| Objective:NG108-15 cells were stimulated by Okadaic acid(OA)to induce tau phosphorylation in Alzheimer’s disease(AD)cell model.The aim of this study was to investigate the effects of stilbene glycoside on phosphorylated Tau protein and the expression of related phosphokinase,phosphatase and phosphokinase regulators in NG108-15 cells induced by Okadaic acid.Methods:The effects of TSG on proliferation of NG108-15 cells which induced by Okadaic acid at 24 h was observed by MTT assay.NG108-15 cells in logarithmic growth stage were taken and divided into normal group,model group,low,medium and high dose groups of TSG(50,100,200 μmol/L).The effect of TSG on apoptosis was detected by AO/EB double fluorescence staining.The expression of CDK5,GSK3β,CK1,MAPK1,JNK,PP2 A,PP2B,PP5,PKA,P35,Tau and P-Tau were detected by western blot.The expression of mRNA of CDK5,GSK3β,PP2 A and PP2 B were measured by RT-PCR.And the expression and distribution of CDK5,GSK3β,PP2 A,PP2B and Tau protein were detected by immunofluorescence.Results:MTT results showed that OA significantly inhibited the proliferation of NG108-15 cells in a dose-dependent manner,When the concentration was 80nmol/L,the cell survival rate decreased significantly(P<0.01).and AO/BE fluorescence staining showed that the cells had apoptosis.With 80nmol/L as the model dose,TSG could enhance the proliferation capacity of OA induced NG108-15 cells in a dose-dependent manner.Compared with the model group,TSG can inhibit the aging of NG108-15 cells by OA,and improve the cell survival rate(P<0.05).(1)Effect of TSG on OA-induced Tau protein phosphorylation in NG108-15 cellsWestern blot analysis showed that the P-Tau content in the model group was significantly higher than that in the normal group(P<0.01).Compared with the model group,the P-Tau content in the middle and high dose groups decreased after TSG administration.<0.01 or P<0.05),in the immunofluorescence assay,the TSG administration group was able to reduce the aggregation of Tau protein compared to the model group.The results indicate that TSG reduces the level of Tau phosphorylation in OA-induced NG108-15 cells and reduces the degree of accumulation of Tau protein.(2)Effect of TSG on OA-induced phosphokinase in NG108-15 cells.Western blot analysis showed that the levels of phosphokinase CDK5,GSK3β,PKA,CK1,MAPK1,and JNK proteins in the model group were significantly higher than those in the normal group(P<0.01),and were administered after TSG administration compared with the model group.The levels of phosphokinase CDK5,GSK3β,PKA,CK1,MAPK1,and JNK were decreased(P<0.01 or P<0.05).RT-PCR results showed that the transcription of CDK5 and GSK3β mRNA in the model group was significantly increased compared with the normal control group(P<0.05 or P<0.01),and TSG significantly down-regulated the expression of CDK5 and GSK3β mRNA in NG108-15 cells induced by OA(P<0.05 or P<0.01).In immunofluorescence assay,compared with the model group,the TSG group could reduce the expression of CDK5,GSK3β,PKA,CK1,MAPK1,and JNK proteins.The results indicate that TSG can reduce the expression of phosphokinase,thereby reducing the phosphorylation level of Tau.(3)Effect of TSG on OA-induced phosphatase in NG108-15 cellsWestern blot analysis showed that the levels of phosphatase PP2 A,PP2B,and PP5 protein in the model group were significantly lower than those in the normal group(P<0.01).After TSG administration,compared with the model group,the phosphatase PP2 A was administered.The content of PP2 B and PP5 protein was significantly increased(P<0.01 or P<0.05).There was no significant difference in PP5 protein content in the low dose group.RT-PCR results showed that the transcription of PP2 A and PP2 B mRNA in the model group was significantly decreased compared with the normal control group(P<0.05 or P<0.01),and TSG could up-regulate the expression of PP2 A and PP2 B mRNA in NG108-15 cells induced by OA(P< 0.05 or P < 0.01).Immunofluorescence results showed that the TSG administration group increased the expression of PP2 A and PP2 B proteins compared with the model group.The results indicate that TSG can increase the expression of phosphatase and enhance the dephosphorylation level of P-Tau.(4)Effect of TSG on OA-induced phosphokinase regulatory factors in NG108-15 cellsWestern blot analysis showed that compared with the normal group,the content of P35 protein in the model group was significantly increased(P<0.01).Compared with the model group,the content of P35 in the drug-administered group decreased after administration of TSG(P<0.01).The results suggest that TSG can down-regulate the level of phosphokinase regulatory factors,thereby reducing the phosphorylation level of Tau protein.Conclusion:TSG can increase the anti-aging ability and survival rate of OA-induced NG108-15 cells and decrease the abnormal phosphorylation level of Tau protein.The mechanism may be related to down-regulating the phosphoprotein kinase and its regulatory factors,up-regulating the related phosphatase,and regulating the balance between phosphorylation and dephosphorylation. |
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