The Effect Of Ns7 On The Motor Ability And Early Motor Neurons Of Spinal Cord In The ALS-SOD1^G93A Mice

Amyotrophic lateral sclerosis（ALS） is a fatal neurodegenerative disease characterized by upper and lower motor neuronal cell progressive death. The 90-95% of ALS cases are sporadic and approximately 5-10% of cases are familial. Those patients who developed ALS normally aged between 50 and 65, and the symptoms of ALS are myasthenia, spasm, convulsion that resulted in muscle injury could cause the premature death in dyspnea and dysphagia of patients. Up to now, riluzole is the sole therapeutic drug authorised by FDA. Unfortunately, there is no significant projected medicative outcome of riluzole.ALS is a kind of motor neuron diseases involving multiple system and its pathogenesis is related to manifold factors. It is considered that oxidative stress is one of important causative factor. Early research found, a large quantitive of DNA, protein and lipid under oxidative damage had been found in nervous tissue through pathological changes. In the clinical pathological examination, carbonyl protein and nitrotyrosine showed increased in the spinal cord and cortex of patients. Moreover the lipid peroxidation had been noticed in neurons, astrocyte and microglia. Research based on the ALS-SOD1^G93A animal model indicated that the free radical content grew along with the progression of disease. According to this factor, applying some antioxidants in ALS remedy to obliterate an excess of free radical had been proved capable of inhibiting the neurodegeneration, putting off the onset time and prolongating the life span of animals as well. Therefore, antioxidants may have medicative effects on ALS and the further research in this aspect will have important theoretical and practical significance to provide novel options for developing new drugs to treat ALS.Iso-suillin（ns7）（3, 6- dihydroxy-（2E, 6E, 10E） 3,7,11,15- hexamethyl hexadecin-2,6,10,14-tetraene） ethyl benzoate), one of the polyisoprene phenolic compounds, was a single compound separated from Suillus luteus by the research group headed by professor Wang Li’an of the College of Life Science, Hebei Normal University. It is the derivant of kiwi fruit rhzomorph. Proven by recent studies, ns7 gets a strong capacity of anti-oxidative activity and tumour-suppression, besides it is more skilled than Vc on eliminating DPPH free radicals outside the body and lessening the malondialdehyde（MDA） content in serum and liver of senile mouse simultaneously. Nevertheless there is no report about the neuroprotective effects of ns7 by means of its powerful anti-oxidative activity. In the present study, the ALS-SOD1^G93A transgenic mice（genetic background of C57BL/6） were used as research objects, and were divided into blank control group（wild-type littermates）, control group(ALS-SOD1^G93A transgenic mouse) and experimental group(ns7 group, ALS-SOD1^G93A transgenic mouse). Ns7 was dissolved by sesame oil. The animals of the blank control group and the control group were given sesame oil（15ml/kg） intragastrically in the 40-90 days after they were born. The animals of the experimental group were given ns7（50mg/kg） intragastrically in the 40-90 days after they were born. The skeletal muscle contraction capacity, the coordination ability and the balance ability of the mice were evaluated through the rotarod test and the hanging wire test. The immunohistochemical techniques and the TEM technique were used to observe the ultrastructural changes of motor neurons in the anterior horn of spinal cord, and the western blot method was used to detect the expression of HSP70, Caspase3 and Bax. To explore the powerful antioxidant of ns7 for protecting motor neurons and delaying ALS pathologic process, the results of the above were statistically analyzed.The experimental results are as follow:1. The duration of rotarod test of the experimental group was extremely significant longer than the control group start from the week 21（P < 0.01） and the time that the experimental group mice could not finish the rotarod test was three weeks later than the control group. After the 21 th week, the duration of rotarod of the blank control group was consistently significantly longer than the control group and the experimental group.2. The duration of the hanging wire test of the experimental group shortened since the 20 th week and was two weeks later than the control group. On the 25 th week, the duration of the experimental group was still six seconds, however the animals of control group had lost the ability of hanging wire completely for three weeks. Consistently, the testing duration of experimental group was extremely significant longer than that of the control group after the 19 th week（P < 0.01）. The duration of the hanging wire test of the blank control group was extremely significant longer than that of both the control group and the experimental group（P < 0.01）.3. The weight of the control group and the experimental group was significantly lower than that of the bank control group since the 21 h week（P < 0.01）, yet there was no significant difference between the control group and the experimental group（P > 0.05）. However, the reduction rate of the average weight of mice in experimental group was slower than of the control group.4. The average life span of mice in both the experimental group and the control group were 169 and 158 days respectively. The average life span of the two groups were significantly different（P < 0.05）. Nevertheless there was no death in blank control group before the 30 th week.5. The number of Ch AT positive motor neurons in cervical spinal anterior horn and the lumbar spinal anterior horn of the mice in blank control group were extremely significant higher than that in both the control group and the experimental group（P < 0.01）. However there were no significant difference between the control group and the experimental group in cervical spinal anterior horn and the lumbar lumbar spinal anterior horn.6. The TEM results shown that there were a large amount of mitochondria around the cell nucleus of motoneurons in the cervical cord and the lumbar cord of the blank control group, and the morphology of these mitochondria appeared plump, the cristae seemed relatively obvious and there was no swell at all. Meanwhile there was a decline in the number of mitochondria around the nucleus of motor neurons in the cervical cord and lumbar cord of the control group and the experimental group, and these mitochondria presented swollen and had vacuole inside. The conditions inside the mitochondria of mice in the experimental group were worse than that in the blank control group but were better than that in the control group.7. The results of western blot shown that there were no significant differences of the expression of HSP70、Caspase3 and Bax protein among three groups in cervical spinal cord and lumbar spinal cord（P > 0.05）.Here are the conclusions after analysing the experiment results:1. From postnatal day 40-90 days, every day in the stomach given at a dose of 50 mg/kg of ns7 efficiently improved the skeletal muscle contractility and the coordination ability and the balance ability of the ALS-SOD1^G93A mice, and it was able to prolong the lifespan of the mice significantly.2. The improvement of the above described function could be relevant with the protection of ns7 to lower the reduction rate of mitochondria, and to improve the performance of motor neurons and to attenuate the apoptosis.Our preliminary studies provided us the fundamental data for further exploration of the protecting effect of ns7 for motor neurons.