Effect Of Chinese Medicine Jianpi Yifei Recipe On Glutamate-induced Spinal Cord Injury

ObjectiveAmyotrophic lateral sclerosis(ALS)is a disabling,lethal neurodegenerative disease characterized by upper and lower motor neuron damage,clinical signs of motor function loss,and neurophysiological manifestations.Respiratory failure occurs at the end of the disease,eventually leading to death.At present,the cause of the disease is unknown,and there are many hypotheses about its pathogenesis.At this stage,glutamate-mediated excitotoxicity is generally accepted;at the same time,the correlation between glutamate-mediated excitotoxicity and mitochondrial dysfunction has been continuously demonstrated.This study established the preparation of Jianpi Yifei Recipe and glutamate induction model,found that Jianpi Yifei Decoction can reduce glutamate-induced astrocyte model glutamate excitotoxicity and increase glutamate-induced mitochondrial function in PC12 cell model.Methods1.Preparation of experimental animals,cells and Jianpi Yifei Prescription lyophilized powder1.1 Animals:Pregnant SD rat,waiting for labor,take newborn rats within 24 hours to extract cortical astrocytes;14-16 d fetal rats to extract spinal anterior horn motor neurons.1.2 Jianpi Yifei prescription Preparation of lyophilized powder2.Effect of Jianpi Yifei prescription on glutamate-induced expression of EAAT2 in astrocytes.Primary culture of astrocytes and neurons;Astrocytes were pretreated with Jianpi Yifei Recipe(low concentration 0.25g/L,medium concentration 0.5 g/L,high concentration 1 g/L)for 24 h,and glutamate(250 μ mol/L)was induced for 4 h.After establishing the cell injury model,the survival of each group of astrocytes was detected by MTT assay.The expression of EAAT2 was detected by IF method and Wb method.The concentration of glutamic acid in each medium was determined by UV colorimetry.Survival of neurons after the supernatant of the culture medium.3.Effect of Jianpi Yifei Prescription on mitochondrial function injury induced by glutamate in PC12 cellsPC12 cells were cultured,Preconditioning with Jianpi Yifei Recipe(low concentration 0.5 g/L,medium concentration 1 g/L,high concentra-tion 2 g/L)for 24 h,glutamate(250 μ mol/L)induction for 4 h to establish PC12 cell injury model After that,the survival of PC12 cells was detected by MTT assay,and the mitochondrial membrane potential,intracellular ROS and calcium ion concentrations of PC12 cells were detected by flow cytometry and immunofluorescence.Results1.Identification and culture of astrocytes and neuronal cellsThe proportion of astrocytes passed to the third generation is more than 96%.The proportion of spinal cord neurons cultured to 6 days was more than 90%.2.Effect of Jianpi Yifei Recipe on glutamate-induced expression of EAAT2 in astrocytes2.1.Glutamic acid group,Jianpi Yifei Recipe(low,medium,high concentration)pretreatment group,normal group astrocyte survival rate were 68%,78%,83%,86%,100%;2.2.Immunofluorescence measurement The mean optical densities of EAAT2 expression in the pretreatment group and the normal group of glutamic acid group and Jianpi Yifei formula(low,medium and high concentration)were 0.61,0.69,0.79,0.87 and 0.94,respectively.The expression of EAAT2 was found to indicate that the expression of EAAT2 protein was significantly increased after the intervention of Jianpi Yifei Recipe(P<0.05,P<0.01).2.3.Determination of the concentration of glutamic acid in the astrocyte culture medium of each group indicated that the concentration of glutamic acid in the culture medium of Jianpi Yifei Recipe group decreased significantly,the difference was statistically significant(P<0.05,P<0.01).2.4.Glutamic acid group,Jianpi Yifei Recipe(low,medium,high concentration)pretreatment group,normal group supernatant after treatment of neurons,the survival of each group of neurons were 72.67,92.33,133.67,143.33 160.67.3.Effect of Jianpi Yifei prescription on mitochondrial function injury induced by glutamate in PC12 cells3.1.The effect of Jianpi Yifei Recipe on the activity of PC12 cells induced by glutamate Compared with the normal group,the survival rate of glutamate model cells was significantly decreased(P<0.01).Compared with the model,the survival rate of the spleen and Yifei prescription was increased from 0.5 g/L after 24 h,and the cell viability increased with the concentration to 4 g/L,and 8 g/L began to decrease.There was no significant difference between the 89/L treatment groups(P>0.05),and there was no significant difference between the control group and the normal group(P>0.05).The 16 g/L group showed that the high con-centration of Jianpi Yifei Recipe had toxic effects on cells.3.2.Detection of spleen and Yifei Recipe on glutamate-induced mitochondrial membrane potential,ROS and intracellular calcium concentration in PC12 cells.Preconditioning with Jianpi Yifei Recipe can increase the membrane potential of PC12 cells(P<0.01)and reduce intracellular ROS production.Inhibits calcium influx.Conclusions1.Within the corresponding dosage range,Jianpi Yifei prescription can reduce the damage of glutamate excitotoxicity on astrocytes,increase the expression of EAAT2,decrease the concentration of extracellular glutamate,and reduce glutamate.Damage to neuronal cells,thereby protecting neurons.2.Within the corresponding dose range,Jianpi Yifei prescription can stabilize the mitochondrial membrane potential of PC12 cells,reduce the production of intracellular reactive oxygen species ROS,maintain intracellular calcium ion concentration,and thus reduce the cytotoxic damage to PC12 cells.