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Role Of SNARE Complexes In Spinal Cord Dorsal Horn In Chronic Inflammatory Pain

Posted on:2020-12-14Degree:MasterType:Thesis
Country:ChinaCandidate:X L DuanFull Text:PDF
GTID:2404330596987769Subject:Pharmacy
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Objective:Soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)-mediated membrane fusion plays an important role in vesicle transport and neurotransmitter release.Recent studies had confirmed that membrane trafficking of postsynaptic receptors,including N-methyl-D-aspartate(NMDA)subtype of glutamate receptors(NMDARs),depends on SNARE proteins-mediated exocytosis.NMDARs plays a key role in pain plasticity and behavioral sensitization,and SNARE complexes are involved in pain modification.The exact composition and function of the SNARE complexes in spinal cord dorsal horn,however,remain largely unclear.The current study aimed to investigate the major components of the SNARE complexes in spinal dorsal horn and the possible role in chronic inflammatory pain.Method:In this study,the localization of SNAP25 in spinal dorsal horn was explored by immunohistochemistry.The interaction between SNARE proteins family in spinal cord dorsal horn was investigated by co-immunoprecipitation and immunoblotting.A model of inflammatory pain was established by intraplantar injection of Complete Freund's Adjuvant(CFA)in rats and pep-SNAP25 that interfered with the formation of the SNARE complexes was inthrathecally injected to investigate the regulation of peripheral inflammation on the assembly of the SNARE core complexes.The possible role of SNARE complexes in the spinal dorsal cord in chronic inflammatory pain were investigated by co-immunoprecipitation,immunoblotting,sulfo-NHS-biotin surface labelling and behavioral tests.Results:(1)Immunohistochemistry results showed that SNAP25 was abundantly distributed in the dorsal horn neurons of the superficial gray matter of the spinal cord and co-distributed with PSD95,while its homologous protein SNAP23 was mainly distributed in the nerve fiber bundle of the white matter of the spinal cord;(2)Syntaxin1,syntaxin2,syntaxin3,syntaxin4,VAMP1,VAMP2,Munc18-1,Munc18-2and Munc18-3 were immunoprecipitated from spinal dorsal horn lysates of rats with anti-SNAP25 specific antibody,and the relative contents of syntaxin4,VAMP2 and Munc18-1 were much higher than their homologs;(3)Also,SNAP25,VAMP2 and Munc18-1 were immunoprecipitated from spinal dorsal horn lysates of rats by anti-syntaxin4 specific antibody,indicating that SNAP25,syntaxin4,VAMP2 and Munc18-1 may constitute the dominant SNARE core complexes in spinal cord dorsal horn;(4)After intraplantar injection of Complete Freund's Adjuvant,the relative contents of syntaxin4,VAMP2 and Munc18-1 were significantly increased by the SNAP25 of immunoprecipitation;(5)Also,after intraplantar injection of Complete Freund's Adjuvant,the relative contents of SNAP25,VAMP2 and Munc18-1 were significantly increased by the syntaxin4 of immunoprecipitation,indicating that peripheralinflammationpromotedtheassemblyof SNAP25/syntaxin4/VAMP2/Munc18-1 core complexes in spinal cord dorsal horn;(6)Intrathecal administration of GABA_A receptor antagonist bicuculline(0.5?g)and NMDA receptor agonist NMDA(1.0?g)were significantly increased the relative content of syntaxin4,VAMP2 and Munc18-1 by the SNAP25 of immunoprecipitation.(7)Intrathecal administration of the NMDA receptor antagonist D-APV(1.0?g)were significantly inhibited the effect of bicuculline on the interaction of SNAP25 with syntaxin4,VAMP2 and Munc18-1,indicating that the assembly of SNARE complexes in spinal cord dorsal horn was closely regulated by neural activity and NMDA receptor activity;(8)Intrathecal injection of pep-SNAP25 that interfered with the interaction between SNARE proteins significantly inhibited membrane expression and synaptic expression of GluN2B subunit-containing NMDA receptors(GluN2BRs)in inflammatory rats,indicating that the SNARE complexes played an important role in the exocytosis of GluN2BRs;(9)Intrathecal injection of pep-SNAP25dose-dependently inhibited the reduction of paw withdrawal thresholds(PWTs)and paw withdrawal latencies(PWLs)induced by CFA,suggesting that disruption of SNAP25/syntaxin4/VAMP2/Munc18-1 complexes effectively inhibited chronic inflammatory pain.Conclusion:In spinal dorsal horn neurons,SNAP25,syntaxin4,VAMP2,and Munc18-1 predominantly formed SNARE complexes.Peripheral inflammation promoted the assembly of this complex,thereby regulating the exocytosis of NMDARs and increasing synaptic expression.Interference with the formation of this complex effectively alleviated mechanical pain hypersensitivity and thermal hyperalgesia caused by Complete Freund's Adjuvant.
Keywords/Search Tags:Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, Inflammatory pain, Spinal cord dorsal horn
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