| ObjectiveTo explore the effect of three methods and three-acupoint on the recovery of sciatic nerve injury(SNI)rats by behavioral study,and to observe the effect of three methods and three-acupoint on the ultrastructure of SNI rats based on morphology.RNA-Seq technology was used to explore the gene expression of three methods and three-acupoint on the key parts of nerve conduction pathway of SNI rats,including nerve injury point,dorsal root ganglia(DRG),dorsal horn of spinal cord,ventral horn of spinal cord and gastrocnemius muscle,and to elucidate the recovery mechanism of peripheral nerve injury from the transcriptome level.MethodsSD rats were randomly divided into three groups:Sham group,Model group and Three methods and three-acupoint group.In the Sham group,the right sciatic nerve was found and exposed without clamping.In the Model group,the right sciatic nerve clamp injury model was established,resulting in sciatic nerve crush injury.Seven days after the model was established,point-pressing,plucking,and kneading methods were administered quantitatively at Yinmen(BL37),Chengshan(BL57),and Yanglingquan(GB34)points on the affected side once a day by the "Massage and tuina manipulation simulator"(Patent No.ZL 20070187403.1)in the Three methods and three-acupoint group,each method and point was stimulated for 1min,and each rat was intervened for 9 min/d(lmin/point/method×3 methods×3 points).Intervention was applied for 10 days,then 1 day of rest,followed by 10 more days of intervention,totally equaling 20 times of intervention.The specific methods are as follows:1.The sciatic functional index(SFI)was used to evaluate the fine movement of the hind limbs of the rats,the muscle strength of the hind limbs of the rats was evaluated by the inclined plate test,the pain function of the rats was evaluated by the accumulated pain score,and the effects of the three methods and three-acupoint on the sensory and motor functions of the SNI rats were explored to clarify the therapeutic effect of tuina on the recovery of peripheral nerve injury.2.The ultrastructures of nerve injury point,ventral horn of spinal cord and gastrocnemius muscle were observed by transmission electron microscope,which provided morphological basis for promoting the functional recovery of SNI rats.3.RNA-Seq technology was used to explore the effect of three methods and three-acupoint on gene expression of nerve injury point,DRG,dorsal horn,ventral horn and gastrocnemius in rats with SNI.GO analysis and KEGG pathway analysis were used to analyze the differential expression genes and further elucidate the mechanism of three methods and three-acupoint to promote the repair of injured nerve.4.Pdzd2,Camk2a,Mdk,C1ql3 and Klf7,which are involved in the regulation of synapses and sensory neurons in DRG of primary neurons,were verified by Real Time-PCR,and the mechanism of tuian promoting the repair of peripheral nerve injury was further clarified.Results1.Behavioral Results1.1 SFI resultsCompared with the baseline,the values of SFI in the Model group and the Three methods and three-acupoint group decreased significantly after 0 times of intervention(7 days after operation);compared with the Model group,the values of SFI in the Three methods and three-acupoint group increased significantly after 10 times of intervention(P<0.05);compared with the Model group,the values of SFI in the Three methods and three-acupoint group increased significantly after 15 times and 20 times of intervention(P<0.01).1.2 Inclined plate test resultsCompared with the Sham group,the angle of oblique plate decreased significantly(P<0.01)in the Model group and the Three methods and three-acupoint group(P<0.01).Compared with the Model group,the angle of inclined plate began to increase in the Three methods and three-acupoint group(P<0.05).Compared with the Model group,the angle of oblique plate in the Three methods and three-acupoint group increased significantly(P<0.01),but there was still significant difference(P<0.01)compared with the Sham group.1.3 Cumulative pain score resultsThe results of cumulative pain score showed that the cumulative pain score of rats in each group was the same and all were 0 before modeling,and the rats in each group landed on the ground after two hind paws and carried weight.After 7 days of model establishment,the cumulative pain score of rats in the Model group and the Three methods and three-acupoint group was significantly higher than that in the Sham group(P<0.05);after 10 and 20 times of intervention,the cumulative pain score of rats in the Three methods and three-acupoint group was significantly better than that in the Model group(P<0.05),but there was still a certain gap between the Three methods and three-acupoint group and the Sham group(P<0.05),suggesting that the three methods and three-acupoint can relieve the spirit to a certain extent the pain sensation of the injured rats.2.Electron microscopic results Results2.1 Electron microscopic results of nerve injury pointCompared with the Sham group,the myelin sheath of the Model group was completely destroyed,even formed myelin ball,accompanied with axonal atrophy and mitochondrial degeneration;the intervention of three methods and three-acupoint could improve the ultrastructure of nerve injury point,and the myelin sheath of nerve fiber in the Three methods and three-acupoint group was relatively intact,without obvious swelling or atrophy of axon,but without vacuole line Granuloma.2.2 Electron microscopic results of ventral horn of spinal cordThe ultrastructure of ventral horn neurons in the Sham group was basically normal;compared with the Sham group,the motor neurons in the ventral horn of the Model group were seriously damaged,with nucleolus,chromatin and mitochondrion contracting and uneven nuclear membrane;compared with the Model group,the neurons in the Three methods and three-acupoint group were less damaged,with deep chromatin staining significantly reduced,and the nuclear membrane was smooth and complete intact without obvious shrinkage and swelling.2.3 Electron microscopic results of gastrocnemius muscleIn the Sham group,myosin filaments were arranged in order,and typical structures such as A-band,I-band,Z-line and M-line were obvious;in the Model group,myofilaments were disordered,A-band,I-band,Z-line and M-line disappeared,myofascicular atrophy was serious,satellite cell necrosis,apoptosis and mitochondrial inactivation were found;in the Three methods three-acupoint group,myofilaments were arranged in order,Z-line and M-line could be clearly observed,and the number of satellite cell necrosis was significantly reduced The structure of mitochondria is relatively complete.3.RNA-Seq ResultsThe OD260/280 value of each sample is between 1.8 and 2.2,and the RIN score is more than 8 before the database can be established and sequenced.The sequencing results of each site are as follows:3.1 Sequencing results of nerve injury pointThe GO function of 221 differentially expressed genes in the SNI group and three methods and three-acupoint group was enriched in the regulation of multicellular tissue process,virus defense response,response to external stimulation,immune system process,response to biological stimulation,defense response,etc.;KEGG pathway analysis showed that it was related to signal transduction pathway,immune system pathway,endocrine system pathway,cell growth and apoptosis pathway.The function of GO is enriched in the regulation of striated muscle cell differentiation,myocyte differentiation,myoblast differentiation,myotube differentiation,striated muscle contraction,muscle organ development,contractile fiber and other biological processes related to myocyte regulation.3.2 Sequencing results of DRGThere were 369 differentially expressed genes in the three groups.The functions of differentially expressed GO included biological regulation process,response to stimulation,active regulation of biological process,presynaptic process involved in chemical synaptic transmission,movement,growth,molecular sensor activity,etc.;KEGG pathway was enriched in Wnt signaling pathway,IL-17 signaling pathway,MAPK signaling pathway,etc.3.3 Sequencing results of the dorsal horn of spinal cordThe dorsal horn of spinal cord showed that there were 61 differentially expressed genes in the Three methods and three-acupoint group compared with the Model group;the three groups of differentially expressed genes GO enriched in signal regulation,synaptic part,signal transduction regulation,cell differentiation regulation and other fuctions;KEGG pathway enriched in MAPK signal pathway,VEGF signal pathway,AMPK signal pathway.Tuina promotes the recovery of pain function of peripheral nerve injury,which is related to the gene expression of spinal dorsal horn.3.4 Sequencing results of the ventral horn of the spinal cordThe ventral horn of the spinal cord showed that there were 217 differentially expressed genes in the three groups,among which 113 were obtained by comparing the Three methods and three-acupoint group with the Model group;the GO function results of the three groups showed that they were related to cell process,response to stimulation,immune system process,negative regulation of biological process,etc.;KEGG analysis was closely related to immune system,viral infection disease pathway,cancer,signal transduction pathway,cut correlation etc.3.5 Sequencing results of gastrocnemius muscleGastrocnemius muscle showed that three methods and three-acupoint could promote the recovery of rats with peripheral nerve injury,and improve the gene expression of gastrocnemius muscle.The GO function of differentially expressed genes includes single organism process of cell process,metabolism process,biological regulation,response to stimulation,multicellular tissue process,positive regulation of biological process,etc.;KEGG was enriched in cytokine cytokine receptor interaction,AMPK pathway,etc.4.Real Time-PCR ResultsCompared with the Sham group,the gene expression of Pdzd2,Camk2a,Mdk,C1ql3 and Klf7 in DRG of the model group increased,and the intervention of three methods and three-acupoint could reduce the expression of these genes in DRG of the nerve injured rats,and the expression trend was consistent with that of RNA-Seq technique.Conclusion1.Tuina can promote the recovery of fine movement,muscle strength of hind limbs and pain sense function of SNI rats,and promote the behavioral recovery of peripheral nerve injury.2.Tuina can promote the integrity of myelin sheath,restore the structure of motor neurons in ventral horn of spinal cord,reconstruct the structure of gastrocnemius muscle,and play a role in the morphological recovery of peripheral nerve injury in SNI rats.3.Tuina promotes the recovery of SNI rats by regulating the gene expression of nerve injury point,ventral horn of spinal cord,gastrocnemius muscle,dorsal horn of spinal cord and DRG.The regulation of each part involves multiple biological processes and signal pathways.4.Tuina may promote axon regeneration,sensory and motor function recovery of SNI rats by reducing the expression of Pdzd2,Camk2a,Mdk,C1ql3,K1f7 and other genes in DRG.These genes may be important therapeutic targets for tuina to promote the repair of peripheral nerve injury. |
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