| Part-I MCP-1/CCR2 enhances spinal NMDA Receptor activity involved in cancer-induced bone pain in RatsObjective Cancer-induced bone pain is a refractory pain encountered clinically,seriously affecting the quality of life of patients,and the mechanism is not fully known.The mechanism of MCP-1 and its receptor(CCR2)in cancer-induced bone pain is not yet clear.The purpose of this study is to investigate the mechanism of MCP-1 /CCR2 in cancer-induced bone pain.Methods A total of 232 healthy female SD rats,of which 180 were subjected to behavioral tests using a ciliary mechanical stimulation needle,SD rats were randomly divided into eighteen groups(n=10):Sham group,CIBP group,Sham operation + PBS solvent group(group Sham+PBS),cancer-induced bone pain + PBS group(group CIBP+PBS),Sham operation + MCP-1Ab MCP-1 neutralizing antibodies group(group Sham+ MCP-1Ab),cancer-induced bone pain + MCP-1Ab group(group CIBP+MCP-1Ab),Sham operation + DMSO solvent group(group Sham+DMSO),cancer-induced bone pain + DMSO group(group CIBP+DMSO),Sham operation +RS102895 CCR2 inhibitor group(group Sham+RS102895),cancer-induced bone pain +RS102895 group(group CIBP+RS102895),Sham operation + DMSO group(group Sham+DMSO),cancer-induced bone pain + DMSO group(group CIBP+DMSO),Sham operation + MK801 NMDA receptor inhibitor group(group Sham+MK801),cancer-induced bone pain +MK801 group(group CIBP+MK801),Sham operation +DMSO+PBS group(group Sham+DMSO+PBS),cancer-induced bone pain+DMSO+PBS group(group CIBP+DMSO+PBS),cancer-induced bone pain+DMSO+MCP-1 group(group CIBP+DMSO+MCP-1),cancer-induced bone pain+MK801+MCP-1 group(group CIBP+MK801+MCP-1),and Von Frey was used to the behavioral test.Another 48 SD rats were randomly divided into the following 12 groups(n = 4):Sham operation group(group Sham),cancer-induced bone pain 5d group(groupCIBP 5d),cancer-induced bone pain 9d group(CIBP 9d),cancer-induced bone pain 14 d group(CIBP 14d),cancer-induced bone pain + PBS group(group CIBP+PBS),cancer-induced bone pain + MCP-1Ab 1h group(group CIBP+MCP-1Ab 1h),cancer-induced bone pain+MCP-1Ab 2h group(CIBP+MCP-1Ab 2h),cancer-induced bone pain+MCP-1Ab 12 h group(CIBP+MCP-1Ab 12h),cancer-induced bone pain +DMSO group(group CIBP+DMSO),cancer-induced bone pain + RS102895 2h group(group CIBP+RS102895 2h),cancer-induced bone pain + RS102895 4h group(CIBP+RS102895 4h),cancer-induced bone pain + RS102895 12 h group(CIBP+RS102895 12h),western blot was used to detect the expression of MCP-1,CCR2,p-NR1,NR1,p-NR2 B and NR2 B in spinal cord of rats.The remaining four CIBP SD rats were were used to detect the expression and localization of CCR2 and NMDA receptor subunits in spinal dorsal horn by immunofluorescence.Results Compared with group Sham,the pain threshold of group CIBP decreased obviously(P<0.01),and the expression levels of MCP-1,CCR2,p-NR1 and p-NR2 B in spinal cord were time-dependent increased versus group Sham(P<0.05,P<0.01);After intrathecal injection of MCP-1Ab,the pain threshold of group CIBP + MCP-1Ab increased obviously versus group CIBP+PBS(P<0.05),the expression levels of p-NR1 and p-NR2 B in spinal cord were showed a significant down-regulation(P<0.01).After intrathecal injection of RS102895,compared with group CIBP + DMSO,the pain threshold of group CIBP + RS102895 increased obviously(P <0.01),the expression levels of p-NR1 and p-NR2 B in spinal cord were showed a significant down-regulation(P<0.01).After intrathecal injection of MK801,compared with group CIBP + DMSO,the pain threshold of group CIBP + MK801 increased obviously(P<0.05).After intrathecal injection of MK801,then the MCP-1 protein was injected intrathecally,compared with group CIBP + DMSO + PBS,the pain threshold was decreased in group CIBP + DMSO + MCP-1(P<0.01),compared with group CIBP+ DMSO + MCP-1,the pain threshold of group CIBP + MK801 + MCP-1 did not continue to decrease(P<0.01),differences were statistically significant.The results of immunofluorescence showed that the expression of CCR2 and p-NR1 on the ipsilateral was significantly higher than contralateral.CCR2 and p-NR1 were co-expressed with spinal dorsal horn neurons.Conclusion MCP-1/CCR2 can induce the phosphorylation of NMDA receptor subunits NR1 and NR2 B in the spinal dorsal horn neurons of cancer-induced bone pain rats,which is involved in the hyperalgesia of bone cancer pain rats.Part-Ⅱ Relationship between C-C chemokine receptor type2 and P38 mitogen-activated protein kinase signaling pathway in the spinal cord of ratsObjective To investigate the relationship between C-C chemokine receptor type 2(CCR2)and P38 mitogen-activated protein kinase(P38MAPK)signaling pathway in the spinal cord of rats and further clarify the mechanism of cancer-induced bone pain(CIBP).Methods A total of 92 healthy female SD rats,of which 60 were subjected to behavioral tests using a ciliary mechanical stimulation needle,SD rats were randomly divided into six groups :sham operation group(group S),cancer-induced bone pain group(group B),sham operation + DMSO solvent group(group SD),cancer-induced bone pain + DMSO solvent group(group BD),sham operation + RS102895 CCR2 inhibitor group(group SR),cancer-induced bone pain + RS102895 CCR2 inhibitor group(group BR),and Von Frey was used to the behavioral test.Another 32 SD rats were randomly divided into the following 8 groups(n = 4): sham operation group(group S),cancer-induced bone pain 5d group(group B5),cancer-induced bone pain 9d group(group B9),cancer-induced bone pain 14 d group(group B14),cancer-induced bone pain + DMSO solvent group(group BD),cancer-induced bone pain + RS102895CCR2 inhibitor 0.5h group(group BR0.5h),cancer-induced bone pain + RS102895CCR2 inhibitor 4h group(group BR4h),cancer-induced bone pain + RS102895 CCR2 inhibitor 12h group(group BR12h).Western blot was used to detect the expression of P38,p-P38 and CCR2 in spinal cord of rats.Results The mechanical withdrawal thresholds in group B decreased obviously versus group S,and the differences were statistically significant(P<0.01).At day 9post-injection,the mechanical withdrawal thresholds in group BD decreased obviously versus group SD,the mechanical withdrawal thresholds in group BR increased obviously versus group BD,the difference was statistically significant(P <0.01).The expression levels of p-P38 and CCR2 in spinal cord of group B5,group B9 and group B14 were showed a significant up-regulation versus group S,the difference was statistically significant(P <0.01).The expression of p-P38 in group BR0.5h、BR4h、BR12h showed a significant down-regulation versus group BD,the difference was statistically significant(P <0.01).Conclusion CCR2 in the spinal cord may be involved in the development of cancer-induced bone pain by activating P38 MAPK signaling pathway in rats. |
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