The Role Of Dorsal Root Ganglion OCT1 In The Pathogenesis Of Neuropathic Pain

BackgroundNeuropathic pain(NP)is an important public health problem;limitations in understanding of its pathogenesis diminish the therapy effect on neuropathic pain in clinic.Epigenetic regulation plays an important role in the process of neuropathic pain.As an important part of epigenetics,DNA methytransferase has been reported to regulate the neuropathic pain.Researches found the increasing of methytransferase subtype 3a(DNMT3a)in the dorsal root ganglia(DRG)could decrease expression of μ opioid receptor(MOR)and gated potassium channel 1.2(Kv1.2),thus induced increasing of the neuron’s excitability.This is an important peripheral mechanism of neuropathic pain pathogenesis.We have found the expression of DRG octamer transcription factor 1(OCT1)was increased in the chronic constriction injury of the sciatic nerve(CCI)model;inhibition of OCT1 alleviated pain behaviors of rats.Using bioinformatics tool we predicted that OCT1 can recognize and combine with DNMT3 a gene’s promoter region.So,we hypothesize that OCT1 promotes DNMT3 a transcription,upregulates the methylation level of the gene’s promoter region,inhibits the expression of MOR and Kv1.2,and thus participants in the development and maintenance of neuropathic pain.The current study will provide a new idea for effective prevention and treatment of neuropathic pain.ObjectiveInvestigating whether OCT1 regulates the process of neuropathic pain and the exact mechanism.Methods(1)Investigating the relationship of OCT1 and neuropathic pain.CCI surgery was performed on the rat.The translation and expression of OCT1 were detected.The location of OCT1 in the rat’s DRG and its co-localization with the pain relating molecules were checked.(2)To research the impact of OCT1 on the rat’s neuropathic pain.I Whether blocking the OCT1 in the rat’s DRG can affect the development of the rat’s neuropathic pain: using the technique of microinjection,OCT1 siRNA was injected into the rat’s DRG,then CCI surgery was performed on the rat.Rat’s paw withdraw threshold to the mechanical stimulation,paw withdraw latency to the heat stimulation,paw withdraw latency to the cold stimulation were observed and detected.II Whether block the OCT1 in the rat’s DRG can affect the maintenance of the rat’s neuropathic pain: the CCI surgery was performed on the rat first,and then the OCT1 siRNA was microinjected into the rat’s DRG.Rat’s paw withdraw threshold to the mechanical stimulation,paw withdraw latency to the heat stimulation,paw withdraw latency to the cold stimulation were observed and detected.III Whether overexpress the OCT1 in rat’s DRG can affect the development and maintenance of the rat’s neuropathic pain: rAAV5-OCT1 was microinjected into the rat’s DRG to overexpress the OCT1,and then the rat’s pain behaviors were observed and recorded.(3)To study the mechanism that how OCT1 regulates the neuropathic pain.I qRT-PCR,Western Blot were used to check the mRNA and protein level of OCT1,DNMT3 a,MOR and Kv1.2 in the method(2)tissues.To confirm whether block the OCT1 in CCI rat can down regulate transcripton and expression of DNMT3 a.To confirm that overexpress the OCT1 in na?ve rat can up regulate DNMT3 a transcripton and expression,down regulate MOR and Kv1.2transcripton and expression.II To verify that blocking OCT1 in rat’s DRG can increase the therapy effect of morphine,and blocking OCT1 in rat’s DRG can decrease neuropathic pain is via the function of MOR.Results:1.The protein level of OCT1 was increased in the injured DRGs but not the contralateral side of DRGs after CCI.OCT1 in rat’s DRG increased from day 3after CCI and lasted till day 14 after CCI.2.OCT1 was distributed in the large,medium,and small cells.OCT1 mainly distributed in the large and medium cells.OCT1 was co-localiztion with neuronal nuclear antigen(NeuN)and glutaminase(GS).OCT1 was found in the same cells withneurofilament-200(NF200),plant lectin B4(IB4)and Calcitonin gene related peptide(CGRP).3.Previous injection of OCT1 siRNA into the DRG could inhibit CCI induced pain behaviors without affecting the locomotor function.Injection of OCT1 siRNA into the DRG after CCI surgery could alleviate CCI induced hyperalgesia and allodynia.4.Mimicking the high expression of OCT1 in the DRG induced rat with mechanical allodynia,thermal hyperalgesia,cold allodynia and spontaneous pain.5.None of the DRG microinjection,siRNA blocking or rAAV5 overexpression could affect the locomotor function.6.CCI surgery increased the mRNA and protein levels of OCT1 and DNMT3 a,and decreased the expression of MOR and Kv1.2 in the injured DRGs.Blocking expression of OCT1 could down regulate DNMT3 a and up regulate MOR and Kv1.2 expression.Overexpression of OCT1 in the DRG could upregulate the mRNA and protein levels of DNMT3 a and downregulate the expression of MOR and Kv1.2.7.OCT1 could co-localize with DNMT3 a,MOR or Kv1.2 in the DRG cells.8.Blokcing of OCT1 inhibited the regulation of MOR and Kv1.2 by DNMT3 a.9.OCT1 regulate the neuropathic pain caused by CCI via function of MOR and Kv1.2,and at last caused central sensitization.Conclusion:Dorsal root ganglia OCT1 can promote the transcription of DNMT3 a,and then down regulates the moleculars of MOR and Kv1.2.Thus,OCT1 promote the development and maintenance of neuropathic pain.