Three-point, Three-point Regulation Of Autophagy Promotes The Recovery Of Motor Function In SNI Rats

[Objective]Based on the comprehensive evaluation of ethology,morphology,histology and molecular biology,we try to explore whether three methods and three points intervention could regulate the autophagy of spinal cord to muscle signal channel in SNI rats.Moreover,from the perspective of PI3K-Akt-mTOR pathway and AMPK-mTOR pathway,we study the upstream molecular pathways of tuina regulating motor neuron autophagy.This research is aim at enriching the mechanism of tuina in promoting the motor function recovery after peripheral nerve injury.[Methods]Sciatic Nerve Injury(SNI)rats were subjected to "Three Methods and Three Points",a kind of qualitative and quantitative tuina intervention.Based on the evaluation of morphology,histology and molecular biology,this study tries to analyses the potential mechanism of tuina promoting the recovery of motor function of SNI rats by regulating autophagy.The study is in progress in the following 3 aspects:1.Sciatic nerve functional index(SFI)was used to observe the changes of subtle movement of lower limbs.Transmission electron microscopy was used to observe the changes of autophagy progress and ultrastructure in spinal ventral horn,nerve injury point and gastrocnemius.2.Gastrocnemius wet weight was used to verify the efficacy of tuina intervention.Moreover,by detecting the expression of LC3 and p62 in nerve injury point and gastrocnemius,we try to explain the effect of tuina intervention deferring the atrophy of muscles by regulating the process of autophagy.3.By intrathecal injection of autophagy agonist,rapamycin,as a control group,inclined plane test was used to observe the changes of myodynamia of lower limbs.We detected the co-expression of LC3 and NeuN in spinal ventral horn by Immunofluorescence double-labeling technique.By using the methods of molecular biology,we detect the expression of p-AMPK,p-Akt and other key upstream autophagy factors in spinal ventral horn.In this part,we try to analyses whether tuina intervention can regulate the autophagy of motor neurons in spinal cord.[Results]1.The first part of this research1.1 SFI:On 0 day post-treatment,scores of SFI of model group were significant lower than sham-operation group(P<0.01).On 10 days post-treatment,scores of tuina group were higher than model group(P<0.05).On 15 days and 20 days post-treatment,scores of tuina group was significant higher than model group(P<0.01),1.2 Transmission electron micrographs in spinal cord ventral horn:On 20 days post-treatment,ultrastructure of neurons in model group changed obviously(small pyknotic nuclei,unevenness nuclear membrane,solidified chromatin around the karyotheca).Meanwhile,neurons in the tuina group were much more orderly than the model group.Nuclear membrane and the deep stained of chromatin changed lighter,compared with model group.The autophagysome and autolysosome in tuina group were increased,there was significant difference compared with the model group(P<0.05,P<0.01).1.3 Transmission electron micrographs in sciatic nerve:On 20 days post-treatment,the myelin sheath of sciatic nerve in model group was severely distorted and the axons were atrophied.Moreover,the mitochondria in the axons showed vacuoles in model group.Meanwhile,in tuina group,the myelin sheath was more regularly and axons were not swollen or atrophied.At the same time,there were more autophagic lysosomes in the axons in tuina group(P<0.05).1.4 Transmission electron micrographs in gastrocnemius:On 20 days post-treatment,in the model group,the muscle fibers arranged in disorder and the typical stripes of muscle disappeared.At the same time,the satellite cell nuclei were very irregular,and a large number of vacuolar mitochondria appeared in the model group.Meanwhile,in tuina group,the myocutaneous arrangement was more striated and the satellite cell nuclei were more regular than the model group.Moreover,there were more autophagosome and autolysosome in the gastrocnemius in the tuina group(P<0.01,P<0.05).2.The second part of’ this research2.1 Gastrocnemius wet weight:On 0 day post-treatment,the gastrocnemius of model group was significant lighter than sham-operation group(P<0.01).On 20 days post-treatment,compared with the model group,the gastrocnemius of tuina group was significant increased(P<0.01).2.2 Changes of expression of LC3 and p62 in sciatic nerve injury point:On 0 day post-treatment,compared with sham-operation group,the expression of LC3 and p62 were significant increased(P<0.05)in model group.On 20 days post-treatment,compared with model group,the expression of LC3 were increased in tuina group(P<0.05),furthermore,the expression of p62 were reduced in tuina group(P<0.05).2.3 Changes of expression of LC3 and p62 in Gastrocnemius:On 0 day post-treatment,compared with sham-operation group,the expression of LC3 and p62 were significant increased(P<0.05)in model group.On 20 days post-treatment,the expression of LC3 were significant increased in the model group(P<0.05).Furthermore,compared with model group,the expression of LC3 were as same as the model group in the tuina group(P<0.05).Moreover,the expression of p62 is significant reduced in the tuina group(P<0.05).3.The third part of this research3.1 Inclined plane test:On 0 day post-treatment,compared with sham-operation group,the results of inclined plane were reduced significantly in the model group(P<0.01).On 4 day post-treatment,compared with solvent group,the angles of rapamycin group were increased(P<0.05).After 6 days of intervention,the angles of the rapamycin group were significantly increased(P<0.01).On 6 days post-treatment,compared with model group,the angles of tuina group were increased(P<0.05),moreover,the angles of tuina group were increased significantly after 8 days of intervention,compared with model group(P<0.01).3.2 LC3/NeuN double immunofluorescent labeling in the spinal cord ventral horn:On 0 day post-treatment,compared with sham-operation group,the expression of LC3/NeuN in model group were significantly increased(P<0.05).On 20 days post-treatment,compared with sham operation group,the expression of LC3/NeuN in model group were still in the stage of increase(P<0.05).Compared with solvent group,the expression of LC3/NeuN in rapamycin group were significantly increased(P<0.01).At the same time,compared with model group,the expression of LC3/NeuN in tuina group were higher(P<0.05).3.3 Expression of p62 in the spinal cord ventral horn:On 0 day post-treatment,compared with sham-operation group,the expression of p62 were significantly increased in model group(P<0.01).There were no statistical difference among model group,solvent group,rapamycin group and tuina group.On 20 days post-treatment,compared with sham-operation group,the expression of p62 in model group is still in the stage of increase(P<0.01).Compared with solvent group,the expression of p62 in rapamycin group were significantly reduced(P<0.01).At the same time,compared with model group,the expression of p62 in tuina group were reduced(P<0.05).3.4 Expression of p-mTOR and p-ULK in the spinal cord ventral horn:On 0 day post-treatment,compared with sham-operation group,the expression of p-mTOR and p-ULK were significantly increased in model group(P<0.01).There were no statistical difference among model group,solvent group,rapamycin group and tuina group.On 20 days post-treatment,compared with sham-operation group,the expression of p-mTOR and p-ULK in model group is still in the stage of increase(P<0.05).Compared with solvent group,the expression of p-mTOR in rapamycin group were significantly reduced(P<0.01),and expression of p-ULK were significantly increased(P<0.01).At the same time,compared with model group,the expression of p-mTOR in tuina group were reduced(P<0.05),and expression of p-ULK were significantly increased(P<0.05).3.5 Expression of p-Akt in the spinal cord ventral horn:On 0 day post-treatment,compared with sham-operation group,the expression of p-Akt were increased in model group(P<0.05).On 20 days post-treatment,compared with solvent group,the expression of p-Akt were reduced significantly(P<0.01).Besides,there were no statistical difference between model group and tuina group.3.6 Expression of p-AMPK in the spinal cord ventral horn:On 0 day post-treatment,There were no statistical difference among sham-operation group,model group,solvent group.rapamycin group and tuina group in the expression of p-AMPK(P>0.05).On 20 days post-treatment,the expression of p-AMPK in the ventral horn of spinal cord had no significant difference among control group,model group,solvent group and rapamycin group(P>0.05).However,compared with model group,the expression of p-AMPK in tuina group were significantly increased(P<0.05).[Conclusion]1."Three Methods and Three Points" intervention can promote the formation of autophagy in spinal cord ventral horn,nerve injury point and gastrocnemius of SNI rats,accelerate the clearance and reutilization of necrotic organelles and maintain the stability of motor neuron ultrastructure.2."Three Methods and Three Points" intervention can slow down the atrophy of denervated muscles.The underlying mechanism may be related to the enhancement of autophagy in the nerve injury point and the gastrocnemius.3."Three Methods and Three Points" intervention can promote the recovery of muscle strength of SNI rats.The underlying mechanism is to promote the AMPK-mTOR autophagy process of neurons in the ventral horn of spinal cord,but this process is not achieved by Akt-mTOR pathway.