Effects Of Sigma-1 Receptor On The Regulation Of P2X₃ Receptor In Dorsal Root Ganglion In Rats With Sciatic Nerve Injury

Objective:1.To explore the role of Sigma-1 receptor and P2X₃receptor activation in neuropathic pain mechanisms;2.The effect of Sigma-1 receptor activation in the dorsal root ganglia of rats with neuropathic pain on the expression and function of P2X₃receptor.Methods:1.128 healthy male SD rats were selected for intrathecal catheterization.The SD rats with successful intrathecal catheterization were randomly divided into eight groups:Sham operation group（group Sham）,neuropathic pain group（intrathecal injection of 50μl saline,group CCI）,Sigma-1 receptor antagonist group（intrathecal injection of BD-1047120nmol/20μl,group BD）,Sigma-1 receptor agonist group（intrathecal injection of Pre-08410nmol/10μl,group Pre）,Sigma-1 receptor antagonist+P2X₃receptor agonist group（intrathecal injection of BD-1047 120nmol/20μl+ATP 100nmol/10μl,group BD+ATP）,P2X₃receptor antagonist group（intrathecal injection of A-317491 100nmol/10μl,group A）,P2X₃receptor agonist group（ATP 100nmo/10μl,group ATP）,P2X₃receptor antagonist+Sigma-1 receptor agonist group（intrathecal injection of A-317491 100nmol/10μl+Pre-084 10nmol/10μl,group A+Pre）.There are 16 rats in each group.On the first day after successful intrathecal catheterization,the CCI model was established in each group.In the group Sham,only the sciatic nerve trunk was isolated without ligation,and continuous intrathecal administration began on the first day after operation,and was given for 14 days.The above experimental groups were injected with corresponding solvent saline or dimethyl sulfoxide respectively to flush the tube.Behavioral changes、MWT and TWL were measured at 1 day before operation and 30 minutes after intrathecal injection of corresponding drugs on the 1st,3rd,7th,10th and 14th day after operation.2.On the 7th and 14th day after operation,8 rats in group Sham,group CCI and group BD were taken from L4-6 dorsal root ganglion,and the protein levels of Sigma-1 receptor and P2X₃receptor in dorsal root ganglion were detected by Western Blot method.3.The expression of Sigma-1 receptor and P2X₃receptor in dorsal root ganglion were detected by immunofluorescence staining.4.The relationship between Sigma-1 receptor and P2X₃receptor in dorsal root ganglion was detected by co-immunoprecipitation.Results:1.Behavioral changes:In the group Sham,no neuralgia manifestations such as lameness,hanging in the air,and dorsiflexion of the operative side were not observed at each time point.In the group CCI,group Pre and group ATP,neuralgia manifestations such as claudication,dangling,and dorsiflexion of the operative side of the lower extremities can be observed 1 day after CCI.In the group BD,group A,group BD+ATP,and group A+Pre,the neuralgia manifestations of lameness,dangling,and dorsiflexion of the lower limbs on the operation side were reduced on the first day after CCI.2.Pain threshold determination:Comparison between groups:Compared with group Sham,MWT and TWL in group CCI,group Pre and group ATP were significantly lower than those in group Sham on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）;Compared with group CCI,MWT and TWL were significantly prolonged in group BD and group A on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）;Compared with group BD,MWT and TWL were significantly lower in group BD+ATP on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）,and MWT and TWL in group A+Pre were significantly lower than those in group A on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）;Compared with group ATP,MWT and TWL in group BD+ATP was increased on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）;Compared with group Pre,MWT and TWL in group A+Pre was significantly higher than those in group Pre on the 1st,3rd,7th,10th and 14th day after operation（P<0.05）.Intra-group comparison:there was no significant difference in MWT and TWL at each time point in group Sham（P>0.05）;group CCI,group BD,group A,group ATP,group Pre,group BD+ATP and group A+Pre on the 1st,3rd,7th day after operation,MWT and TWL were lower with time,and there was no significant difference in other time points.3.Western Blot detection indicate:Compared with group Sham,the protein content of Sigma-1 and P2X₃receptor in L4-6 dorsal root ganglion in group CCI increased significantly on the 7th and 14th day after operation,while the protein content of Sigma-1and P2X₃receptor in L4-6 dorsal root ganglion in group BD decreased significantly on the7th and 14th day after operation compared with group CCI.4.Immunofluorescence detection indicates that Sigma-1 receptor and P2X₃receptor were co-expressed in DRG neurons.5.Co-immunoprecipitation detection indicates that Sigma-1 receptor and P2X₃receptor were co-precipitated in DRG neurons.Conclusion:1.The expression of Sigma-1 and P2X₃receptor in dorsal root ganglion of CCI model rats were significantly up-regulated,which were involved in the occurrence and development of neuropathic pain.2.Sigma-1 receptor may be involved in the occurrence and development of neuropathic pain by regulating P2X₃receptor,while the specific regulatory mechanism needs further studied.