Based On The Ubiquitin-proteasome System To Explore The Mechanism Of Jianpi Tongluo Prescription In The Treatment Of Amyotrophic Lateral Sclerosis

Objective:To explore the role of the ubiquitin-proteasome system in the pathogenesis of Amyotrophic Lateral Sclerosis,and to discover effective,safe and reliable traditional Chinese medicine compounds.Looking forward to providing new theoretical and scientific basis for the treatment of ALS.Method:In this study,SOD1^G93A transgenic mice were used as the research objects,and the therapeutic effects of Jianpi Tongluo prescription on the early,middle and late stages of ALS were investigated by fluorescent staining,transmission electron microscopy,HE and Nissl staining.The experiment was divided into three periods:early,middle and late onset,and each period was divided into 4 groups,namely the blank group with male wild-type mice;model group,Jianpi Tongluo prescription group,and Riluzole group,all were male ALS-SOD1^G93A transgenic mice.The mice were administered from the 30th day of age.The Jianpi Tongluo prescription group and the Riluzole group were given the extract of Jianpi Tongluo and riluzole respectively according to the dose,and the blank group and the model group were given the same amount of normal saline by gavage,once a day.The mice were administered until the age of the mice（30+30）days,（30+70）days,and（30+110）days respectively.During the experiment,weight measurement,rod experments and step measurement were carried out every 14 days.At the end of the experiment,serum and spinal cord of SOD1^G93A transgenic mice were collected to detect the content of 26SPSM and NFL in serum and the expression of NEDD4-1,IGF-1β,20S proteasome,ubiquitin,Akt and SOD1 proteins in spinal cord.Results:The weight measurement results of SOD1^G93A transgenic mice showed that the weight in each group remained stable in the early stage of disease.In the middle stage of disease,except for the blank group,the weight of mice in the other three groups began to decrease.Compared with the model group,the weight of the mice in the Jianpi Tongluo prescription group and the Riluzole group decreased slowly,and the degree of weight loss was smaller.The results of motor function showed that the motor function of the mice in the blank group remained stable.The step length and stick time of the mice in the model group were significantly shortened with the development of the disease,the footprints were chaotic,and the dragging and walking traces were obvious,indicating that their motor function was impaired.Compared with the model group,the motor function of the mice in the Jianpi Tongluo prescription group and the Riluzole group was slightly impaired,and the time on the stick and the step length were significantly increased,and the footprint gait was improved.The staining results and electron microscope observation showed that the spinal cord neurons of the model group were significantly changed and the number of neurons was reduced,the vacuoles were obvious,the Nissl bodies were granular and decreased in number,the axons were shortened,the nuclear membrane was deformed or partially ruptured.The number of organelles decreased,and the morphology of mitochondria and endoplasmic reticulum changed significantly.Compared with the model group,the lumbar spinal cord neurons and Nissl bodies of the mice in the Jianpi Tongluo prescription group and the Riluzole group were improved in morphology and number,and the ultrastructure of the cells was also improved.The results of neuron counting showed that the number of lumbar spinal cord motor neurons of mice in the model group decreased significantly with the development of the disease,while the administration of Jianpi Tongluo prescription and riluzole could significantly increase the number of spinal cord motor neurons.With the development of the disease,the serum 26S proteasome and NFL contents of mice in the model group gradually decreased and increased.The change trend of the contents of each index in the Jianpi Tongluo prescription group and the Riluzole group was the same as that in the model group,but the degree was obviously moderated.At each time point,the content of 26S proteasome in the two groups of mice was higher than that in the model group,and the content of NFL was lower than that in the model group.The results of Western blotting showed that at the end of the disease,the expressions of NEDD4-1,Akt and SOD1 in the spinal cord of the model group were significantly higher than those in the blank group,while the expressions of IGF-1β,20S proteasome and ubiquitin were significantly increased.After the administration of Jianpi Tongluo prescription and riluzole,the expression levels of these proteins showed a significant downward trend.Conclusion:Jianpi Tongluo prescription can delay the weight loss of SOD1^G93A transgenic mice,improve their motor function,and reduce the loss of lumbar spinal cord motor neurons in SOD1^G93A transgenic mice.By up-regulating the expression of IGF-1β,20S proteasome,and ubiquitin,and down-regulating the expression of NEDD4-1,Akt,and SOD1.In order to reduce the damage of ubiquitin-proteasome system function in SOD1^G93A transgenic mice,reduce the probability of degenerative neuropathy in spinal motor neurons,and delay the development of the disease.