| ObjectiveAmyotrophic lateral sclerosis?ALS?is a neurodegenerative disease that invades the upper and lower motor neurons.Most of the patients survived only3-5 years after onset of the disease,and most of them died of respiratory failure.The pathogenesis of ALS has not been elucidated up to now.Previous basic research found that Jianpi Yifei Prescription may play an anti-inflammatory role by inhibiting NF-?B signaling pathway,thus delaying the progression of the disease.p38 mitogen-activated protein kinase?p38MAPK?is one of the major members of the mapk family.p38MAPK is involved in the neuroinflammatory response mediated by p38MAPK pathway.It was found in the mutant sod1 transgenic mice model,p38MAPK is one of the major members of the mapk family.Neuroinflammation caused by active microglia and astrocytes causes the death of motor neurons.To further explore the role of neuritis and neuronal apoptosis in the pathogenesis of ALS,the article to study the effects of different concentrations of Jianpi Yifei Prescription on the body weight,survival period and motor function,detection of phosphorylated p38MAPK protein and expression of inflammatory factors in mouse spinal cord by immunofluorescence and Western blotting of hsod1-G93A transgenic mice.Methods1.Animal groupingTwenty-four hSOD1-G93A transgenic mice,aged 8 weeks,were randomly divided into 4 groups with 6 mice in each group.namely model group,low concentration Jianpi Yifei Prescription group(2.86g·kg-1),medium concentration Jianpi Yifei Prescription group(5.72g·kg-1),high concentration Jianpi Yifei Prescription group(11.44g·kg-1),6 normal wild-type mice at the age of 8 weeks in the same litter were set up as the normal control group.The model group and normal group were given normal saline,and the drug group were given different concentrations of Jianpi Yifei Prescription once a day.2.Effect of Jianpi Yifei Prescription on behavior of ALS miceThe weight of mice was measured and recorded,three times a week,the onset time and survival time were determined by weydt score,and the motor function was evaluated by tail suspension test,traction test and treadmill test.3.Effect of Jianpi Yifei Prescription on Immunofluorescence?IF?expression of microglia,astrocytes and p38 protein in Spinal Cord of als miceAt the end of mice,embedded,fixed and sectioned,Then IF staining was carried out,comparative analysis of each mouse microglia activation marker and astrocyte count,and p38,the difference of the nuclear/plasma ratio of P 38 to p 38 fluorescence intensity.4.Effect of Jianpi Yifei Prescription on the expression of p38?p-p38?TNF-??COX-2 and iNOS in Spinal Cord of ALS miceAfter the end stage mice were selected,Western blotl was used to detect the expression of p38?p-p38?TNF-??COX-2 and iNOS.To analysis the date in difference of protein expression in each group.5.Effect of Jianpi Yifei Prescription on apoptosis of Spinal Cord neurons in als miceTo detection he differential expression of NeuN in the anterior horn of lumbar spinal cord of each group by immunofluorescence.To detecte the apoptosis of spinal cord cells in different groups of mice.Results1.On behavior of ALS miceThe results of body weight in ALS mice showed that the model group and the drug group was basically stable during 8-12 weeks,and there was nosignificant difference between them.From 13-14 week,the weight of als mice in the model group and the low concentration Jianpi Yifei group began to decrease,while the weight of the middle and high concentration Jianpi Yifei group was basically stable.From 15-16 week,model group and low concentration Jianpi Yifei group decreased rapidly,while the middle and high concentration Jianpi Yifei group to decrease slowly.Compared with the model group and the low concentration Jianpi Yifei group,the weight loss of ALS mice in the middle and high concentration Jianpi Yifei group was less than that in the lowconcentration group,and with significant differences.The onset time and survival time of ALS mice were shorter than that of drug group.Compared with model group,the onset time and survival time of low concentration Jianpi Yifei group were prolonged by 3 days and 4 days,but there was no statistical difference.However,the middle and high concentration Jianpi Yifei group were prolonged by 13 and 15 days,and the survival time was prolonged by 11 and 17 days.With significant differences.The results of motor function in als mice suggest,At the 9-14 week,the number of drops in the traction test and the treadmill test in each group was stable.From the 15th week,the model group began to drop increased.At the16th week,the fall times of low concentration Jianpi Yifei group began to increase,while the middle and high group were basically stable.Compared with model group,the difference was statistically significant.From the 17-18th week,the number of middle and high concentration Jianpi Yifei group began to increase,while the model group and the low concentration Jianpi Yifei group began to be intolerant the experiment.From 20 weeks,the middle and high group were also intolerant to the experiment.2.0n Immunofluorescence expression of microglia,astrocytes and p38The results of the study suggest that Compared with the normal group,the number of activated glial cells in the model group and the low concentration Jianpi Yifei group increased significantly.Compared with the model group,the number of glial cell activation in the middle and high concentration Jianpi Yifei group decreased significantly,and the difference was statistically significant.Compared with the normal group,the difference of the nuclear/plasma ratio between the model group and the low concentration group of p38MAPK?p-p38MAPK fluorescence intensity was statistically significant?P<0.01?.Compared with the model group,With significant differences in the middle and high concentration groups?P<0.01?.3.0n the expression of p38?p-p38?TNF-??COX-2 and iNOSCompared with the normal group,the expression of p38?p-p38?TNF-??COX-2 and iNOS protein in the model group and the low concentration group was significantly different from that in the control group?P<0.05,P<0.01?.Compared with the model group,the expression of p38?p-p38?TNF-??COX-2 and iNOS protein in the middle and high concentration group was significantly different from that in the model group?P<0.01?.4.On apoptosis of Spinal Cord neurons in ALS miceThe results of Neun show that compared with the normal group,the neurons in the model group and the low concentration group decreased significantly.Compared with the model group,the number of neuron positive cells in the middle and high concentration Jianpi Yifei group increased significantly.The results of TUNEL suggest that compared with the normal group,the number of apoptotic cells in the spinal cord anterior horn cells and their percentage in the total number of cells increased significantly in the model group and in the low concentration group.Compared with the model group,the number of apoptosis and the percentage of apoptotic cells in the group of medium and high concentration Jianpi Yifei Prescription decreased significantly.Conclusion1.Jianpi Yifei Prescription can delay the weight loss,improve the motor function,prolong the onset time and shorten the survival time of of hsod1-G93A transgenic mice.2.Jianpi Yifei Prescription inhibited the excessive activation and proliferation of microglia and astrocytes in a concentration-dependent manner.Down-regulation the expression of p38MAPK?p-p38MAPK?TNF-??COX-2and INOS protein.The results suggest that Jianpi Yifei Prescription may inhibit p38MAPK protein phosphorylation by mediating p38 pathway,reducing the activation of glial cells and regulating the expression of TNF-??COX-2?INOS inflammatory factor,to alleviate neuroinflammation and play a neuroprotective role.3.In this study,we found that there were neuronal loss and apoptosis in the anterior horn of spinal cord in ALS mice.It is possible that p38MAPK pathway can inhibit the phosphorylation of p38,thus inhibit the release of inflammatory mediators,inhibit apoptosis and protect neuron cells. |
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