| [Objective]In order to study whether the manipulation of Tuina,represented by the three methods and three points,can influence the cAMP-PKA signaling pathway and thereby promote the recovery of injury in SNI rats,this study takes a rat sciatic nerve injury model(Sciatic Nerve Injury,SNI)as an example.Comprehensive evaluation of behavior,morphology,and molecular biology,to observe the repair of sensory function and nerve injury in SNI rats,to explore the effect mechanism of Tuina,and to provide scientific basis for the treatment of peripheral nerve injury by Tuina.[Methods]SD rats were divided into three groups:normal group,sham operation group,model group and three points group.SNI model was established by sciatic nerve clamping in model group and the Tuina of three methods and three points group.Three methods and three points group selected BL37,BL57 and GB34 and used "Tuina manipulation simulator" to operate point method,pull method and kneading method for intervention,each point for 1 min.The recovery of sensory function(pain and temperature perception)in rats was evaluated by the light heat tolerance threshold test.The sciatic nerve function index was used to evaluate the nerve recovery and fine motor recovery of rats;HE staining,Nissl staining and immunofluorescence technique were used to observe the sciatic nerve and L4-L6DRG neuron morphology;the expression of cAMP,PKA and CREB in dorsal root ganglia(DRG)was detected by the technique of motor molecular biology;the L4-L6 DRG and nerve were observed by the technique of immunofluorescence Meta morphology.Through the comprehensive analysis of behavioral,morphological and molecular biological results,to evaluate the effect of Tuina on the recovery of sensory function and Fine movement of hind limbs in SNI rats,and to explain the mechanism of Tuina promoting the repair of peripheral nerve injury.[Results]1.Photothermal pain tolerance threshold-Tuina can promote the recovery of sensory function(pain and temperature perception)in SNI ratsThere was no significant difference in the PTT of left foot(normal side)of SNI rats.The difference of right foot(affected side)was obvious.There was no difference between the the sham operation group and normal group on the 7th day after the model establishment.The model group was higher than the normal group and the sham operation group(P<0.05),indicating that the clamping injury caused the sciatic nerve injury,the continuity of nerve fibers was interrupted,and the pain conduction function was damaged.After 20 days of massage,the lifting time of SNI rats in three methods and three points group was significantly shorter than that in model group(P<0.05)2.Sciatic nerve function index(SFI)-Tuina can improve the fine movement and injured nerve recovery of SNI ratsOn the 7th day after modeling,there was no significant difference in the experimental results between the normal group and the sham operation group,and the SFI performance in the model group was lower than that in the sham operation group(P<0.05).At this time,it can be seen that after sciatic nerve injury,the hind limbs of the affected side of the rats are unable to bear the force and severely lame.After 20 days of intervention,the model group was lower than the sham operation group(P<0.05),and the three methods and three points group was significantly higher than the model group(P<0.05)3.Sciatic nerve HE staining-Tuina can improve the structure of sciatic nerve in SNI ratsThe sciatic nerve fibers in the normal group and the sham-operated group were normal in structure,and the fibroblasts were arranged in an orderly manner;the nerve fibers in the model group were arranged loosely and loosely,the fiber filaments were broken,and the nerve fiber filaments were thinned or reduced;In the model group,the nerve fiber cells are arranged in an orderly manner,the structure is dense,and the overall structure is generally normal.4.Nissl stain-Tuina can promote sciatic nerve and DRG morphological recovery in SNI ratsSciatic nerve:the normal group showed lighter staining of the nerve fibers,and the structure of the transverse and longitudinal sections of the fibers was intact and normal;the nerve fiber cells in the sham-operated group were regularly arranged and the structure was dense;the longitudinal and transverse sections of the nerve fibers in the model group were both It can be seen that the fibers are loosely arranged,the structure is disordered,and the nerve fiber filaments are broken in the longitudinal section;the nerve fiber cells in the three methods and three points group are arranged regularly,and some of the nerve fiber filaments are thin in structure and light in staining.DRG:Most of the neurons in the normal group had clear Nissl particles and abundant numbers,and the structure of neuronal cells was normal;in the sham-operated group,most of the neurons had clear Nissl particles and abundant numbers,and the structure of neuron cells was normal;a small number of neuronal cells The Nissl body disintegrated until it was completely dissolved and disappeared,the cytoplasm was lightly stained,and the cells were swollen;the Nissl body particles in the model group were reduced in all neuronal cells,and the Nissl body was lightly stained;the Nissl body was more The body disintegrated into fine dust-like particles,and then completely dissolved and disappeared;due to the disappearance of Nissl body,the cytoplasm was lightly colored,the cell body was swollen,and the cell changed from a multipolar shape to a circle;most neurons in the three-method three-point group The granules are clear,abundant,and the structure of the neuron cells is normal;a small amount of neuronal bodies in the local area of the neuron body disintegrate into fine dust-like granules,and gradually expand outward,the cytoplasm is lightly stained,and the cells are swollen.5.Immunofluorescence staining-Tuina can protect and promote SNI rat sensory neuronsThe neurons in the DRG of the normal group were mostly round or triangular,and the nucleoli were centered,with clear staining and full morphology;the neurons in the sharn-operated group were similar in shape to the normal group;the model group showed a decrease in the number of neurons and increased or broken morphological fold There was a slight edema phenomenon,the diameter increased,and the nucleolus part shifted or disappeared;the three-method three-point group had clear staining and increased number after 20 days of intervention.6.WB massage can promote the recovery of sciatic nerve by promoting the expression of key proteins in the pathway6.1 expression of cAMP in DRGThere was no big difference between the normal group and the sham operation group on the 7th day after the establishment of the model.Compared with the normal group,the expression of the model group was significantly reduced(P<0.05);at the 20th day after the intervention,compared with the model,the expression level of cAMP in the three methods and three points group was significantly increased(P<0.05),and the difference between the three methods and three points group and the model group was significant(P<0.05).6.2 expression of PKA in DRGThere was no big difference between the normal group and the sham operation group on the 7th day after the establishment of the model.Compared with the normal group,the PKA expression in the model group decreased a lot(P<0.05);after 20 days of Tuina,compared with the model group,the PKA expression level in the three methods and three points group increased significantly(P<0.05),and there was no significant difference between the three methods and three points group and the normal group.6.3 expression of CREB in DRGThere was no big difference between the normal group and the sham operation group on the 7th day after the establishment of the model.Compared with the normal group,there was no significant difference between the model group and the model group.After 20 days of intervention,there was an increase in the expression of the three points group,but there was no statistical difference.These results suggest that Tuina can improve the expression of cAMP,PKA and CREB in DRG,and then promote the recovery of peripheral nerve injury.[Conclusion]Tuina represented by three methods and three points,can improve the expression of cAMP,PKA and CREB in DRG.It can be seen that Tuina can activate the pathway by increasing three key proteins in cAMP-PKA pathway,improve the morphology of neurons in DRG of SNI rats,restore their ultrastructure,promote the recovery of sensory function of SNI rats,as well as the recovery of fine movement of hind limbs.Thus can promoting the repair of peripheral nerve.It is suggested that one of the mechanisms of Tuina's treatment of peripheral nerve injury is to activate cAMP-PKA signal pathway. |
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