role of epigenetics in chronic pain of TMJOA and KOA.With a view to revealing the mechanism of TMJOA and KOA pain occurrence and development from the perspective of cell-molecule-nucleolus-behavior.Research MethodsFirst part:(1)Establishment of TMJOA animal model and detection of pain behaviorThe TMJOA inflammatory pain model was induced by the injection of CFA into the bilateral temporomandibular joint(TMJ)of mice.The Von Frey wire was used to measure the mechanical pain threshold of the trigeminal nerve distribution area,the mouse pain expression scale(Mouse Grimace Scale,MGS)and Dolognawmeter were used to evaluate the occlusal strength.These three tests were combined to perform the pain behavior test.(2)Study on the mechanism of TNF-? involved in TMJOA painWe first used Western blotting and immunofluorescence staining methods to observe the effects of CFA injection on the expression and distribution of TNF-? and its receptor TNFR1 in mouse TG and Sp5C.Using C57BL/6 wild-type(WT)and TNF-? knockout(KO)mice,behavioral tests were performed to verify the magnitude of TMJOA pain threshold in mice lacking TNF-? expression.Finally,Sp5C was injected with TNF-? antagonist R-7050 to detect the pain threshold of CFA-TMJ mice.Second part(3)DNA methylation and demethylation status of the TNF-? promoter regionMethylated DNA immunoprecipitation was used to explore the DNA methylation and demethylation status of the TNF-? promoter region in the trigeminal nociceptive system(in TG and Sp5C).(4)Demethylase TET1 participates in the regulation of TNF-? expression and the mechanism of TMJOA painThe expression of major regulatory enzymes related to epigenetics in TG and Sp5C in mice were observed by immunoblotting and immunofluorescence.Finally,shTET1-Lentiviral was injected into TG to verily its role in CFA-TMJ pain.Third part(5)Establishment of KOA animal model and detection of pain behaviorCFA was injected into the knee joint of rats to establish a chronic KOA inflammatory pain model.The mechanical pain threshold was measured using Von Frey,and the knee arthritis characteristics of KOA rats were determined by HE staining.(6)PKC regulates NaV1.9 expression and its mechanism with KOA painThe expressions of NaV1.9 and PKC in DRG were observed by immunoblotting and immunofluorescence.Then,we used PKC inhibitor(GF-109203X)and activator(PMA)in DRGs neurons cultured in vitro and observed their effects on NaV1.9 expression by RT-PCR.The effect of intrathecal injection of GF-109203X and PMA on the expression of NaV1.9 in L4-6 DRG neurons in vivo was also observed by immunofluorescence.At the same time,we tested the effects of PKC inhibitors and activators before and after administration on the nociceptive behavioral response thresholds of thermal,cold,and mechanical thresholds in rats.Fourth part(7)Study on the mechanism of long non-coding RNA involved in KOA painIn the KOA study,we continued to explore whether epigenetic regulation is involved in the transmission of KOA pain.First,CFA or saline was injected into the knee joints of rats to establish a chronic KOA inflammatory pain model.On the seventh day,two groups of rats L4-6 DRG neurons were hybridized with rat IncRNA chip,washed,fixed and scanned to obtain the chip map and read the value to get the original data.GeneSpring GX v12.1 software(Agilent Technologies)was used to perform Quantile normalization on the raw data and subsequent data processing.We obtained statistically significant differentially expressed IncRNAs or differentially expressed mRNAs between the two groups of samples.We verified some of the IncRNAs with the greatest changes by QPCR and performed correlation analysis.Research ResultsFirst part1.In the TMJOA study,CFA(10 ?l)induced TMJOA inflammatory pain in WT mice,which began on day 1 after CFA and lasted for at least 10 days.2.After CFA injection,TNF-? in TG and Sp5C of WT mice was up-regulated.CFA-induced TMJOA pain behavior was significantly inhibited in TNF-? KO mice.Immunofluorescence staining showed that CFA injection can not only enhance the colocalization of TNF-? and Ibal(microglia marker)in TG and Sp5C,but also induce the overexpression of TNF-? in Sp5C neurons.Sp5C injection of TNF-?antagonist can reduce pain hypersensitivity caused by CFA-TMJ injection.Second part3.Through DNA dot-bolt test in TMJOA study,we found that CFA-TMJ did not change the expression levels of 5mc and 5hmc of the whole genome DNA,but DNA methylation immunoprecipitation analysis proved that DNA methylation in TNF-?promoter region was significantly reduced in TG after CFA injection,DNA demethylation was significantly increased,and there was no significant change in DNA methylation of the TNF-? promoter region in Sp5C.4.Further investigation found that CFA-TMJ did not change the expression of the demethylases DNMT1,3A,3B,and histone deacetylases HDAC1,3,but the demethylase TET1 increased in TG after CFA injection.Immunofluorescence staining showed that TET1 was co-standarded with Neun(a neuronal cell marker),but not with Ibal and GFAP(astrocyte markers).Injecting shTET1 into TG can alleviate CFA-TMJOA pain in mice.Third part5.The results of the KOA study showed that compared with the Sham group,the mechanical,thermal,and cold-stimulated foot withdrawal thresholds of the CFA-KOA group were reduced.6.The protein and mRNA expression of Nav1.9 and PKCa increased in DRG of KOA rats.Immunofluorescence experiments found that Nav1.9 preferentially co-localized with IB4+small neurons in KOA rats.In cultured DRG neurons,PMA increased Nav1.9 expression,while GF-109203X prevented the effect of PMA.PMA increased Nav1.9 expression in untreated rats,while GF-109203X decreased Nav1.9 expression in KOA rats.PMA can cause mechanical and cold hyperalgesia in untreated rats.GF-109203X attenuates mechanical and cold hyperalgesia in the KOA pain model.Fourth part7.In the KOA study,we analyzed in detail the expression changes of lncRNAs in the model group and the control group of DRGs.Compared with the normal saline group,there were 181 lncRNAs and 275 mRNAs have significantly different expression in DRG of the CFA induced KOA rats.We verified some of the IncRNAs with the greatest changes by QPCR,and obtained lncRNAs associated with sodium channels through Go Pathway analysis,proving that the increase in sodium channel phase proteins caused by KOA may be related to the differential expression of IncRNAs,which needs further verification.Research Conclusion1.Our results suggest that TNFa in the trigeminal nociceptive system(TG and Sp5C)plays a critical role in CFA-induced inflammatory TMJOA pain.Increased TET1 may increase demethylation stutue of the TNF-alpha promoter region to promote TNF-? expression in the TG,further increase pain in CFA-TMJOA model.2.Nav1.9 might be upregulated by PKC ? in DRG,which contributes to pain hypersensitivity in CFA-induced chronic knee joint inflammation model of KOA pain.Further revealing the expression pattern of IncRNA in KOA peripheral nervous system and predicting the potential functions and targets of this phenomenon,indicating that lncRNA may be a new biomarker and a new therapeutic target for the diagnosis or treatment of KOA.