| ObjectiveTo observe the changes of protein and mRNA expression of pain-related chemokine CX3CL1 and its receptor CX3CR1,mRNA of CCL2 in spinal dorsal horn of rats with sciatic nerve clamp injury,and to explore whether the mechanism of "three handing-three acupoint"on the improvement of pain function in rats with sciatic nerve injury is through chemokine related to pain disturbance of nerve injury.Method74 male Sprague-Dawley rats were randomLy divided into normal group(n=12),sham group(n=24),model group(n=25)and three handing-three acupoint group(n=13).The experimental animal model was made by the method of sciatic nerve clamping injury in the model group and three handing-three acupoint group,and only the sciatic nerve was exposed in the sham group.On the 7 days after modeling,the three handing-three acupoint group intervened with the three neuromuscular related acupoints of Yin men,Yang ling quan and Cheng shan with the simulated point method,plucking method and kneading method of massage manipulation simulator,and grasped restraint in normal group,sham group and model group.The photothermal pain tolerance threshold of normal group,sham group,model group and three handing-three acupoint group was measured at 7 days and 20 days after intervention,and the cumulative pain scores of sham group,model group and three handing-three acupoint group were measured at 7 days after modeling,10 days and 20 days after intervention.7 days after modeling and 20 days after intervention,fresh samples of spinal dorsal horn were taken,the protein and mRNA expression of CX3CL1 and its receptor CX3CR1 in spinal dorsal horn were detected by Western blotting and RT-PCR,and the mRNA of CCL2 was detected by RT-PCR.20 days after intervention,the microglia in spinal dorsal horn were fixed by paraformaldehyde solution perfusion and fixed,and the microglia in spinal dorsal horn were observed by immunofluorescence.Result1.Three handing-three acupoint can improve the pain function of SNI rats.1.1 Photothermal pain tolerance threshold7 days after modeling,there was significant difference between the sham group and the model group and the normal group(P<0.05),and the sham group and the model group were higher than the normal group,and there was significant difference between the model group and the sham group(P>0.05).And the model group was higher than the sham group.20 days after intervention,the model group was higher than the normal group and the sham group,and there was significant difference(P>0.05).The three handing-three acupoint group was higher than the normal group,but lower than the model group,the difference was statistically significant(P>0.05).1.2 cumulative pain score7 days after modeling,the score in the model group was significantly higher than that in the sham group(P<0.05),and the score in the model group was significantly higher than that in the sham group(P<0.05),and the score in the three handing-three acupoint group was significantly higher than that in the sham group(P<0.05),but lower than that in the model group(P<0.05).20 days after intervention,the score of the model group was higher than that of the sham group,and there was significant difference(P<0.05).The score of the three handing-three acupoint group was significantly higher than that of the sham group,but lower than that of the model group(P<0.05).From the horizontal point of view of the three time points,the model group showed a gradual upward trend,while the three handing-three acupoint group showed a downward trend.2.Effects of three handing-three acupoint on the expression of CX3CL1 and its receptors CX3CR1 and CCL2 in spinal dorsal hornThe results of detection of CX3CL1 and its receptor CX3CR1 protein and mRNA of mRNA and CCL2 in spinal dorsal horn:2.1 Western blotting7 days after modeling,there was no significant difference in the expression of CX3CR1 protein among the three groups(P>0.05).20 days after intervention,there was a significant upward trend in the model group compared with 7 days,but there was no significant difference among the three groups(P>0.05).The expression of protein in the three handing-three acupoint group was lower than that in the model group and close to the normal group and sham group,but there was no significant difference in statistics.7 days after modeling,the expression of CX3CL1 protein in each group had no significant difference(P>0.05),but after 20 days of intervention,the protein expression in the sham group had a downward trend,while that in the model group had an upward trend,but there was no significant difference among the each group(P>0.05).2.2 RT-PCR7 days after modeling,compared with the model group,the mRNA expression of CX3CR1 in the sham group was higher than that in the model group,but there was no significant difference(P>0.05).20 days after intervention,the expression of mRNA in the three handing-three acupoint group decreased compared with the model group,but there was no statistical difference(P>0.05).7 days after modeling,compared with the model group,the mRNA expression of CX3CL1 in the sham group was higher than that in the model group,but there was no significant difference(P>0.05).After 20 days of intervention,the expression of mRNA in the three handing-three acupoint group decreased compared with the model group,but there was no statistical difference(P>0.05).7 days after modeling,the expression of CCL2 mRNA in the model group was higher than that in the sham group,and there was no statistical significance(P>0.05).After 20 days of intervention,the mRNA of CCL2 in the model group was higher than that in the sham group,while that in the three handing-three acupoint group was lower than that in the model box sham group,and the difference was statistically significant(P>0.05).3.Morphological observation of microglia in spinal dorsal hornThe spinal dorsal horn of the affected side was magnified 400 times under the fluorescence microscope.The microglia in the spinal dorsal horn of the three handing-three acupoint group were not activated or partially activated,with high branching and rod shape,small cell body and longer processes.20 days after intervention,the microglial processes in the spinal dorsal horn of the model group were shorter than those of the three handing-three acupoint group,which were completely or partially activated,and some of the cell bodies were enlarged.ConclusionThree handing-three acupoint can improve the pain function of rats with sciatic nerve injury,but it is not realized by regulating the expression of chemokine CX3CL1/CX3CR1,which may be related to CCL2,but it needs to be further verified.Three handing-three acupoint can affect the activation of microglia in the dorsal horn of the spinal cord. |
