The Role Of Spinal MrgC-G?i-NR2B-nNOS Signal Pathway In The Bone Cancer Pain

Backgroud Pain is a common clinical feature in cancer patients.Approximately 75%-95%of the cancer patients suffer from pain during the development of cancer.Bone cancer pain is characterized as ongoing background pain,breakthrough pain and allodynia.World Health Organization(WHO)published the three step analgesic ladder in 1982,nevertheless the side effects of opioids limit its clinical application and about 45%cancer pain patients do not receive satisfactory relief.Therefore,exploring the mechanisms under bone cancer pain is of great significance to clinical interventions.Studies have demonstrated that a novel G protein-coupled receptor MrgC,played an important role in the regulation of pain via altering synaptic plasticityas.MrgC was specifically distributed on the neurons of dorsal root ganglia and spinal cord dorsal horn.The unique distribution of MrgC indicates that it might be a potential target for prevention and treatment of cancer pain.However,the underlying mechanisms of MrgC in bone cancer pain still remains elusive.This study intends to investigate the mechanism of MrgC-Gai-NR2B-nNOS signaling pathway in the mice model of bone cancer pain to provide scientific evidence to prevent and treat bone cancer pain.Methods(1)C3H/HeJ male mice and NCTC 2472 fibrosarcoma cells were used to establish bone cancer pain model.The number of spontaneous flinches/2 min(NSF)and paw withdrawal mechanical threshold(PWMT)were used to assess pain behaviours performance.To determine the success of model establishment,H-E staining of tissue sections were used to observe the growth of tumor and the degree of bone destruction.(2)Western blotting and immunofluorescence histochemistry were used to investigate the expression and activity of spinal MrgC,Gai,NR2B and nNOS during the development of bone cancer pain.(3)The effect of single or repeated intrathecal administration with different dose of MrgC agonist BAM8-22(0.8 nmol/5?I,2.4 nmol/5 ?l,8.0 nmol/5 ?l)on the NSF and PWMT were further investigated.Western blotting and immunofluorescence histochemistry were used to detect the expression of MrgC,Gai,NR2B and nNOS in the L3-5 spinal cord segments.(4)The inhibitors or agonists of MrgC-Gai-NR2B-nNOS signaling pathway were used to examine the mechanisms underlying bone cancer pain.Western blotting,immunofluorescence histochemistry and calcium imaging were applied to examine the expression and activity of MrgC,Gai,NR2B,nNOS in the L3-5 spinal cord segments and the concentration of Ca2+ in neurons.Results(1)Inoculation of NCTC 2472 fibrosarcoma cells into the right femur of the mice induced progressive spontaneous flinches and mechanical hyperalgesia(P<0.05)since day 7 to day 14.The HE staining of the right femur of mice in tumor group showed the invasion of tumor cell into bone marrow and bone destruction,which suggested the successful establishment of bone cancer pain model in mice.(2)Compared with Sham group,the expression of MrgC,Gai,NR2B and nNOS were up-regulated since 7 d to 14 d after inoculation,which matched the results of pain behaviors in time phase.(3)Single or repeated intrathecal administration of middle or high dose of the selective MrgC agonist BAM8-22(2.4 nmol/5 ?1,8.0 nmol/5 ?l)improved bone cancer pain behaviors.The analgesia effect of BAM8-22 appeared on 1 h after single intrathecal injection,reached the peak on 2 h after injection,decreased gradually on 12 h and disappeared on 24 h after injection.To mimic the clinical management for cancer pain patients,successive intrathecal administration from day 14 to day 21 were adopted.Data showed that pain behaviors were enhanced on 2 h after drug administration(P<0.05)and the analgesic effect were more apparent in high dose group(8.0 nmol/5 ?l),while lose-dose BAM8-22(0.8 nmol/5 ?l)did not produce a favorable outcome(P>0.05).Intrathecal administration with MrgC antagonist,anti-MrgC,produce the opposite effect.(4)Intrathecal administration with BAM8-22 up-regulated the expression of spinal MrgC and Gai,down-regulated the expression of p-NR2B,t-nNOS and p-nNOS and inhibited the concentration of Ca2+in neurons.Intrathecal administration of anti-MrgC had opposite effects corresponding to changes of pain behaviors.(5)On 14 d after inoculation,activating or inhibiting MrgC-Gai-NR2B-nNOS signaling pathway via application of BAM8-22,alternative antagonist of MrgC,anti-MrgC and ifenprodil,changed the pain behaviors on 2 h after injection and regulated the expression and activity of downstream proteins in this signaling pathway.Conclusions(1)Spinal MrgC played an important role in the development of bone cancer pain.(2)Activation of MrgC attenuated pain behaviors induced by bone cancer via activating Gai and inhibiting the expression and activity of NR2B/nNOS.(3)Regulation of MrgC-Gai-NR2B-nNOS signaling pathway regulated the expression and activity of MrgC,Gai NR2B and nNOS,inhibited the concentration of Ca2+ in neurons,and reduced bone cancer pain effectively.