| Objective: By establishing a spinal nerve ligation(SNL)rat model to simulate neuropathic pain,we observed the changes of paw withdraw thermal latency and paw withdrawal mechaical threshold in rats after the intervention of pivot meridian massage to clarify the analgesic effect of pivot meridian massage.To detect the expression levels of MiRNA-146 a and TLR4 signaling pathways in dorsal root ganglion of rats after pivot meridian massage intervention,and the expression level of TLR4 signaling pathway after overexpression of MiRNA-146 a,so as to explore the mechanism of pivot meridian massage analgesia.This study elaborated the mechanism of pivot meridian massage analgesia from the molecular biology level,in order to provide a scientific theoretical basis for the analgesic effect of pivot meridian massage.Methods: 56 female rats were randomly divided into Blank control group,sham operated group,model group,sham massage group,pivot meridian massage group,agomir-146 a group and negative control(NC)group with 8 rats in each group.After successful insertion of intrathecal cannula in agomir-146 a group and negative control group rats,SNL rat models were established together with model group,sham massage group and pivot meridian massage group.The exposed spinal nerve in sham-operation group was not ligated,and the Blank control group was not treated.After successful modeling,the MiRNA-146 a overexpression group received 10 ?l of lentiviral agoMiRNA-146 a that could up-regulate the expression level of MiRNA-146 a,and the NC group gave an equal dose of the negative control reagent agoMiRNA-146 a negative control.Massage manipulation simulator was used to massage once a day for 14 days in pivot meridian massage group,while sham massage group was used to restrain rats with cloth bags and gently touch their hind limbs for 14 days.Blank control group,sham operation group and model group were not intervened and observed for 14 days.Before and 1,3,7 and 14 days after the establishment of the model,the paw withdraw thermal latency and paw withdrawal mechaical threshold of rats in each group were measured.The expression levels of MiRNA-146 a,TLR4,IRAK1,TRAF6,TNF-alpha and IL-6 in Blank control group,sham operated group,model group,sham massage group and pivot meridian massage group were detected on the 14 th day after modeling.The expression levels of MiRNA-146 a,IRAK1 and TRAF6 in agomir-146 a group and NC group were detected.Result: 1.Weight change of rats: There was no significant difference in body weight of rats in each group before and after the first and third days of modeling(P > 0.05).On the 7th and 14 th day of modeling,there was no significant difference in body weight between Blank control group and sham operated group(P > 0.05).The body weights of the blank control group and the sham operation group were significantly different from those of the model group,the sham massage group and the pivot meridian massage group(P<0.05).There was no significant difference in body weight between model group and sham massage group(P > 0.05).The body weights of the pivot meridian massage group were significantly different from those of the model group and the sham massage group(P<0.05).And there was a significant difference between the over-expression group of MiRNA-146 a and the NC group(P < 0.05).2.Changes of paw withdraw thermal latency and paw withdrawal mechaical threshold in rats: There was no significant difference in paw withdraw thermal latency and paw withdrawal mechaical threshold before modeling in each group(P > 0.05).On the 1st,3rd,7th and 14 th day after modeling,there was no significant difference in paw withdraw thermal latency and paw withdrawal mechaical threshold between Blank control group and sham operated group(P > 0.05).There was no significant difference in paw withdraw thermal latency and paw withdrawal mechaical threshold between model group and sham massage group(P > 0.05).The paw withdraw thermal latency and paw withdrawal mechaical threshold of the blank control group and sham operation group were significantly different from those of the model group,sham massage group and pivot meridian massage group(P < 0.05).On the 1st and 3rd day after the establishment of the model,The paw withdraw thermal latency and paw withdrawal mechaical threshold of the pivot meridian massage group were no significantly different from those of the model group and the sham massage group(P > 0.05).And there was no significant difference in the paw withdraw thermal latency and paw withdrawal mechaical threshold between the MiRNA-146 a over expression group and the NC group(P > 0.05).On the 7th and 14 th day after the establishment of the model,the paw withdraw thermal latency and paw withdrawal mechaical threshold of the pivot meridian massage group were significantly different from those of the model group and the sham massage group(P < 0.05),There were significant differences in the paw withdraw thermal latency and paw withdrawal mechaical threshold between the over-expression group of MiRNA-146 a and the NC group(P < 0.05).3.QPCR results: On the 14 th day after modeling,there was no significant difference in the expression levels of MiRNA-146 a,TLR4,IRAK1 and TRAF6 between Blank control group and sham operated group(P > 0.05);There was no significant difference in the expression levels of MiRNA-146 a,TLR4,IRAK1 and TRAF6 between model group and sham massage group(P>0.05);The expression levels of MiRNA-146 a in Blank control group and sham operated group were higher than those in model group,sham massage group and pivot meridian massage group(P > 0.05).The expression levels of TLR4,IRAK1 and TRAF6 in Blank control group and sham operated group were lower than those in model group,sham massage group and pivot meridian massage group(P < 0.05);The expression levels of MiRNA-146 a in model group and sham massage group were lower than those in pivot meridian massage group(P < 0.05);The expression levels of TLR4,IRAK1 and TRAF6 in model group and sham massage group were higher than those in pivot meridian massage group(P<0.05);The expression levels of IRAK1 and TRAF6 in MiRNA-146 a over expression group were lower than those in NC group(P < 0.05).4.ELISA results: On the 14 th day after modeling,there was no significant difference in the expression of TNF-?and IL-6 between blank control group and sham operation group(P>0.05);There was no significant difference in the expression of TNF-?and IL-6 between model group and sham massage group(P>0.05);The expression of TNF-?and IL-6 in blank control group and sham operation group was lower than that in model group,sham massage group and pivot meridian massage group(P < 0.05);The expression levels of TNF-?and IL-6 in model group and sham massage group were higher than those in pivot meridian massage group(P < 0.05).CONCLUSION: Up-regulation of the expression of MiRNA-146 a and down-regulation of the activation of TLR4 signaling pathway and reduction of the release of inflammatory factors may be one of the effective mechanisms of pivot meridian massage in the treatment of neuropathic pain. |
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