EIF4G2 Contributes To Neuropathic Pain Through Down Regulation Of The Mu Opioid Receptor In Mouse Primary Sensory Neurons

Objective:Neuropathic pain(NP)is a type of intractable pain syndrome caused by the lesion or disease of somatosensory system,which badly diminished patients’ quality of life.Many diseases that damage peripheral or central nervous system(such as diabetes,herpes zoster,stroke,spinal cord injury,nerve injury)induced neuropathic pain,and it is estimated over 90 million patients in China are suffering from such pain condition,which brings a huge burden on social economy system.Current treatments for these pain conditions are limited due to the unclarity of its mechanism,thus,investigation the mechanism of NP and provide new therapeutic targets carries a great deal of significance.As we all know,peripheral nerve injury leads to abnormal expression of receptors,ion channels,enzymes and other molecules in dorsal root ganglia(DRG),altered neuronal excitability and induced peripheral sensitization,which plays a key role in the development and maintenance of neuropathic pain.For example,peripheral nerve injury downregulates the expression of the μ-opioid receptor(MOR),leads to the diminishing of MOR-controlled neurotransmitter release from the primary afferents,which limited opioid analgesic efficacy for the treatment of neuropathic pain.After the process of transcription and post-transcription,genes turn into mature mRNA,and are translated into polypeptide chains on ribosomes in the cytoplasm.The eukaryotic initiation factor 4 gamma 2(eIF4G2)is a "bridge" signaling molecule for protein translation initiation,which can recruit translation initiation factors such as eIF4A and eIF4E to form a translation initiation complexes and regulate protein synthesis.eIF4G2 is involved in two major modes of protein translation,ie.capdependent and cap-independent protein translation and has a variety of biological functions that regulate cell proliferation,apoptosis,neurodevelopment and other processes.Like MOR,most of the signaling molecules involved in neuropathic pain are proteins,we speculate that their expression is likely to be regulated by eIF4G2,but it is not clear whether this is actually the case.Therefore,the present study hypothesize that peripheral nerve injury activates eIF4G2,which disrupt the initiation of protein translation,leading to the downregulation of MOR expression that contribute to neuropathic pain.In the present study,we mainly used the spinal nerve ligation(SNL)mice model and combines methods including animal behavior tests,primary DRG neuron culture,molecular biology,immunofluorescence,DRG microinjection and RNA interference technology to(1)clarify the changes of peripheral nerve injury on eIF4G2 expression and its distribution in DRG;(2)explore the role of eIF4G2 in the development and maintenance of neuropathic pain,evaluate its implications as an analgesic target;(3)elucidate whether eIF4G2 regulate MOR expression contribute to neuropathic pain.This study may clarify the underlying mechanisms of NP and provide a new avenue for its management.Chapter 1.Peripheral nerve injury induces changes in the expression and distribution of eIF4G2Materials and Methods:(1)The SNL and CCI mouse model were used.Paw mechanical withdrawal frequency(PWF)to 0.07g and 0.4g von frey stimulation,paw thermal withdrawal latency(PWL-heat)and paw cold withdrawal latency(PWL-cold)on the injured side(Ipsi)and contralateral side(Contral)were assessed at 3,7,10 and 14 days after surgery;(2)the mRNA and protein expression of eIF4G2 in the spinal dorsal horn and DRG at different time points after SNL were evaluated by RT-qPCR and Western blot;(3)the protein expression of eIF4G2 in the Ipsi DRG of CCI model were evaluated by western blot;(4)the DRG of naive mice were collected,double immunofluorescence was used to assess the co-expression of eIF4G2 and different types of neuronal markers(NF200,IB4,CGRP)or satellite glial cell markers(GS),and the percentage of positive cells and cross-sectional cell area were counted;(5)on the 7th day after SNL,DRG from the ipsilateral and contralateral side were collected,eIF4G2 positive neurons and distribution patterns were compared between the two sides.Results:(1)Compared with basal values,PWF on the Ipsi side of SNL and CCI mice significantly increased at all postoperative time points,mechanical hyperalgesia occurred;PWL-heat and PWL-cold significantly decreased,heat and cold hyperalgesia induced,while pain thresholds in the sham group remained constant,indicating successful preparation of SNL and CCI models;(2)At 3,7,and 14 days after SNL,both eIF4G2 mRNA and protein expression in the DRG on the Ipsi side increased significantly and reached the peak on day 7,with a 2.3-fold increase in mRNA expression and a 1.9-fold increase in protein expression;however,there was no significant change in protein expression in the Contral side DRG and the Ipsi side spinal dorsal horn;(3)on day 7 after CCI,the eIF4G2 protein expression increased with 1.7-fold compared to basal values,verifying that peripheral nerve injury caused increased eIF4G2 expression;(4)in naive mouse DRG,eIF4G2 was mainly expressed in neuronal cytoplasm,not satellite glia,with co-localization with NF-200,IB4 and CGRP;79%of eIF4G2-positive neurons were co-labeled with NF-200,13.6%colabeled with IB4,and 14%co-labeled with CGRP;40.5%of eIF4G2 positive neurons were large diameter neurons(cell area>600 μm2),42.1%were medium diameter neurons(cell area 300-600 μm2),and 17.4%were small diameter neurons(cell area<300 μm2);(5)In SNL model,the distribution of eIF4G2 on the small-diameter neurons was increased on the Ipsi side.(Ipsi vs.Contral,20.9%vs.13.6%).Chapter 2.The role of DRG eIF4G2 in neuropathic painMaterials and Methods:(1)On day 5 before SNL,eIF4G2 small interfering RNA(siRNA)or its negative control(scrRNA)was pre-microinjected into DRG,the changes of mechanical and thermal pain threshold at different time points after surgery were detected;on day 5 after SNL,the DRG was collected and Western blot was performed to detect the effect of siRNA on eIF4G2 protein expression;the spinal dorsal horn was collected and Western blot was performed to detect the expression of ERK,pERK and GFAP;(2)on day 7 after SNL,the mice had developed nociceptive hypersensitivity,postmicroinjection of eIF4G2 siRNA or scrRNA was applied,the changes of PWF,PWLheat and PWL-cold were observed;on day 14 after SNL,the DRG was collected and Western blot was performed to detect the effect of siRNA on eIF4G2 protein expression;the spinal dorsal horn was harvest and Western blot was performed to detect the expression of ERK,pERK and GFAP;(3)the DRG of naive mice was microinjected with AAV-eIF4G2 and its negative control(GFP),the changes of PWF,PWL-heat and PWL-cold were observed from the 1st to the 8th week after virus injection;at the 8th week,conditioned place preference(CPP)test was used to determine spontaneous pain.The expression of ERK,pERK and GFAP in the spinal dorsal horn was detected by Western blot.Results:(1)eIF4G2 siRNA pretreatment attenuated SNL-induced mechanical,thermal and cold hypersensitivity;Western blot results showed that eIF4G2 expression with 0.46-fold reduce in the siRNA group,and pERK1/2 and GFAP expression in the spinal dorsal horn were inhibited,indicating that eIF4G2 is involved in the development of neuropathic pain;(2)eIF4G2 siRNA post-microinjection had a therapeutic effect on the SNL induced neuropathic pain,pERK1/2 and GFAP expression in the spinal dorsal horn were suppressed,suggesting that eIF4G2 is involved in the maintenance phase of neuropathic pain;(3)mice treated with AAVeIF4G2 showed increased PWF,decreased PWL-heat and PWL-cold starting from 3 weeks and lasted to 8 weeks after DRG microinjection;the CPP test indicated that mice in the eIF4G2 overexpression group showed significant preference in lidocainepaired chambers;Western blot showed that pERK1/2 and GFAP were activated in the spinal dorsal horn of AAV-eIF4G2 treated mice DRG.Chapter 3.eIF4G2 contribute to neuropathic pain through downregulate of muopioid receptor in DRGMaterials and Methods:(1)Double immunofluorescence was used to assess the co-expression of eIF4G2 and MOR;(2)Western blot to detect MOR expression changes after DRG microinjected with eIF4G2 siRNA or scrRNA;(3)Western blot to detect MOR expression changes after DRG microinjection with AAV-eIF4G2 or GFP;(4)primary DRG neurons were cultured,transfected with AAV-eIF4G2 or(and)siRNA,Western blot was performed to detect the expression of eIF4G2 and MOR;(5)eIF4G2 siRNA was pre-microinjected into DRG on day 5 before SNL,and the maximal possible analgesic effect of morphine was detected on day 3 after SNL;on day 4 after SNL,methylnaltrexone(MNTX)was injected intraperitoneally,the changes of PWL-heat were observed.Results:(1)In naive mouse DRG,23.8%of eIF4G2-positive neurons were co-labeled with MOR;(2)MOR protein expression in DRG of mice microinjected with eIF4G2 siRNA was higher than in the scrRNA group(0.83-fold change vs.0.43-fold change),indicating that inhibition of eIF4G2 increases MOR expression;(3)DRG microinjected of AAV-eIF4G2 downregulated MOR expression with 0.42-fold decrease;(4)overexpression of eIF4G2 in primary cultured DRG neurons downregulates MOR,then transfection of eIF4G2 siRNA reverses this decrease,further verifying eIF4G2 negative regulate to MOR;(5)on day 3 after SNL,the%MPAE of mice pre-microinjected with eIF4G2 siRNA was significantly increased(scrRNA group vs.siRNA group:54.5%vs.81.4%);on day 4 after SNL,MNTX reversed the analgesic effect produced by inhibition of DRG eIF4G2.Conclusion:1.In peripheral nerve injury induced neuropathic pain mice,eIF4G2 expression was dynamically increased only in the nerve injury related DRG.In naive mouse DRG,eIF4G2 was distributed in large,medium and small diameter neurons,peptidergic and non-peptidergic neurons,whereas after nerve injury,the expression of eIF4G2 in small diameter neurons was increased.The present study identified eIF4G2 as a novel target involved in neuropathic pain.2.DRG knockdown of eIF4G2 attenuates nociceptive hypersensitivities and spinal central sensitization during the development and maintenance phases of neuropathic pain.DRG overexpression of eIF4G2 induced evoked and spontaneous pain in na?ve mice and also lead to spinal central sensitization.This study revealed eIF4G2 may serve as an important therapeutic target for neuropathic pain.3.eIF4G2 has a spatial basis of interaction with MOR,and both in vivo and in vitro experiments suggest negative regulation of MOR by eIF4G2 as a mechanism for neuropathic pain.Inhibition of eIF4G2 enhances the analgesic effect of morphine on neuropathic pain via upregulate MOR.The present study enriched the posttranscriptional mechanisms regulating the expression of peripheral opioid receptors and provided new ideas for better use of opioids for neuropathic pain.