Mechanism Research On Ephrinb/EphB Signaling Contributes To Spinal Nociceptive Processing Via Calpain-1 And Caspase-3

【Background】Pain has always been a major problem in the medical field and one of the most urgent problems in clinical work.It can be routinely divided into inflammatory pain,neuropathic pain and injury-induced pain according to their neurobiological characteristics.The classification is clear,but the specific mechanism and effective treatment is still not clearly defined.In recent years,studies have shown that the subfamily of receptor tyrosine kinase(RTKs)ephrins and their receptor Ephs play a role in many aspects of central nervous system development,such as synaptic remodeling,axonal guidance and tissue plasticity.The diversity of Ephs receptors is due to its interaction with ligand ephrins through the interaction of cross-cell membranes to form complex signal systems involved in a wide range of tissue formation processes,especially the development and maintenance of dendritic spines,and take important role in the nociceptive pain and neuropathic pain.This action is adapted to the majority of excitatory glutamate postsynaptic potentials in the brain,not only in the control of the cytoskeleton of actin in dendritic spines,but also in the trafficking of glutamate receptors involved in the formation of synaptic plasticity.In addition,EphB receptors have also recently been identified to be associated with pathophysiology of Alzheimer’s disease and neuropathic pain,and thus become a target for therapeutic intervention.In addition to the involvement of ephrins/ephs signaling system in the process,there is also a large number of calpain activation during the development of nociceptive pain.Studies have shown that activated calpain initiates massive influx of Ca2 + and promotes the degradation of skeletal and plasma membrane proteins,affecting the phosphorylation of calpain and participated in the process of neurodegeneration and synaptic plasticity.Taken together,we hypothesized that the ephrin / eph signaling pathway regulates spinal nociceptive pain by regulating changes in calpain in the spinal cord that are involved in synaptic plasticity.Our study aimed to observe the effects of intrathecal injection of receptor tyrosine kinase subfamily EphB1 and its ligand ephrinB2-Fc(Fc-tag)on spinal cord pain in mice and its effect on the expression of calpain-1 by animal behavior assay,western blot(WB),immunohistochemistry(IHC)and immunofluorescence(IF).To explore whether the ephrinB / EphB signaling pathway can participate in the process of spinal nociceptive pain through the interaction between calpain-1 and its downstream molecules.【Methods】1.Male Kunming mice were randomly divided into two parts,one part were established nociceptive model by intrathecal injection of ephrinBs ligand in spinal cord in mice,the other part were establish chronic constriction injury(CCI)pain reversal model by intrathecal injection of EphBs receptors.The changes of behavior in two groups of different model mice were measured by using mechanized withdrawal threshold and thermal withdrawal latency.During the course of behavioral testing,the spinal cord tissues of some mice were taken out,and the expressions of calpain-1 and caspase-3 in the spinal cord were detected by Western blotting and immunohistochemistry.2.The mice were intrathecal injection with ephrinBs and calpain-1 specific inhibitor MDL28170,according to the injection sequence mice were divided into MDL28170 pre-treatment group and MDL28170 post-treatment group.The behavioral changes of the two groups were observed by using the mechanized withdrawal threshold and thermal withdrawal latency,and the expression of calpain-1 and caspase-3 in the course of behavioral changes were detected by WB and IHC respectively.3.Spinal cord nociceptive model was established by intrathecal injection of ephrinBs ligand.After the model was successfully made by behavioral assay,spinal cord tissues of mice were taken and localization of expressions of calpain-1 and caspase-3 was detected by immunofluorescence.【Results】1.Through the first part of the experiment we found: intrathecal injection of ephrinB2-Fc resulted in a time-and dose-dependent mechanical hyperalgesia and thermal allodynia and increased expression of calpain-1 and caspase-3 in the spinal cord(P <0.001);The injection of the ligand ephrinB1-Fc showed only obvious mechanical hyperalgesia but not thermal allodynia(P <0.01).Compared with the saline control group,intrathecal injection of EphB2-Fc significantly reduced the pain response of CCI mice(P <0.05).2.Through the second part of the experiment we found: intrathecal injection MDL28170 pre-treatment or post-treatment were significantly reversed mechanical hyperalgesia and thermal allodynia responses induced by ephrinB2-Fc injection and decreased the expression of calpain-1 and caspase-3 in the spinal cord(P <0.01).3.Immunofluorescence staining revealed that both calpain-1 and caspase-3 were co-expressed with NeuN(neuronal markers)rather than GFAP(astrocyte marker)and Iba-1(oligodendrocyte marker).【Conclusion】 This study leads to the following conclusion:1.The receptor tyrosine kinase subfamily ephrinBs/EphBs participated in spinal nociceptive processing.Intrathecal injection of ligand ephrinBs produced mechanical hyperalgesia and thermal allodynia in a time-and dose-dependent manner in mice;intrathecal injection of receptor EphBs reduced or even reversed the pain response in CCI mice.Immunofluorescence staining revealed that both calpain-1 and caspase-3 were co-expressed with NeuN(neuronal markers)rather than GFAP(astrocyte marker)and Iba-1(oligodendrocyte marker),Indicating that the mechanism may be related to regulating the expression of calpain-1 and caspase-3 in neurons.2.EphrinBs/EphBs involved in the pain of spinal nociceptive processing mainly through the regulation of calpain-1 and caspase-3 expression changesin the spinal cord.The intrathecal injection of ephrinBs induced mechanical hyperalgesia and thermal allodynia,which is directly related to the increased expression of calpain-1 and caspase-3 in the spinal cord.This effect can be antagonized by MDL28170,a specific inhibitor of calpain-1,and induced significantly decreased of caspase-3 expression.3.Immunofluorescence staining showed that both calpain-1 and caspase-3 were only co-expressed with NeuN(neuronal marker),indicating that the mechanism may be related to the changes of calpain-1 and caspase-3 expression in neurons.Receptor tyrosine kinase subfamily,ephrinBs / EphBs signaling pathway,mainly involved in the occurrence and development of spinal nociceptive pain through the action of calpain-1and caspase-3 in spinal cord.