EphrinB2/EphB2 Signaling Pathway Is Involved In The Mechanism Study Of Bone Cancer Pain By Regulating Spinal Cord Synaptic Proteins And Inflammatory Signaling

Objective: Breast cancer is prone to bone metastasis,which causes bone tissue destruction and peripheral nerve damage,leading to central sensitization and severe pain.Studies have shown that Ephrin and its Eph receptors play an important role in synaptic plasticity and synaptic transmission,and Ephrin/Eph signaling abnormalities are closely related to the production and maintenance of various types of pain.In this study,we elucidated the role of Ephrin B/Eph B signaling pathway in bone cancer pain from the neural and inflammatory mechanisms of spinal central sensitization and explored the possibility of Ephrin B/Eph B as a therapeutic target for cancer pain.Methods: The model of cancer-induced bone pain was established by injecting MRMT-1 breast cancer cells into the left tibia of SD rats.The successful establishment of the model was determined by tibial X-ray,HE staining of tibia sections,and mechanical pain detection.14 days after the modeling,Ephrin B /Eph B signals were blocked by intrathecal injection of EphB2-Fc,and then changes in pain behavior of CIBP animals were analyzed to determine the analgesic effect of EphB2-Fc.Spinal cord tissue was collected to observe the morphological changes of synapses in CIBP rats by transmission electron microscopy.HE staining and Nissl staining were used to analyze the levels of inflammatory cell infiltration and neuronal protein synthesis in the spinal cord of CIBP rats.Proteomics,immunofluorescence and western blotting were used to analyze the expression changes of synaptic related proteins and inflammatory signaling proteins in the spinal cord of CIBP rats,and to further determine the regulatory effects of EphB2-Fc on the expression of these proteins.Results:Part I:（1）The model of CIBP was successfully constructed: CIBP rats showed the sensitivity of mechanical pain increased,tibial bone tissues injured（X-ray）and bone trabecular loss（HE staining）.（2）Changes of synaptic structure and neuronal function in CIBP rats: Transmission electron microscopy showed that the density of presynaptic activation region increased,the Nissl staining showed the protein synthesis ability of neurons decreased.（3）EphrinB2/EphB2 signal enhancement in CIBP rats: the expressions of EphrinB2,phosphorylated EphB2 and total EphB2 were increased in the spinal cord tissue.（4）Abnormal regulation of synaptic protein expression in CIBP rats: up-regulated expression of presynaptic membrane and synaptic vesicles related proteins,up-regulated expression of postsynaptic excitatory receptors,down-regulated expression of postsynaptic inhibitory receptors,up-regulated expression of calcium signaling and mitochondria mitogenesis related proteins.（5）EphB2-Fc alleviates bone cancer pain by inhibiting EphrinB2/EphB2 signaling: Intrasheath injection of EphB2-Fc reduced mechanical pain sensitivity in CIBP rats,down-regulated EphrinB2/EphB2 signaling in spinal cord,and restored synaptic related protein expression regulation.PartⅡ:（1）Inflammatory infiltration increased and activation of astrocytes and microglia in the spinal cord of CIBP rats;（2）NLRP3 inflammasome activation and inflammatory factor expression were increased in CIBP rats: The expressions of NLRP3,ASC,Activated-caspase-1,COX2,IL-1β and TNF-α were increased.（3）EphB2-Fc treatment inhibits spinal inflammation in CIBP rats: intrathecal injection of Eph B-Fc down-regulates the expression of inflammatory proteins associated with the spinal cord.Conclusion: EphB2-Fc plays an analgesic role in CIBP by inhibiting the EphrinB2/EphB2 signaling pathway in the spinal cord,regulating the balance of excitatory and inhibitory synaptic signals in the spinal cord and reducing inflammatory responses.