| Backgroud: Motor neuron disease(MND)is a chronic progressive neurodegenerative disease that selectively affects spinal anterior horn cells,brainstem motor neurons,cortical pyramidal cells,and pyramidal tracts.Studies have shown that envelope glycoprotein syncytin-1 encoded by hunman endogenous retrovirus(HERV)-W family env gene has a abnormally high level in the muscle of patients with MND.But whether it has correlation with MND motor neuron injury is not yet conclusive.In addition,related studies have shown that high expression of syncytin-1 in skeletal muscle can induce activation of glial cells in spinal cord,leading to oxygen free radical accumulation and mitochondrial damage.Autophagy-lysosomal pathway(ALP)is one of the main ways to repair and eliminate abnormal proteins and damage organelles.Participation in cell differentiation,apoptosis and activation of glial cells can induce the activation of autophagy to maintain the steady state of the body.OBJECTIVE: To study the injury of motor neurons in spinal cord anterior horn,cerebral cortical which is induced by abnormally high expression of syncytin-1 and observe the changes of autophagy.Methods: Eight weeks old C57 BL / 6J male mice were divided into 4 groups.After anesthetized by 10% chloral hydrate(1ml per 100mg),the left anterior tibial anterior muscle was exposed,200 μl of recombinant plasmid p CMV-tag2B-syncytin(1.5 μg / μl)and 200 μl of empty plasmid p CMV-tag2B(1.5 μg / μl)was injected by microinjector into the experimental groups and control groups respectively.In the experimental group 1 and the control group 1,the weight of the mice was measured at 0,30 and 60 days after the injection of the plasmid,and the motor function was evaluated by the climbing test and the m NSS score.The resting potential of the gastrocnemius muscle,the light contraction potential,the potential of strong contraction were measured,and the nerve conduction velocity were recorded on the 60 th day after implantation of the plasmid.The morphological changes of the muscle fibers were observed by HE staining on the gastrocnemius muscle on 60 th day after implantation.The number and morphology of the motor neurons were observed by Nissl staining.The expressions of autophagy index P62 and microtubule-associated protein 1 light chain 3b(LC3B)were observed by immunofluorescence staining in spinal cord anterior horn and cerebral cortic-al.The astrocyte(GFAP)and microglia(Iba1)were observed by immunohist-ochemical staining and the changes of cell morphology.The expression of inducible nitric oxide synthase(i NOS),tumor necrosis factor-α(TNF-α),LC3 B,P62,syncytin-1 were measured by western blot.Expression of i NOS,TNF-α,LC3 b,P62,syncytin-1 m RNA in spinal cord and cerebral cortex motor area were also detected.The mice in the experimental group 2 and the control group 2 were evaluated for body weight and motor function at 0,30,60,90 and 120 days after plasmid implantation.The electromyography was performed on the 120 th day after plasmid implantation.Repeated HE staining,immunofluorescence,immunohistochemistry,western blot,q RT-PCR detection after performance of electromyography.Results:There is no significant difference in body weight and motor function scores between experimental group 1 and control group 1,experimental group 2 and control group 2(P> 0.05).In the experimental group 2,the electromyogram showed that there was fibrillation potential in gastrocnemius at rest,the amplitude of the action potential was increased and time limit of MUAP was prolonged in the light contraction state.Denervation showing as simple phases was observed in the strong contracted muscles.Nerve conduction studies showed motor nerve conduction velocity slowing down.By HE staining,gastrocnemius muscles of the experimental group 2 showed that part of muscle fibers were atrophic,and some were polygonal,round atrophy which were manifestations of typical nerval damage.The results of Nissl staining showed that the number of motor neurons in the spinal cord anterior horn and cerebral cortex motor area of the experimental group 2 was significantly lower than that in the control group 1,2 and experimental group 2.Immunohistochemistry staining showed the activation and increased numbers of astrocytes and microglia in the spinal cord anterior horn and cerebral cortex motor area in the experimental group 2.The results of immunofluorescence showed that the expression of LC3 B and P62 was higher in the spinal cord anterior horn and cerebral cortex motor area in the experimental group 2 than other groups.The expression of syncytin-1 in the gastrocnemius muscles,spinal cord anterior horn and cerebral cortex motor area in the experimental group 2 was significantly higher than that in the control group 1,2 and experimental group1(P <0.05),The expression of TNF-α,i NOS,LC3 B,P62 and protein in the spinal cord anterior horn and cerebral cortex motor area in the experimental group 2 was significantly higher than that in the control group 2(P <0.05),while the experimental group 1 and the control group 1 had no significant difference in the expression of TNF-α,i NOS,LC3 B,P62 and syncytin-1 protein(P> 0.05).The level of TNF-α,i NOS,LC3 B,P62 and syncytin-1 m RNA in the spinal cord of the experimental group 2 was significantly higher than that in the control group 2(P <0.05).The experimental group 1 and the control group 1 showed no significant difference in the level of TNF-α,i NOS,LC3 B,P62 and syncytin-1 m RNA(P> 0.05).Conclusion: 1.The expression level of syncytin-1 in the skeletal muscles was positively correlated with the time of plasmid implantation relatively.2.The mice that syncytin-1 highly expressed in skeletal muscle had a higher level of syncytin-1 in the spinal cord enlargement and cerebral cortex motor area relatively,and the level of syncytin-1 was positively correlated with the time of plasmid implantation.3.The mechanisms of motor neuron injury in mice,which induced by the elevated levels of syncytin-1 in the skeletal muscle,included glial cell activation,oxidative stress,immune inflammatory response and so on.The degree of damage aggravated with the time of plasmid implantation.4.The expression of autophagy markers was significantly higher against the injury of motor neurons in mice with elevated levels of syncytin-1 in skeletal muscle.ASince autophagic activation has protective effects on motor neurons,this may suggest that the activation of autophagy was not sufficient to remove abnormal proteins and damaged organelles completely,and could not eliminate motor neuron damage completely. |
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