| Background and Objective:Some neurological diseases,such as hemiplegia after stroke,spinal injury,Parkinson's disease and so on,may have marked and durable muscular hypertonia and bring lots of burdens and pains to the patients. To remit the hypertonia effectively is a key point determining the patients'prognosis. Nowadays, few muscle relaxant, such as baclofen, has been widely accepted for the treatment of muscular hypertonia, but the effect isn't steady and has obvious side effect at the same time, So their application in clinic is limited greatly. At present,facial spasm is one of common dysmyotonia neurological diseases. In clinic,borulinus toxin type A has been proved as a effective drug which can be appliedonly to local lesion, so its application has been limited greatly. Therefore, to search new drugs and methods is a difficult question to improve the treatment of muscular hypertonia in clinic.Among the snake venom, neurotoxin is the most toxic component, which results in flaccid paralysis and respiratory failure in animals.According to different biological activity, neurotoxin can be divided into two types: Type one is the presynaptic neurotoxin, which can inhibit the release of acetylcholine during the nervous pulse conduction. Type two is the postsynaptic neurotoxin, which can bind to the acetylcholine receptor of the end plate, so block the nerve conduction. We want to modify the neurotoxin to reduce the toxicity, then apply it to muscular hypertonic animals and expect to find a new method to cure muscular hypertonia in clinic. The objective of our experiments is as follows: 1. To extract the neurotoxin from the snake venom. 2. To reduce the toxicity by the chemical modification. 3.The modificated neurotoxin is applied to rats suffered from spinal injury, then we evaluate the improvement of muscular hypertonia.Methods:DEAE-Sephadex A-50 chromatography and Sephadex G-50 gel filtration were used to separate the snake venom,then the neurotoxin wasconfirmed by evaluating the various curve peaks. After the neurotoxin was collected, the concentration of the protein was measured by using Folin-phend method. The purity of the neurotoxin was evaluated by SDS-polyacrylamide gel electrophoresis and mice'experiments,moreover,the median lethal dose of the neurotoxin was calculated. The amino- and sulfhydryl- in the neurotoxin were chemically modified,then the toxicity of the modified neurotoxin was learned about by mice' experiments. We applied the modified neurotoxin to rat models suffered from spinal injury to learn about whether muscular hypertonia was improved or not.Results: The second curve peak was confirmed to be neurotoxin by mice' experiments and the physiological recorder. Folin-phend method was used to measure the concentration of neurotoxin,then we can know the result was 0.0138g/ml. Its purity was more than 80% by calculating stain sensibitity since single band was seen in the electrophoregram of SDS-PAGE. The result was accorded with mice'experiments.The median lethal dose of neurotoxin was 1.11mg/kg by calculating. The toxicity of neurotoxin was reduced by using acetic anhydride and NEM to modify neurotoxin. At last,muscular hypertonia was improved.Conclusions:1 Neurotoxin was extracted from the snake venom successfully by using DEAE-Sephadex A-50 chromatography and Sephadex G-50 gel filtration.2 After neurotoxin was modified by using acetic anhydride and NEM, its neurotoxicity was reduced greatly,especially by using NEM.3 The modified neurotoxin was applied to the rats(suffered from spinal injury and had marked muscular hypertonia). These rats'muscular hypertonia and the rats'survival time were greatly improved, especially by using NEM. The chemical modification of neurotoxin had marked effect on attenuation... |
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