The Role Of Macrophage-colony Stimulating Factor In The Pathogenesis Of Bone Cancer Pain

Background:At present,the mechanism of cancer pain is still unclear,and more and more scholars believe that central sensitization plays an important role in the process of cancer pain.The activation of microglia also participates in the formation of central sensitization.It has been demonstrated that macrophage-colony stimulating factor receptor(M-CSFR)is specifically expressed in microglia in the central nervous system(CNS).Macrophage-colony stimulating factor(M-CSF),the ligand of M-CSFR,is increased in injured neurons and participates in the pathological events after nerve injury.However,the relationship between M-CSF and bone cancer pain are still incompletely understood.The current study aim to investigate the role of M-CSF in the pathogenesis of bone cancer pain and further carry out interventions targeted on M-CSFR based on the establishment of C3H/HeN mice model.This study would provide new insights to clinical medications for bone cancer painMethods:Inoculation of osteosarcoma cells in the femur medullar of mice was used to build the model of bone cancer pain.Pain behaviors was assessed by paw withdrawal mechanical threshold(PWMT)and the number of spontaneous flinches(NSF)to evaluate the success of osteosarcoma cells inoculation.Western blotting was performed to examined the expression of microglia marker Iba-1,M-CSF,M-CSFR,IL-34 and inflammatory factors in the spinal cord.Immunofluorescence labeling was performed to examined the expression of Iba-1 and M-CSFR in the spinal cord.On day 14 after operation,the mice were intrathecally administered either PLX3397 or vehicle continually for 7 days,once a day.Pain behaviors were tested to evaluate the analgesic effect of PLX3397.Western blotting examined the expression of Iba-1,pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α)and phosphorylated mitogen-activated protein kinases(MAPKs)in the spinal cord.Immunofluorescence labeling examined the expression of Iba-1 in the spinal cord.Naive mice were intrathecally treated with mouse recombinant M-CSF three times,each time with an interval of 12 hours.We further investigated the effect of recombinant M-CSF on nociceptive behaviors,microglial activation,phosphorylation of MAPKs and production of pro-inflammatory cytokines in the spinal cord.Results:1.Intramedullary inoculation of osteosarcoma cells into the bone marrow cavity of right femur in mice resulted in down-regulated PWMT and up-regulated NSF on day 14 after inoculation.2.Western blotting demonstrated that the expression of spinal M-CSF up-regulated on day 4 after inoculation and M-CSFR level increased on day 10,without altering IL-34 protein level.3.Repeated intrathecal administration with the M-CSFR inhibitor PLX3397 attenuated pain behaviors,spinal microglial activation,MAPKs phosphorylation and pro-inflammatory cytokines production.4.Repeated intrathecal administration of recombinant M-CSF induced pain behaviors,spinal microglial activation and morphological changes,phosphorylation of MAPKs and pro-inflammatory cytokines up-regulation.Conclusions:These findings suggest that upregulation of spinal M-CSF and M-CSFR levels may play an important role in bone cancer pain.The interaction between M-CSF and M-CSFR may have a connection with microglial activation.Interruption of the interaction between M-CSF and M-CSFR can effectively suppress the activation of microglia,MAPKs phosphorylation and pro-inflammatory cytokines production,thereby attenuating bone cancer-induced pain behaviors.