Effects Of Ginkgolide B On JNK Protein And Apoptosis In Amyotrophic Lateral Sclerosis Cell Model

ObjectiveAmyotrophic lateral sclerosis,a fatal of Neurodegeneration disease,is characterized by progressive degeneration of motor neurons in the brain and spinal cord,resulting in muscle weakness,paralysis,and death.There are many hypotheses,such as genetic mechanism,oxidative stress,excitatory glutamate toxicity,accelerated apoptosis,autoimmune mechanism,environmental factors and aging.Ginkgolide B,a PAF receptor antagonist that not only has anti platelet aggregation effects,many studies have shown that Ginkgolide B has anti-inflammatory,anti-apoptosis,energy metabolism,astrocyte protection,anti-excitotoxicity and immunoregulatory effects.JNK signaling pathway is one of the main MAPK family members,and participates in neuronal apoptosis and neuroinflammation.SOD1 can regulate JNK/SAPK activity,and JNK/SAPK pathway may play a neuroprotective role in ALS motoneurons.In order to investigate the protective effect.of Ginkgolide B on amyotrophic lateral sclerosis cell model and its mechanism,the effects of different concentrations of Ginkgolide B on Cell Survival were investigated The apoptosis rate of each group was detected by flow cytometry,and the expression of Caspase-3,p-JNK,c-Jun and p-c-Jun proteins was detected by Western blot(WB)The mechanism of action of Ginkgolide B on amyotrophic lateral sclerosis(ALS)cell model was explained from the view of anti-apoptotic protective neurons.Methods1.The establishment of amyotrophic lateral sclerosis cell modelCell,Lentivirus:NSC34 cell,lentivirus with overexpression of h SOD1WT、h SOD1G93A and empty plasmid:NSC34 cells with logarithmic growth were divided into four groups,each group was treated with h SOD1WT、h SOD1G93A and empty plasmid respectively,The expression of green fluorescence was observed under inverted Fluorescence microscope.After purinomycin screening,the morphological changes of the cells in each group were observed under inverted Fluorescence microscope,and the expression of SOD1 protein in each group was detected by W-b method.2.The effect of GinkgolideB on amyotrophic lateral sclerosis cell modelThe survival of hSOD1G93A-NSC34 cells treated with different concentrations of GinkgolideB(25,50,75,100 μg·mL-1)for 48 h was determined by MTT Assay,and the optimal concentration of Ginkgolide B was selected.3.The effect of Ginkgolide B on Apoptosis of amyotrophic lateral sclerosis cell modelThe following experimental groups were normal group,model group,model group+75 μg·mL-1 GB group,model group+SP600125 group,model group+75 μg·mL-1 GB+SP600125 group,The apoptosis of each group was detected by Flow cytometry(Apoptosis rate%=early apoptosis rate%+late apoptosis rate%)4.Effects of GinkogolideB on the expression of Caspase-3,p-JNK,p-c-Jun,and c-Jun in amyotrophic lateral sclerosis cell modelThe protein expressions of Caspase-3,p-JNK,c-Jun and p-c-Jun were detected by Western blotting after 48 hours of drug culture.Results1.The establishment of amyotrophic lateral sclerosis cell modelThe establishment of amyotrophic lateral sclerosis cell model showed that the cells of each group had green fluorescence expression after infection with lentivirus,compared with the normal NSC34 cell group,There was no obvious change in cell morphology in the wild type group and the empty plasmid group,but in the mutant group,the cell bodies became round,the synapses became shorter and decreased.Compared with the normal group,the expression of SOD1 protein in the empty plasmid group had no significant difference(P<0.05),but the expression of SOD1 protein in the wild type group and the mutant group had significant difference(P<0.01),Compared with the mutant group,the expression of SOD1 protein in the wild type group was not significantly different.2.The effects of different concentrations of Ginkgolide B on the survival rate of amyotrophic lateral sclerosis cells.The results showed that the survival rate in the ALS cells treats with different concentration of GinkgolideB 25 μg·mL-1,50 μg·mL-1,75 μg·mL-1,100μg·mL-1 was65%,75%,83%,79%respectively Compared with the normal group,the cell survival rate of the model group decreased significantly(P<0.01).When the concentration of Ginkgolide B was 25 μg·mL-1,there was no significant difference in cell survival rate between the model group and the Control Group The cell survival rate increased significantly(P<0.01),especially when the concentration was 75 ug·mL-1.3.Effect of GinkgolideB on Apoptosis of amyotrophic lateral sclerosis cell modelThe results of flow cytometry showed that compared with normal group,the apoptosis rate in the model group was significantly increased,and the early apoptosis rate was significantly higher than that of Normal Group(P<0.01)Compared with model group,apoptosis rate in model group+GB Group,model group+SP600125 group,model group+GB+SP600125 group decreased significantly,and apoptosis rate in early stage decreased significantly(P<0.01),compared with model group+GB Group The apoptosis rate of model group+SP600125 group had no significant difference,the apoptosis rate of model group+GB+SP600125 group decreased(P<0.01),the result showed that Ginkgolide B could reduce the apoptosis degree of amyotrophic lateral sclerosis cell model。It is suggested that the protective effect of anti-apoptosis may be mediated by JNK signaling pathway.4.Effects of GinkgolideB on the expression of p-JNK,c-Jun,p-c-Jun,Caspase-3 protein in amyotrophic lateral sclerosis cell model by Western BlotThere were significant differences in p-JNK,p-c-Jun/c-Jun,Caspase-3 protein expression in model group(P<0.01).Compared with model group,p-JNK,p-c-Jun/c-Jun and Caspase-3 protein expression in model group+GB,model group+SP600125,model group+GB+SP600125 were significantly different(P<0.05,P<0.01,P<0.01).The expression of p-JNK,p-c-Jun/c-Jun,Caspase-3 protein in model group and SP600125 group was not significantly different from that in model group and SP600125 group p-JNK,p-c-Jun/c-Jun,Caspase-3 protein expression was significantly different from that in model group(P<0.05).It was suggested that the expression of p-JNK,p-c-Jun/c-Jun,Caspase-3 protein decreased after administration of Ginkgolide B and SP600125 The expression of p-JNK,p-c-Jun/c-Jun and Caspase-3 decreased obviously。Conclusion1.Amyotrophic lateral sclerosis model was established successfully2.Ginkgolide B can improve the survival rate of amyotrophic lateral sclerosis cell model3.Different concentrations of Ginkgolide B could improve the cell survival rate。When the concentration of Ginkgolide B was 75 μg·mL-1,the cell apoptosis was reduced and the expression of Caspase-3 protein was inhibited。These results suggest that Ginkgolide B may play a neuroprotective role by inhibiting cell apoptosis.4.In this study,we found that in the amyotrophic lateral sclerosis cell model,Ginkgolide B may inhibit the phosphorylation of c-Jun and Caspase-3 by mediating JNK signaling pathway,thus reducing the phosphorylation level of c-Jun and the activity of Caspase-3 plays the role of anti-apoptosis,the protection of neurons.