| [Objective]The experiment research the Spinal cord and dorsal root ganglion neurons about Bcl-2, caspase-3 gene of sciatic nerve injury (SNI) rat,from mitochondrial/cytochrome C mediated apoptotic pathway inquiry tuina influencesneurons apoptosis of SNI rats, further analysis of the mechanism of tuina to promote peripheral nerve injury.[Methods]Use 91 SD rats whice were divided into five groups.they were control group, sham operation group, model group, mecobalamine control group and tuina group, made the SNI model by nerve clamping method, mecobalamine control group was injection methylcobalamin into intraperitoneal, tuina grouptreated by "tuina technique Simulator"which treated by imitating Stiring,Pointing and Kneading Manipulation, After the intervention,Acupoints selected Yinmen,Chengshan and Yanglinquan, each point worked 1 minute,total of 9 minutes. After the tuina intervention,we test Inclined plane test, SFI, light and heat pain tolerance threshold of the rats motion and sensory function;the spinal cord, sciatic nerve morphology was analyzed and used TUNNEL staining observed neuronal apoptosis,while observing the sciatic nerve electrophysiology and changes of gastrocnemius wet weight,Iook for evidence of repair of the nerve injury; Finally,the Bcl-2, caspase-3 gene content in the spinal cord, dorsal root ganglia, sciatic nerve were detected to explain the mechanism howtuina to promote peripheral nerve regeneration and repair.[result]1. Behavioral-tuina can can promote SNI rats movement,sensory function recovery.1.1 Inclined plane test, SFI Results:After 7 days, the rats inclined plane test model, SFI scores were significantly lower than the control group (P<0.05), illustrates the preparation of SNI model were success, rats showed motor dysfunction. After Intervention 7,20 times, the tuina group, the mecobalamine control group inclined plane test, SFI scores with the model group were significantly higher (P<0.05), and the intervention of 20 times were significantly higher than the 7 times intervention, indicating tuina intervention effective, and the effect of gradually lifting,tuina can promote SNI rats motion functional recovery.1.2 light and heat pain tolerance threshold Results:7 days after modeling, model group light and heat pain threshold was significantly higher than the control group (P<0.05), illustrates the preparation of SNI model were success. After Intervention 7,20 times, the model group light and heat pain threshold was significantly higher than the control group (P<0.05), the tuina group, mecobalamine control group light and heat pain tolerance threshold compare with model group were significantly lower(P<0.05), the tuina group, mecobalamine control group light and heat pain tolerance threshold compare with model group were significantly higher (P<0.05), indicating that after 20 times treatments the SNI rats sensory function returned to normal.2 Morphology,function-Tuina can improve the rat spinal cord, nerve morphology andreduce number of neuronal apoptosis, improve nerve conduction velocity, increased gastrocnemius wet weight.2.1 spinal cord HE staining:Experiments show that the spinal cordHE staining morphology has changed:7 days after modeling, model group, the presence of spinal cord neuronal apoptosis, cell body swelling. After treatment 7 times, morphology mecobalamine control group and tuina group improved. After treatment,20 times, model group morphological changes has also been restored, but still significantly worse than the control group, morphology mecobalamine control group and tuina group greatly improved, but significantly better than the model group, suggesting that tuinacan improve large manipulation rat morphological manifestations.2.2 Sciatic nerve HE staining:Experiments show that the sciatic nerve HE staining morphology has changed:7 days after modeling, model group, sciatic nerve myelin seen scattered and disintegration ofaxonal and other symptoms. After treatment 7 times, morphology mecobalamine control group and tuina group improved. After treatment,20 times, model group morphological changes has also been restored, but still significantly worse than the control group, morphology mecobalamine control group and tuina group greatly improved, but significantly better than the model group, suggesting that tuinacan improve large manipulation rat morphological manifestations.2.3 TUNNEL staining:After 7 days, Number of model group sciatic neuron apoptosis was significantly higher than the control group (P<0.05), indicating that after modeling began the neuronal apoptosis phenomenon. After Intervention 7,20 times, the number of neuronal apoptosis of model group is still higher than the control group (P<0.05), the mecobalamine control group, tuina group number of apoptotic neurons compared with model group decreased significantly (P<0.05), indicating that tuina interventions that could reduce the number of apoptotic neurons and promote nerve regeneration.2.4Gastrocnemius wet weight Results:7 days after modeling, model group gastrocnemius wet weight was significantly lower than the control group (P<0.05), described gastrocnemius atrophy after modeling.After Intervention 7,20 times, model group gastrocnemius wet weight is lower than the control group (P<0.05), mecobalamine control group, the tuina group gastrocnemius wet weight of model group was significantly higher (P<0.05), indicating tuina can promote the recovery of the gastrocnemius wet weigh, prevent muscle atrophy.2.5Neurophysiological Results:7 days after modeling, motion model group, sensory nerve conduction velocity was significantly lower than the control group (P<0.05), Description after modeling nerve conduction velocity reduced. After Intervention 7,20 times, model group nerve sensory nerve conduction velocity lower than the control group (P<0.05), mecobalamine control group, tuina group motion, sensory nerve conduction velocity compared with model group rose significantly higher (P<0.05), explained tuina can promote the recovery of injured nerve,after treatment 20 times mecobalamine control group, tuina group motion, sensory nerve conduction velocity are still lower than the control group (P<0.05), but high model group (P<0.05), and after 20 times stronger than the effect of the tuina group 7 times, indicating tuina after the intervention of neurological function is gradually restored.3 Immunohistochemical results-tuina can promote the Bcl-2 gene expression in the spinal cord, dorsal root ganglia, sciatic nerve, reducing the expression of caspase-3 gene.3.1 Bcl-2 ImmunohistochemistryBcl-2 gene immunohistochemical expression and results in the spinal cord, dorsal root ganglia, sciatic nerve.After 7 days after modeling, model group Bcl-2 in the spinal cord, dorsal root ganglia, sciatic nerve was significantly higher than the control group (P<0.05), indicating after modeling can improve gene expression.after tuina intervention 7,20 times, the model group, the control mecobalamine group, tuina group Bcl-2 in the spinal cord, dorsal root ganglia, sciatic nerve wassignificantly higher than the control group (P<0.05), the tuina group, mecobalamine control group Bcl-2 expression compare with model group were significantly higher (P<0.05), and the treatment of the 7,20 times both better than the model group, indicating tuina interventions have a therapeutic effect; tuina group Bcl-2 expression was no significant difference between mecobalamine control group (P>0.05), indicating tuina and mecobalamine can improve the Bcl-2 gene expression.3.2 casepase-3 Immunohistochemistrycasepase-3 immunohistochemical expression and results in the spinal cord, dorsal root ganglia, sciatic nerve.After 7 days after modeling, model group casepase-3 in the spinal cord, dorsal root ganglia, sciatic nerve was significantly higher than the control group (P<0.05), indicating after modeling can improve gene expression. after tuina intervention 7,20 times the model group, the mecobalamine control group, tuina group casepase-3 was significantly higher than the control group (P<0.05), the tuina group, mecobalamine control group casepase-3 compare with model group were significantly higher (P<0.05), and the treatment of the 7,20 times both better than the model group, indicating tuina interventions have a therapeutic effect; tuina group casepase-3 expression was no significant difference between mecobalamine control group(P>0.05), indicating tuina and mecobalamine can reduce the casepase-3 gene expression.4. Western blot results-tuina can promote the expression of Bcl-2 gene、reducing the expression of caspase-3 genein the spinal cord. After modeling 7 days, the expression of model groupBcl-2, casepase-3 gene in the spinal cord was significantly higher than the control group (P<0.05), indicating after modeling can improve gene expression. After tuinaintervention 7,20 times the model group, the mecobalamine control group, tuina group of Bcl-2, casepase-3 gene expression in the spinal cord was significantly higher than the control group (P<0.05), the tuina group, mecobalamine group of Bcl-2 gene expression in the spinal cord compared with model group was significantly higher (P<0.05); casepase-3 gene expression in the spinal cord compared with model group was significantly lower (P<0.05), described the intervention tuinacan therapeutic SNI, Can promote the expression of Bcl-2 gene, inhibits the expression of caspase-3 gene,can play an anti-apoptotic role in the organization of neuronal apoptosis, and thus exert a neuroprotective effect, there is no difference in comparison mecobalamine control group and massage group (P>0.05), indicating mecobalamine and tuina interventions can play a neuroprotective effect.[Conclusion]1. Tuina can promotetherecovery of SNI rats inclined plane test, SFI, light and heat pain tolerance threshold, indicating that tuina can improve motorand sensory function ofSNI rats.2. Tuina can increase the number of damaged axons myelinated nerve, the nerve regeneration and target organ built establish connection, reducing the degree of cell swelling, promote nerve regeneration key parts-"neurons" functional recovery. Tuina reduces the number of apoptotic nerve cells and improve the movement of the sciatic nerve, sensory nerve conduction velocity, can also increase the wet weight of the gastrocnemius, promote injury recovery.3. Tuina can increase the expression of Bcl-2 gene, decreasing the expression of caspase-3 gene in the spinal cord, dorsal root ganglia, sciatic nerve, and thus play its anti-apoptotic role to promoting the regeneration of nerve damage, which may promote nerve tuina one of the mechanism of injury and repair.Tuina intervention can promote the SNI rats movement, sensory functional recovery, can also improve the neuronal morphology and improve nerve conduction velocity, reduced the number of apoptotic neurons, promote gastrocnemius muscle recovery, increase ventral horn of spinal cord, dorsal root ganglia, sciatic expression of Bcl-2 gene, decreasing the expression of caspase-3 gene, and thus play its anti-apoptotic effects. |
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