The Mechanism Of Neuroligins Involving In The Development Of Bone Cnacer Pain

Objective:Bone cancer pain is one of the most intractable chronic pain,which occurs in patients from either primary bone tumors or metastases,and the mechanism is extremely complex and widely involved.Neuroligins(NLs)are postsynaptic cell-adhesion molecules implicated in synaptic maturation and function.Methods and Results:(1)Illumina HiSeqTM revealed bone cancer pain related genes in lumbar Spinal cord and DRG.Methods:Female SD rats weighting in 180～200 g were randomly divided into Sham group and Carcinoma group,after the construction of bone cancer pain model,the paw withdrawal threshold(PWT)was determined.21 days later,the L4～L6 right spinal cord dorsal horn and dorsal root ganglion(DRG)were harvested for high-throughput sequencing.Results:The mRNA expression profiles in carcinoma-baring rats were compared with sham.In DRG,there were 1274 differential expressed genes,including 684 up-regulated genes and 590 down regulated genes.In spinal dorsal horn,we identified 2098 differential expressed genes,among which 1632 genes up regulated and 466 genes down regulated.(2)Overexpress NL1 and NL2 in spinal dorsal horn significantly prevent the bone cancer induced mechanical allodynia.Methods:Lentivirus individually overexpressing NL1,NL2 or NL3 was constructed and injected into the spinal cord of carcinoma baring rats.Western blot,RT-PCR and Multiple immunostaining were used to detect the expression and distribution of NL1,NL2 and NL3,Combined with behavioral analysis to determine whether NL1,NL2 and NL3 were involved in the formation of bone cancer pain.Results:Individually overexpressing NL1 and NL2 in spinal dorsal horn attenuated the mechanical allodynia of rats with carcinoma inoculation,whereas the paw withdrawal mechanical threshold(PWMT)of carcinoma-baring rats with NL3-overexpressing almost unchanged.The frequency and amplitude of sEPSCs all significantly decreased,and the amplitude and frequency of sIPSCs also decreased obvirusly.(3)NL1 and NL2 involved in BCP through GABAergic neuron and glutamatergic neuron,respectively.Methods:Using multiple immune staining to reveal the cell type of NL1 and NL2.Whole-cell patch-clamp technology records the action potential and post-synaptic currents of primary cultured neurons and acute spinal slices.Western blot,RT-PCR and Immunofluorescent stain were used to detect the expression of VGAT and GABAR-γ2 in spinal cord of rats in each group.Results:NL1 was located at excitatory synapse of GABAergic neuron.Whole-cell patch-clamp recording showed that the frequency and amplitude of sEPSCs all significantly decreased in carcinoma baring-rats,and reversed by up-regulation of NL1.NL2 was located at inhibitory synapse of glutamatergic neuron.The amplitude and frequency of sIPSCs decreased in carcinoma baring-rats,and reversed by NL2-overexpressing in spinal cord.The expression of VGAT and GABAR-γ2 decreased in spinal dorsal horn of carcinoma-bearing rats,and this decrease obviously reversed by up-regulation of NL2 in spinal cord of rats with carcinoma inoculation.Conclusion:1.NL1 and NL2 involved in the occurrence and development of bone cancer pain.2.NL1 played a crucial role in excitatory synapses of GABAergic neurons in bone cancer-induced pain:the reduction of NL1 resulted in the decrease of the excitability of GABAergic neurons,which leaded to the disinhibition of superficial excitatory neurons,further caused the hyperexcitability of central nervous system,and finally leaded to hyperalgesia and hyperalgesia of rats.3.In bone cancer-induced pain,the reduction of NL2 both decreased the release of neurotransmitter in presynaptic membrane and reduced the inhibitory receptors in postsynaptic membrane,directly and strongly reduced the inhibition of nervous system.