The Role Of P65 In Persistent Postoperative Pain

Background and ObjectivePersistent postoperative pain occurs after some common surgeries,for instance,opening heart surgeries,inguinal hernia repairings and cesarean surgeries.The accident is between 4% and 13%,its duration ranging from 3 to 30 months.Persistent postoperative pain is an important clinical problem and severely reduces the patient’s quality of life,while the mechanism has not been clear yet.At present,the role of ion channel receptors in pain has been taken more and more seriously,such as the potassium ion channel receptors and sodium ion channel receptors.Nav1.7 is one of the sodium receptor subtypes.It is mainly expressed in spinal cord dorsal root ganglion neurons in the peripheral nervous system and responsible for the incoming damaging-signal.But the role Nav1.7 plays in persistent postoperative pain is not clear.The NF-kappa B expressing in multiple types of cells is the important nuclear transcription factor.After the NF-kappa B activates in the nucleus involved in regulation of a variety of genes by combining with the promoter region of target gene.P65 is a common subtype of NF-kappa B and involved in the regulation of inflammatory mediators in the occurrence and development of pain.This study aims to investigate the role of Nav1.7 and p65 play in persistent postoperative pain and explore whether p65 is involved in the regulation of Nav1.7 in the persistent postoperative pain model.MethodsFirst of all,we detected the expression of Nav1.7 and p-p65(Phosphorylation of p65)in ipsilateral L4-6 DRGs of SMIR rats by western-blot and immunostaining,and then we intrathecally injected with ProTx-II(Nav1.7 blocker)or PDTC(p65 inhibitor)while detected the change of Nav1.7 expression and mechanical withdrawal threshold,finally chromatin immunoprecipitation assay method was used to detect whether could p-p65 bind in the Nav1.7 gene promoter region directly.The data were analyzed statistically by GraphPad Prism 5.0 statistical software.Results 1 The SMIR model rats mediated mechanical hyperalgesia of ipsilateral hind paw plantar.Between incision group(incision but no retraction)and naive group,PWT(paw withdrawal threshold)were no statistical difference.Ipsilateral PWT in SMIR group was significantly decreased from 5d after the SMIR surgery to 20 d compared with the incision group,while no significant difference exists in contralateral PWT between incision group and SMIR group.Compared with naive group,paw withdrawal latencies of bilateral hind paw plantar in SMIR group rats were not significantly changed,indicating that SMIR model was successfully established.2 Up-regulation of Nav1.7 in DRG of SMIR rats was involved in the hyperalgesia.We performed western blotting analysis,which revealed that Nav1.7 expression in the ipsilateral DRG of the SMIR rats was significantly up-regulated on 10 d and last until 20 d.To ascertain whether the up-regulated Nav1.7 plays a very important role in the mechanical hyperalgesia of SMIR rats,behavior change of SMIR rats were detected by i.t injection of ProTx-II.ProTx-II was found to effectively alleviate mechanical hyperalgesia of ipsilateral paws induced by SMIR,but had no effect on contralateral paws.3 Nav1.7 was expressed in positive cells of CGRP,NF-200 and IB-4.To further determine the cell-types of Nav1.7 expression in the DRG neurons,we used double immunofluorescence staining method.The result showed that Nav1.7 was expressed in positive cells of CGRP,NF-200,IB-4 but not in cells of GFAP(a specific marker of astrocyte).Cell diameter was measured by ImageJ software,we found that about 80% Nav1.7 positive cells are small diameter neurons,17% are medium diameter neurons,and only 3% are large-diameter neurons.4 P-p65 was over expressed in DRG of SMIR rats.As it has been reported,p-p65(phosphorylated NF-κB)involved in process of neuropathic pain and inflammatory pain.In the study,the role p-p65 played in persistent post-surgical pain was detected.We found that PDTC,an inhibitor of NF-κB,can prevent ipsilateral paw hyperalgesia induced by SMIR rats,but had no effect on contralateral paw.Western blotting result showed nucleoprotein p-p65 in rat ipsilateral L4-L6 DRGs at 10 d and 15 d after SMIR surgery increased obviously comparing with incision group;however the level of p-p65 in group pretreated with PDTC 0.5ug was down-regulated comparing with SMIR group.5 P-p65 contributed to the persistent post-surgical pain through up-regulated expression of Nav1.7 in DRG of SMIR rats.As it is known,p-p65 is a significant transcription factor that regulates transcription of multiple target genes.To further investigate whether p-p65 participates in the regulation of Nav1.7 in the persistent post-surgical pain model,PDTC was i.t injected to detect the expression change of Nav1.7.The result indicated that compared with incision group,Nav1.7 was increased and PDTC could prevent the up-regulation of Nav1.7 induced by SMIR at 10 d and 15 d after surger.Next we did double immunohistofluorescence stain.p-p65 was found to have co-localized with CGRP,NF-200 and IB-4.ChIP assay indicated p-p65 could combine with SCN9 A proximal promoter region for primer 2,primer 7,primer 8,primer 9.Conclusion:p-p65 induced up-regulated voltage-gated sodium channel Nav1.7 contributed to the PPP.