The Effects Of Pain Behavior And Molecular Mechanism Of Ninjurin2 In Rats With Neuropathic Pain

BackgroundNeuropathic pain is a pain directly caused by disease or damage of the somatosensory nervous system,which is a chronic pain.Prolonged pain conditions increase the incidence of anxiety and depression in patients.At present,the research on the pathogenesis of neuropathic pain is still in the exploration stage,The therapeutic effects of clinically available drugs are limited and mostly accompanied by significant side effects.Post-herpetic neuralgia is the typical clinical neuropathic pain.Previous studies have suggested that the occurrence of neuropathic pain is mainly related to central sensitization and peripheral sensitization.Neuronal inflammatory response,NF-κB and pro-inflammatory factors produced during oxidative stress,and activation of glial cells are involved in neuropathic pain processes.The study found that ninjurin 2 is highly up-regulated after nerve injury in ischemic stroke;In vitro culture of cells revealed that ninjurin 2 is up-regulated in Schwann cells of the distal ganglion after peripheral nerve injury,and promotes neurite outgrowth in dorsal root ganglion neurons through homophilic cell interactions between neuronal axons and Schwann cells;Studies have also shown that NINJ 2 protein may be a novel regulator of endothelial inflammation and activation,it has a pro-inflammatory effect in vascular endothelial cell activation and inflammation.However,whether ninjurin 2 is involved in neuropathic pain has not been studied.Based on this experimental design,the relationship between ninjurin 2 and neuropathic pain and its related molecular mechanisms were studied.ObjectiveThis experiment intends to use the rat sciatic nerve branch selective ligation and cutting model(Spared Nerve Injury,SNI),Western-blot technique was used to observe the expression of NINJ 2 and NF-κB in the spinal cord of SNI rats,Immunofluorescence double staining was used to detect the expression of NINJ 2 protein,nerve cells and NF-κB in the spinal cord of SNI rats.Gene silencing of NINJ 2 protein expression using intrathecal drug injection technology,observing its effect on pain behavior in SNI rats,and the expression changes of NF-κB and pro-inflammatory factors IL-1β,IL-6 and TNF-α in the spinal cord.To explore the role of NINJ 2 protein in the formation and progression of neuropathic pain,its possible molecular mechanisms,and explore new therapeutic targets for neuropathic pain.Methods 1 Male SD rats were divided into two groups randomly: model group(SNIgroup,n=8)and sham operation group(Sham group,n=8).Paw withdrawal threshoid(PWT)was measured continuously on before the SNI surgery,and the 1st,3rd,5th,7th,10 th,and 14 th day after surgery.2 Male Sprague-Dawley rats were divided into two groups randomly: model group(SNI group,n=15)and sham operation group(Sham group,n=12).The expression of NINJ 2 protein and NF-κB in the spinal cord before operation and 3rd,7th and 14 th after operation were measured by Western-blot technique.Immunofluorescence double staining was used to detect the colocalization of NINJ 2 protein,NF-κB and nerve cells in the spinal dorsal horn of rats after 14 days.3 Male Sprague-Dawley rats were divided into four groups randomly: Sham+saline group(n=8),SNI+saline group(n=8),SNI+siRNA group(n=8)and SNI+scrRNA group(n=8).).The corresponding drugs were intrathecalally injected into the sheath tube,and the model was surgically performed 1 week later.The PWT of the rats were measured 3 days,1 day before surgery,1st,3rd,5th,7th,10 th,and 14 th days after surgery.The expression levels of NINJ 2 protein and NF-κB in the spinal dorsal horn of rats in each group were detected by Western-blot technique.And the expression levels of proinflammatory cytokines IL-1β,IL-6 and TNF-α in the spinal cord of rats in each group were measured by enzyme-linked immunosorbent assay(ELISA).Results 1 SNI surgery causes hyperalgesia in ratsSNI surgery caused mechanical hyperalgesia in the hind paw of therat.Pain behavioral results showed no significant difference in preoperative basic PWT between the two groups;PWT began to decrease significantly in the SNI group 3 days after surgery,reached the lowest value on the 7th day after surgery,and lasted 14 days after surgery.SNI surgery significantly reduced rat PWT,and the experimental neuropathic pain model was successfully constructed.2 The expression of NINJ 2 and NF-κB in the spinal cord of rats after SNIwasup-regulated;the expression of NINJ 2 protein and neuronal markers NeuN and NF-κB were co-expressed in the spinal cord.Western-blot results showed that the expression levels of NINJ 2 and NF-κB in the spinal cord of the SNI group increased at 3rd,7th and 14 th after operation,and the expression level was the highest on the 7th postoperative day.The protein expression level and pain behavior were the highest.There was a certain correlation between changes,and the trend of NF-κB was close to that of NINJ 2 protein.The expression levels of NINJ 2 protein and NF-κB in the spinal cord of Sham group were not significantly changed.Immunofluorescence double staining revealed that NINJ 2 protein was co-expressed with neuronal markers-NeuN and NF-κB.3 Intrathecal injection of lentivirus to interfere with ninjurin 2 expression affects pain behavior in SNI rats and regulates the expression of NINJ2 protein,NF-kB and pro-inflammatory factors in the spinal cord of SNI ratsPWT results showed that pre-intrathecal injection of NINJ 2 siRNA significantly increased PWT levels in SNI rats,and PWT in SNI+siRNA group increased at 3rd,5th,7th,10 th,and 14 th days after surgery.The spinal cord of the rats was taken 14 th days after surgery.The results of Western-blot showed that the expression of NINJ 2 and NF-κB in the spinal cord of SNI rats decreased compared with the SNI+Saline group(P <0.05).NINJ 2 protein may regulate the expression of NF-κB in neuropathic pain.The ELISA results showed that compared with the SNI+Saline group,the expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in the SNI+siRNA group were significantly down-regulated,indicating that the NINJ 2 protein plays a proinflammatory role in neuropathic pain.ConclusionThese results suggest that the selective ligation and sciatic nerve injury of rat sciatic nerve can up-regulate the expression of NINJ 2 protein in rat spinal cord.NINJ 2 protein may regulate the expression of NF-κB,pro-inflammatory factors IL-1β,IL-6 and TNF-α.Involved in the process of neuropathic pain,NINJ 2 protein plays a pro-inflammatory role in the neuroinflammatory response of neuropathic pain.