The Roles And Mechanism Of PIM1 In Regulating Neuropathic Pain In Dorsal Root Ganglion

BackgroundNeuropathic pain is a spontaneous,persistent and chronic pain induced by primary and secondary damages to the peripheral or central nervous system.It is characterized by spontaneous pain,abnormal hyperalgesia and even hypersensitivity.Dorsal root ganglion(DRG)is a major place that contain incoming sensory neuron's cell bodies,which mediate the activities of DRGs through nerve signals transmission.PIM1 is a serine/threonine kinase which belongs to PIM family.Until now,there is little reports on the expression and roles on DRGs.Therefore,it is of great significance to explore the effects of PIM1 on the excitability of neurons in DRGs to elucidate the pathogenesis of neuropathic pain and provide the potential therapeutic targets for drug development in treatment of neuropathic pain.MethodsPART?.Identify the distribution and expression of PIM1 in DRGs during neuropathic pain.(1).PIM family m RNA expression was measured in the neuropathic pain model established by L4 spinal nerve ligation(SNL).(2).PIM1 m RNA and protein in the ipsilateral L4 DRG was detected in the neuropathic pain model.(3).The localization and the distribution of PIM1 in the DRGs was identified by immunofluorescence.PART ?.Investigate the effects of PIM1 inhibition on the behavior of SNL induced neuropathic pain model.(1).Screen the sequence that knockdown PIM1 in the neuro-2a cell lines.(2).Investigate the effects of PIM1 knockdown in vivo on the paw withdrawal latency induced by mechanical stimulation,paw withdrawal frequency induced by thermal and cold stimulation when PIM1 in the L4 DRG was inhibited through adeno-associated virus microinjection into the DRG.PART ?.Investigate the potential mechanisms of PIM1 inhibition in regulating neuropathic pain.(1).The co-expression of PIM1 and CXCR4 in the DRGs was identified by immunofluorescence staining.(2).To investigate the effects of PIM1 on the CXCR4 and phosphorylated CXCR4 protein expression in vivo and vitro study.(3).To investigate the effects of CXCL12/CXCR4 binding which transferred into cytoplasm.Results1.Compared with Sham group,the PIM1 m RNA in the ipsilateral L4 DRG was increased significantly.While the PIM2 m RNA was decreased and there was no significantly difference about the PIM3 m RNA at 7 days after SNL surgery.2.Compared with 0 day,the PIM1 protein in the ipsilateral L4 DRG was increased persistently at 3 day after SNL surgery,while there were no significant changes on the control side.However,there were little variation on the control or ipsilateral L3 DRG at scheduled time points after SNL surgery when compared with 0 day.3.Immunofluorescence staining showed that PIM1 was expressed in the neurons of Neu N~+neuron cells,instead of GS~+satellite glial cells.Furthermore,the cross sectional area of 46%PIM1~+neurons were between 0 and 300?m~2,30%PIM1~+ neurons were between 300 and 600?m~2 and 24%PIM1~+neurons were between 600 and 2000?m~2.Besides,22.8%PIM1~+neurons were co-expressed with CGRP,14.6% PIM1~+neurons were co-expressed with IB4 and 32.4%PIM1~+neurons were co-expressed with SP.4.Microinjection of p AAV-sh PIM1 into DRG inhibited PIM1 protein upregulation trend induced by SNL surgery.Furthermore,compared with SNL+Scramble group, microinjection of p AAV-sh PIM1 into the DRGs rescued the behavior change induced by SML surgery on the ipsilateral side.Including downregulating SNL induced paw withdrawal frequency to mechanical stimuli,upregulation SNL-induced paw withdrawal latencies to thermal/cold stimulation.However,there was no significantly behavior change in the control side.5.Immunofluorescence staining showed that PIM1 and CXCR4 were co-expressed in the DRG neurons.In vitro study,PIM1 overexpression significantly increased the phosphorylated CXCR4 protein,but have little significant effect on CXCR4 protein expression.Knockdown PIM1 significantly inhibited the phosphorylated CXCR4 expression.But have little effects on CXCR4 protein expression.In vivo study, microinjection p AAV-sh PIM1 into DRG significantly inhibited the phosphorylated CXCR4 but have little effects on CXCR4 expression.In addition,the confocal laser microscopy showed that PIM1 inhibitor SMI-4a significantly inhibited the ratio of CXCL12 to CXCR4 binding into neuro-2?cell lines.ConclusionPIM1 protein in the DRG was increased significantly which was involved in the occurrence and maintenance of neuropathic pain after SNL surgery.Inhibition PIM1 may improve neuropathic pain induced by SNL surgery by regulating CXCL12/CXCR4pathway and inflammatory responses.The results suggested that PIM1 maybe a potential therapeutic target for neuropathic pain.