The Study On The Signal Pathways Of EphrinB-EphB In Pain

Part 1 PKA is required for the modulation of spinal nociceptive information related to ephrinB-EphB signalingObjectives:EphB receptors and their ephrinB ligands are implicated in modulating of spinal nociceptive information processing. Here, we investigated whether protein kinase A (PKA), acts as a downstream effector, participates in the modulation spinal nociceptive information related to ephrinB-EphB signaling.Methods:PKA catalytic subunit (PKAca) expression, cAMP-response element binding protein phosphorylation (p-CREB), thermal hyperalgesia, and mechanical allodynia were analyzed in male, adult C57BL/6 mice. A PKA inhibitor, H89, was used to investigate the contribution of PKA to spinal nociceptive information related to ephrinB-EphB signaling.Results:Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal PKAca and p-CREB. Pre-treatment with H89, a PKA inhibitor, prevented the activation of CREB by ephrinB2-Fc. Inhibition of spinal PKA signaling prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Furthermore, blockade of the EphB receptors by intrathecal injection of EphB2-Fc reduced formalin-induced inflammatory, chronic constrictive injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, which were accompanied by decreased expression of spinal PKAca and p-CREB.Conclusions:Overall, these results confirmed the important involvement of PKA in the modulation of spinal nociceptive information related to ephrinBs-EphBs signaling. This finding may have important implications for exploring the roles and mechanisms of ephrinB-EphB signaling in physiologic and pathologic pain.Part 2 EphrinB-EphB signaling regulates spinal pain processing via PKCyObjectives:Spinal ephrinB-EphB signaling is involved in the modulation of pain processing. The aim of the present study was to investigate whether protein kinase C-y (PKCy) acts as a downstream effector in regulating spinal pain processing associated with ephrinB-EphB signaling in mice.Methods:Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filaments test, respectively. PKCy expression were determined by Western blot analysis. PKCy-targeting small interfering RNA (siRNAs) were used to investigate the contribution of PKCy to spinal nociceptive information related to ephrinB-EphB.Results:The intrathecal injection of ephrinB2-Fc, an EphB receptors activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PKCy. Knockdown of spinal PKCy prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptors blocker, suppressed formalin-induced inflammatory, CCI-induced neuropathic, and TCI-induced bone cancer pain behaviors, in addition to reducing the activation of spinal PKCy. Finally, the intrathecal injection of MK801, an N-methyl-D-aspartate (NMDA) receptor blocker, prevented the pain behaviors and spinal PKCy activation induced by ephrinB2-Fc.Conclusions:Overall, the results confirm the important role of PKCy in the regulation of spinal pain processing associated with ephrinB-EphB signaling.Part 3 Involvement of the PLCyl in spinal pain processing associated with ephrinB-EphB signalingObjectives:Spinal ephrinB-EphB signaling is involved in the modulation of pain processing. Here, we investigated whether phospholipase C-yl (PLCyl) acts as a downstream effector in regulating spinal pain processing associated with ephrinB-EphB signaling.Methods:Thermal hyperalgesia and mechanical allodynia were measured using radiant heat and von Frey filaments test. The expression of PLCyl was determined by western blot. To investigate the contribution of PLCyl to spinal nociceptive information related to ephrinBs-EphBs, PLCyl-targeting siRNA was used.Results:The intrathecal injection of ephrinB2-Fc, an EphB receptors activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PLCγ1. Knockdown of spinal PLCyl prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptors blocker, suppressed formalin-induced inflammatory and CCI-induced neuropathicr pain behaviors, in addition to reducing the activation of spinal PLCγ1.Conclusions:These results confirm the important role of PLCyl in the regulation of spinal pain processing related to ephrinB-EphB signaling.Part 4 COX-2 is required for the modulation of spinal nociceptive information related to ephrinB-EphB signalingObjectives:EphB receptors and their ephrinB ligands are implicated in modulating spinal nociceptive information processing. Here, we investigated whether cyclooxygenase-2 (COX-2), acts as a downstream effector, participates in the modulation of spinal nociceptive information related to ephrinB-EphB signaling.Methods:Thermal hyperalgesia and mechanical allodynia were measured by using radiant heat and von Frey filaments test, respectively. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of spinal COX-2 mRNA. Spinal COX-2 and extracellular signal-regulated kinase (ERK) protein were determined by western blot analysis.Results:Intrathecal injection of ephrinB2-Fc caused thermal hyperalgesia and mechanical allodynia, which were accompanied by increased expression of spinal COX-2 mRNA and protein. Inhibition of spinal COX-2 prevented and reversed pain behaviors induced by the intrathecal injection of ephrinB2-Fc. Blockade of EphB receptors by intrathecal injection of EphB2-Fc reduced complete Freund’s adjuvant (CFA)-induced inflammatory pain behaviors, which were accompanied by decreased expression of spinal COX-2 mRNA and protein. Furthermore, treatment with U0126, a mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor, suppressed spinal ERK activation and COX-2 mRNA and protein expression induced by intrathecal injection of ephrinB1-Fc.Conclusions:These results confirmed the important involvement of COX-2 in the modulation of spinal nociceptive information related to ephrinBs-EphBs signaling.