| Objective: Brachial plexus root avulsion often occurs in traffic accidents and obstetric paralysis,which can lead to a large number of neurons dead in the spinal cord.And then patients will be troubled with movement and sensory disorders of upper limb.In order to investingate the pathogenesis of this disease,we investigated the protective and regenerate effect of Puerarin on the neuronal death induced by brachial plexus root avulsion and its mechanism.Methods: There are 252 adult male SD rats,which with a weight of 250±20g,were randomly divided into normal group,model group,low,medium and high dose treatment group.Model group and treatment group underwent right C5-C8 spinal cord nerve anterior root avulsion operation.When the rats was wake,we give them daily intraperitoneal injection after operation.The control group were given saline,treatment groups were given 50 mg/kg/d,100 mg/kg/d and 200 mg/kg/d puerarin solution.After the operation 1,2,4,6,8 weeks we take out the spinal cord tissue from these rats.There are 12 rats were killed each point of time.We observed the morphology and quantity of spinal cord motoneurons by Nissl staining.We detected the expression of NGF and GAP-43 m RNA by real time fluorescence quantitative PCR.We detected the expression of NGF and GAP-43 protein though Immunohistochemistry and Western Blot.Results: The Nissl staining results showed that: compared with the normal group,the model group spinal motoneurons have poor shape at each time point of,the gap between cells was increased,the array of cells was disorganized,the dendrites of motor neurons were significantly reduced and the number of neurons reduced obviously.The statistical results were statistically significant(P<0.001).Compared with the model group,puerarin treatment groups of spinal cord anterior horn motor neurons` morphology and arrangement were improved,dendrites of motor neurons were increased than the model group and the survival rate of neurons was significantly increased,the difference was statistically significant(P<0.05).The results of immunohistochemistry showed that: compared with the normal group,the expression of NGF positive neurons in the spinal cord anterior horn motor neurons of the model group was significantly reduced at 1-8 weeks and the statistical results were statistically significant(P<0.001).Compared with the model group,the NGF positive neurons expression of puerarin treatment group of spinal cord anterior horn motor neurons began to increase since 2nd week.Then the NGF positive neuronsmaintained at high expression until 8th week,the expression trend was obviously and the difference is more significant.Compared with the normal group,model group began to express GAP-43 positive of the motor neurons at 1st week after grafting,peaked at 2nd week,decreased after 4th week and recovered to the normal level at 8th week.Compared with the model group,the expression of GAP-43 positive neurons in puerarin treatment group reached a high level at 1st week after operation and reached a peak at 2nd week,then decreased slightly and decreased to a low level at the end of 8th week.The results of Western Blot showed that: compared with the normal group,the expression of NGF protein in spinal cord anterior horn motor neurons of the model group was significantly reduced at 1-8 weeks,and the statistical data were statistically significant(P<0.001).Compared with the model group,the NGF protein expression amount in treatment group was higher than the model group at 1-8 weeks.The50mg/kg/d dose group was statistically significant than model group only in 1st week(P<0.05).Between 100mg/kg/d dose group and model group,the difference was statistically significant in 4-8 weeks(P<0.05).The difference between 200mg/kg/d dose group and the model group were statistically significant only in 8th week(P<0.05).Combined with the results of the previous study,the experimental results of GAP-43 protein expression in the spinal cord anterior horn motor neurons in each treatment group were in accordance with the results of the Western Blot and immunohistochemical results,which is not to be mentioned here.Real time fluorescent quantitative PCR results showed: compared with the normal group,we detected the expression of NGF m RNA began to decrease since 4th week of model group,6th week to the lowest,but the expression level was increased in 8th week.Compared with the model group,the NGF m RNA expression of Puerarin treatment group began to increase at 6th week,the difference is statistical significance(P<0.05).The expression amount of NGF m RNA was higher than others in 8th week,the difference was statistically significant(P<0.01).Compared with the normal group,the expression amount of GAP-43 m RNA in the model group peaked at 1st week after operation,decreased slightly in 2nd weeks and decreased to a low level at the end of 4th week.Compared with the model group,the GAP-43 m RNA expression of puerarin treatment group was sharply increased since 1st week and reached the peak in 2nd week,then began a slow decline,In 8th week,the GAP-43 m RNA expression is still higher than the model group.Conclusion: Puerarin can protect spinal cord anterior horn motoneurons after brachial plexus root avulsion,and its mechanism may be related to the expression of NGF andGAP-43 m RNA and protein in spinal motoneurons. |
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