PKA Is Involved In Neuropathic Pain Through The P38MAPK Pathway To Mediate Spinal Cord Cell Apoptosis

Purposes: To investigate the expression of protein kinase A(PKA)in neuropathic pain and its possible mechanism.Methods: The expression profile data set GSE24982 of neuropathic pain was downloaded from the GEO database,and differentially expressed genes(DEGs)were selected by the R software limma package,and the cluster profile package was used to perform gene ontology and KEGG pathway enrichment analysis on DEGs.The protein-protein interaction network was constructed using the STRING database,and the cytoHubba plug-in of Cytoscape software was used to screen the hub gene.Then,male SD rats weighing 200-220 g were selected to prepare the neuropathic pain model by the method of spared nerve injury(SNI).The mechanical pain threshold of rats was measured by von Frey wire to determine whether the model was successful.The expression of key genes,p-p38 MAPK,p38MAPK,TNF-?,IL-1?,caspase-3,caspase-9,bax and bcl-2 were detected by western blot.Expression and localization of key genes by immunofluorescence single staining.Immunofluorescence double staining showed the colocalization of key genes with spinal astrocytes,microglia and neurons.ELISA was used to detect the content of TNF-? and IL-1?,and TUNEL was used to detect the apoptosis of spinal cord cells.After PKA and p38 MAPK inhibitors were injected into the intrathecal of rats,the expression of key genes,p-p38 MAPK,p38MAPK,TNF-?,IL-1?,caspase-3,caspase-9,bax and bcl-2 were detected again by western blotting.TNF-? and IL-1? were detected by ELISA.Apoptosis of spinal cord cells was detected by TUNEL.Results: In this study,449 DEGs and 20 hub genes were screened,among which PKA was identified as the key gene involved in neuropathic pain.SNI can induce the decrease of mechanical pain threshold(P < 0.05),the increase of PKA expression inspinal cord(P < 0.05),the activation of p38 MAPK pathway(P < 0.05),and the increase of TNF-?,IL-1?,caspase-3,caspase-9,bax,bcl-2 protein expression(P <0.05).Immunofluorescence single staining showed that PKA expression in spinal cord increased,and mainly located in the dorsal horn of SNI ipsilateral spinal cord.Immunofluorescence double staining showed that PKA was co-localized with neurons,astrocytes and microglia.TUNEL double staining showed that apoptosis cells were mainly neurons.Intrathecal injection of PKA inhibitor could not only alleviate the mechanical hyperalgesia of SNI rats(P < 0.05),but also decrease the expression of TNF-?,IL-1?,caspase-3,caspase-9,bax,bcl-2 protein(P < 0.05),and inhibit the activation of p38 MAPK pathway(P < 0.05).However,although p38 MAPK inhibitor could reduce the expression of TNF-?,IL-1?,caspase-3,caspase-9,bax and bcl-2(P< 0.05),it had no effect on PKA expression(P > 0.05).Conclusion: PKA is significantly up-regulated in the spinal cord of SNI-induced neuropathic pain rats,and is involved in the induction and maintenance of neuropathic pain.The mechanism may be realized by activating the p38 MAPK pathway to mediate spinal cord cell apoptosis.