| ObjectiveAmyotrophic lateral sclerosis(ALS)is a rare neurological disease,characterized by muscle weakness,atrophy of muscles,dysphagia,difficulty of swallowing and respiratory failure.It is lack of special treatment.This study proposed the hSODl-G93A transgenic mice,giving the treatment of Jianpi yifei Recipe,and by evaluating weight changes,motor function and survival duration to observe the curative effect of Jianpi yifei Recipe.Application of immunofluorescence is used to determine the detection of lumbar spinal anterior horn neurons nucleoprotein(Neuronal Nuclei,NeuN),microglia integrin alpha M(CD11b)and nuclear transcription factor NF-κB p65.Then the anti-inflammatory effects of Jianpi yifei Recipe on ALS mice and related mechanism was under preliminary discussion.Methods1.HSOD1-G93A transgenic model mice were randomly divided into:normal saline group,low concentration of Jianpi yifei Recipe group,middle concentration of Jianpi yifei Recipe group and high concentration of Jianpi yifei Recipe group.Wild type mice were set for Control group.Each group has 6 mice.Model mice were given saline or corresponding concentration of Jianpi yifei Recipe.Control group were given same amount of saline.Treatment was given once per day.2.Determination of body weight,onset time and survival duration by Weydt scores,motor function evaluation by tail suspension test,hanger test and rota-rod test were made.Statistical analysis of differences between groups was done.3.Immunofluorescence staining was applied to observe anterior horn NeuN positive cells counting,CD11b positive activated microglia and nuclear/cytoplasm ratio of NF-κB p65 fluorescence intensity(Fn/Fc).Statistical analysis of differences between groups was done.Results1.WeightCompared with normal saline group,difference of weight in low concentration group had no statistical significance(P>0.05).Difference in the middle concentration and high concentration group statistically significant(P<0.05,P<0.01).2.Onset time and survival duration Onset time of saline group,low concentrationgroup,middle concentration group and high concentration group were(88.83 +/-4.12),(101.33 + 5.75),(108.83-6.68),(113.50-10.74)days,respectively.Compared with saline group,onset time in low concentration group was no significant difference.Onset time of middle concentration group and high concentration group was significantly delayed,statistically significant(P = 0.986,0.035,0.001).Survival duration of saline group,low concentrationgroup,middle concentration group and high concentration group were(125.17 +/-3.71)and(132.17 + 2.93),(138.50-5.05),(143.83-3.97)days.Compared with saline group,Survival duration in low concentration group,middle concentration group and high concentration group was extended with statistically significant difference(P-0.017,0.000,0.000).3.Motor functionHanger test:9-14 weeks,saline group and the low concentration,the concentration and high concentration of spleen and lung group model mice fell in a low level of steady state,with no significant difference.15 to 16 weeks of age,saline group drop frequency increase rapidly,at the same time Jianpi yifei Recipe groups are basically stable.17 weeks of age,saline group ended the test.And Jianpi yifei Recipe group drop frequency increased rapidly.Compared with saline group,low concentrationgroup,middle concentration group and high concentration group drop less,with difference statistically significant(all P<0.01).Rota-rod test:Compared with saline group,low concentration group,middle concentration group and high concentration group drop less,with differences statistically significant(P<0.05).4.NeuN positive cell countCompared with saline group,low concentration group,middle concentration group and high concentration group has more NeuN positive cell count,with significant difference(P<0.01).5.CDllb immunofluorescence expressionActivated microglia count:Compared with saline group,low concentration group,middle concentration group and high concentration group has less CDllb positive activated microglia counts,with difference statistically significant(P<0.05,P<0.01,P<0.01).Proportion of activated microglia:Compared with control group,proportion of activated microglia in saline group was increased significantly(P<0.01).Compared with saline group,proportion of activated microglia in low concentration group has no significant difference(P = 0.052),and middle concentration group and high concentration group has higher proportion of activated microglia with statistically significant difference(P<0.01).6.Nuclear/cytoplasm ratio(Fn/Fc)of NF-κB p65 fluorescence intensityCompared with saline group,Fn/Fc of NF-κB p65 has no difference in low concentration group,with no significant difference(P = 0.995).Fn/Fc of NF-κ B p65 in middle concentration group and high concentration group were significantly decreased,and difference was statistically significant(P<0.01).Conclusion1.After disease onset,hSOD1-G93A transgenic mice has serious weight loss,motor function decline and shorter life span.Application of Jianpi yifei Recipe may delay the onset time,prolong the survival period,keep the body weight and improve the motor function.2.After disease onset,hSOD1-G93A transgenic mice has serious anterior horn nerve cells loss,more activation of microglia and more nuclear translocation of NF-κ B p65 in spinal cord.High concentrations of Jianpi yifei Recipe can reduce the spinal cord anterior neurons loss。Middle and high concentration of concentration of can inhibit the microglia activation and inhibit the NF-κB p65 nuclear translocation process in model mice.3.The functional mechanism of Jianpi yifei Recipe may be through inhibiting the NF-κB signaling pathways,reduce the microglia activation,thus playing the role of nerve inflammation resistance and reduce the neuron damage and loss,finally slow disease progression. |
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