Effects Of PKC/NF-κB On The Regulation Of P2X₃ Receptor In Dorsal Root Ganglion In Rats With Sciatic Nerve Injury

Objective:To observe the effects of intrathecal injection of protein kinase C（PKC）inhibitor bisindolylmaleimide I（GF109203X）and nuclear factor-kappa B（NF-κB）inhibitor pyrrolidine dithiocarbamate（PDTC）on the thermal withdrawal latency（TWL）and mechanical withdrawal threshold（MWT）of sciatic nerve chronic constrictive injury（CCI）rats and the expression levels of PKC,NF-κB and P2X₃ receptor（P2X₃R）in dorsal root ganglia（DRG）.To explore the mechanism of PKC/NF-κB on P2X₃R and their relationship in dorsal root ganglia of rats with sciatic nerve injury.Methods:Eighty successful SPF grade healthy male SD rats with intrathecal tube,7weeks old and weighing 180-200g,were randomly divided into five groups:sham operation group（group Sham）,neuropathic pain group（group CCI）,GF109203X solvent group（group DMSO）,PKC inhibitor group（group GF）and NF-κB inhibitor group（group PDTC）with 16 rats in each group.On the first day after successful intrathecal catheterization,rats in group Sham were isolated from the trunk of the sciatica without ligating.The rat model of neuropathic pain was established by CCI surgery in the group CCI,GF,DMSO,PDTC.The rats in the five groups were given continuous intrathecal administration on the first day after operation.The group PDTC was injected with NF-κB inhibitor PDTC 20μg/10μl（saline as solvent）,10μl saline flushing tube after injection twice a day.In group GF,PKC inhibitor GF109203X 0.36μg/10μl（with 1%dimethyl sulfoxide as solvent）was injected intrathecally,10μl dimethyl sulfoxide flushing tube after injection once a day.In group DMSO,20μl dimethyl sulfoxide was injected once a day.In group CCI,20μl saline was injected twice a day.The TWL and MWT were measured at 1day before operation,1d,3d,7d,10d,14d after operation,30 minutes after intrathecal administration,respectively.Specimens were taken from each group on the 7th and 14th day after operation,and the levels of p-PKCα,p-NF-κB p65 and P2X₃R protein in dorsal root ganglion were detected by Western Blot.Results:The TWL and MWT decreased significantly in group CCI and group DMSO on the 1st,3rd,7th,10th and 14th day when comparing with the group Sham（P<0.05）.The TWL and MWT increased on the 1st,3rd,7th,10th and 14th day after CCI surgery in group GF and group PDTC when comparing with the group CCI（P<0.05）.The TWL and MWT increased on the 1st,3th,7th,10th and 14th day after CCI surgery in group GF when comparing with the group DMSO（P<0.05）.The protein levels of p-PKCα、p-NF-κB p65 and P2X₃R in the L_4-6-6 DRGs in the group CCI and group DMSO were significantly increased on the 7th and 14th day after CCI surgery when comparing with the group Sham（P<0.05）.The protein levels of p-PKCα、p-NF-κB p65 and P2X₃R in the L_4-6DRGs in the group GF and group PDTC were significantly decreased on the 7th and 14th day after CCI surgery when comparing with the group CCI（P<0.05）.The protein levels of p-PKCα、p-NF-κB p65 and P2X₃R in the L_4-6-6 DRGs in the group GF were significantly decreased on the 7th and 14th day after CCI surgery when comparing with the group DMSO（P<0.05）.Conclusion:The up-regulation of p-PKCα,p-NF-κB p65,P2X₃R expression in the dorsal root ganglion of CCI rats were involved in the occurrence and development of neuropathic pain.Phosphorylated PKCαand phosphorylated NF-κB p65 regulated with each other.Phosphorylated NF-κB p65,PKCαhave a mutual regulation relationship with P2X₃ receptor respectively,while the specific regulatory mechanism needs further research.