Tuina Regulates The Activity Of Spinal Cord Glial Cells And The Analgesic Mechanism Of Related Pro-inflammatory Factors To Inhibit Central Sensitization

The pain caused by peripheral nerve injury is a common clinical symptom.The clinical efficacy of Tuina for treating such symptoms is exact.However,the mechanism of the effect of Tuina on analgesia is still unclear.Previous studies have shown that central sensitization plays an important role in the pathogenesis and maintenance of neuropathic pain.When there is nerve damage,glial cells will be activated and pro-inflammatory factors will be released,they all contribute to central sensitization.This study adopted a variety of neurobiological research methods such as pain behavior,HE staining,in vivo electrophysiology,immunofluorescence chemistry,Western blotting,enzyme-linked immunosorbent assay,etc.by replicating the chronic constriction injury of the sciatic nerve(CCI).Through observing the analgesic effect of Tuina on CCI model and the effects of spinal field potential,astrocytes,microglia,microglia M1 receptor,interleukin-1β and tumor necrosis factor α expression explore the mechanism of spinal cord analgesia in the treatment of neuropathic pain with Tuina.Part Ⅰ: The analgesic effect of Tuina on CCI model ratsObjective: To study the analgesic effect of sputum method on CCI model rats and the effect on the wet weight and morphology of gastrocnemius muscle.Methods: Replicate and validate the CCI model: A total of 24 rats in this experiment were equally distributed into three groups,which were blank group,sham operation group and CCI group.The rats in the sham-operated group and the CCI group were performed the corresponding surgical operation,and the paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)were observed before surgical and,3,7,10,14 days after modeling.The analgesic effect of Tuina on CCI model rats: A total of 32 rats in this experiment were equally distributed into four groups,which were blank group,sham operation group,CCI group,CCI+Tuina group.In this experiment,the the CCI+Tuina group was added on the basis grouping of the experiment one,and 8 animals in each group.The CCI model + massage group applied a massage technique once a day,and the remaining groups operated the same as experiment one.The behavioral detection method and time node are the same asexperiment one.After the gastrocnemius muscle was taken,the wet weight was weighed,and the morphological changes of myocytes were observed under 20-fold and40-fold light microscope.Results:(1)PWT and PWL were significantly decreased in the CCI group,and the differences were statistically significant at each time point compared with the blank group and the sham operation group(P<0.05).(2)The PWT and PWL values of the CCI+Tuina group were significantly decreased on the 3rd day after modeling,and the difference was statistically significant compared with the blank group and the sham operation group(P<0.05);The PWL value continued to increase.PWT and PWL were significantly different from the CCI model group on days 10 and 14(P<0.05).(3)14days after model establishment,the recovery rate of the wet weight of the ipsilateral gastrocnemius muscle in the model group and the model + massage group was significantly decreased,and the difference was statistically significant compared with the blank group and the sham operation group(P<0.05);The recovery rate of the wet weight of the ipsilateral gastrocnemius muscle in the massage group was higher than that in the model group,and the difference between the groups was statistically significant(P<0.05).(4)The morphology of rat gastrocnemius muscle was observed under light microscope.The cross-sectional area of gastrocnemius muscle in model group and CCI+Tuina group was significantly smaller than that in blank group and sham operation group.The difference between the two groups was statistically significant(P<0.05).The cross-sectional area of muscle cells in the CCI+Tuina group was higher than that in the model group,and there was no significant difference between the groups(P>0.05).Conclusion: The pain behavior of CCI model is stable.The method of Tuina can reduce the PWT and PWL of CCI model rats.The Tuina method can improve the gastrocnemius atrophy of CCI model rats.Part Ⅱ: Effects of Tuina on the field potential of spinal dorsal horn in CCI model ratsObjective: To study the effect of Tuina on the spinal dorsal horn field potential of CCI model,and to explore the spinal cord analgesia mechanism of CCI model by Tuina method.Methods: The grouping and intervention methods are the same as the experiment2 of Part Ⅰ.On the 14 th day after modeling,the field potential of the spinal dorsal horn of each group was recorded using electrophysiological techniques.Results:(1)On the 14 th day after model establishment,the field potential of the spinal dorsal horn in the CCI group was significantly increased after the tonic stimulation,which were significantly different from those of the blank group and the sham operation group at each time points(P<0.05).(2)Compared with the model group,the field potential of the spinal dorsal horn in the CCI+Tuina group was statistically significant difference(P<0.05),except for 60 minutes after the tonic stimulation.Conclusion: CCI modeling leads to long-term potentiation in the dorsal horn of the spinal cord,and Tuina can suppress this phenomenon.Part Ⅲ:Effects of Tuina on the expression of glial cells and related proinflammatory factors in spinal dorsal horn of CCI model ratsObjective: To study the effects of Tuina on the expression of astrocytes,microglia,microglia M1 receptor,IL-1β and TNF-α in spinal dorsal horn of CCI model rats by Tuina method,and to explore whether the inhibition effect of central sensitization by Tuina method is related to glial cells and proinflammatory factors.Method: This section is divided into three sections.Section Ⅰ: Glial cells are involved in mediating neuropathic pain in rats.SD rats were randomly divided into three groups: CCI group,intrathecal saline group,and intrathecal glial cell blocker.The glial cell blocker was administered through the sheath tube.Observe the PWT and the PWL changes of each group.Section II: Effect of Tuina on the expression of glial cells in spinal dorsal horn of CCI model rats.Grouping and intervention methods are the same as part II.The expression of glial cells and microglia M1 receptor in the spinal dorsal horn of rat spinal cord was observed by immunofluorescence and Western blot at 14 days after model establishment.Section III: Effects of Tuina on the expression of IL-1β and TNF-α in the spinal dorsal horn of CCI model rats.The experimental animals are the same as Section II.In this experiment,the effects of Tuina on the expression of IL-1β and TNF-α in the spinal dorsal horn of CCI model rats were observed by immunoenzyme-linked immunosorbent assay.Results:Section I:(1)PWT and PWL of the three groups of rats decreased on the first day after modeling,and there was no significant difference between the three groups(P>0.05).(2)The PWT and PWLof the CCI group and intrathecal saline group were continuously decreased from the first day to the end of the experiment,there was no significant difference between the two groups(P>0.05).(3)The PWT and PWLof intrathecal injection blocker group was significantly lower than that of the model group and the intrathecal saline group,the difference was statistically significant(P < 0.05)on the 3rd,5th,and 7th days after model establishment.Section II:(1)On day 14 after model establishment,the expression of astrocytes,microglia and microglia M1 receptor CD68 in the right spinal dorsal horn of CCI group and CCI+Tuina group was significantly higher than that of the blank group.The difference was statistically significant(P < 0.01).(2)The content of astrocyte protein in the CCI+Tuina group was lower than that in the CCI group,and the difference between the groups was statistically significant(P<0.01).(3)The content of CD68 protein in the CCI+Tuina group was lower than CCI group.The difference between the groups was statistically significant(P<0.05).Section III:(1)The percentage of IL-1β and TNF-α protein in the right spinal dorsal horn of the CCI group,CCI+Tuina group was significantly higher than that of the blank group and the sham operation group,the differences were statistically significant(P < 0.01).(2)The percentages of IL-1β and TNF-α protein in the CCI+Tuina group were significantly lower than those in the CCI model group,and the difference between the groups was statistically significant(P<0.05).Conclusion:(1)Intrathecal injection blockers reduce the PWT and PWL of CCI model rats,glial cells participate in neuropathic pain caused by CCI modeling.(2)CCI model can increase the content of astrocytes,microglia and CD68 protein in the spinal cord dorsal horn,Tuina can inhibit this phenomenon.(3)CCI modeling can lead to an increase in the percentage of IL-1β and TNF-α protein in the dorsal horn of the spinal cord.Tuina can suppress this phenomenon.