Intervention And Mechanism Of Stilbene Glucoside On Abnormal Phosphorylation Of Tau Protein In APP / PS1 / Tau Transgenic Mice

Objective: In this study,APP / PS1 /Tau triple transgenic dementia(3 × Tg AD)mice with high expression of human tau gene mutation were used as the research objects to observe the effects of stilbene glycoside on phosphorylated Tau protein and the expression of relevant protein phosphokinase,protein phosphatase and phosphokinase regulators in 3 × Tg AD mice.To investigate the effect of stilbene glucoside on abnormal phosphorylation of tau protein and its mechanism in APP / PS1 / Tau transgenic mice,and to provide experimental basis for the development of early intervention drugs for Alzheimer’s Disease(AD).Methods: Forty-five eight-month-old male 3×Tg-AD mice were randomly divided into model group,positive control group,TSG low-dose group,TSG medium-dose group,TSG high-dose group,with 9 mice in each group..In addition,nine eight-month-old male 57 BL / 6J mice were selected as normal control group.The corresponding drugs were given by gavage for consecutive 60 days.The Spatial learning and memory ability was detected by Morris Water Maze;the changes of Nissella body in cerebral cortex and hippocampus were observed by Nissl staining method;the expression and distribution of phosphorylation Tau protein(Ser404 site)and Tau protein in brain tissue were detected by Immunofluorescence(IF)staining method.The expression and distribution of P39 expression in brain tissue was detected by Immunohistochemical(IHC)staining method.The mRNA change of Brain tissue CyclinDependent Kinase 5(CDK5),Extracellular Regulated Protein Kinases(ERK1/2),C-Jun NTerminalkinase(JNK),Protein phosphortase 2B(PP2B),Protein Phosphatase 1(PP1)were detected by real time quantitative Reverse Transcription Polymerase Chain Reaction(q RT-PCR).The protein change of phosphorylation Tau protein(Thr205 site),CDK5,ERK1/2,JNK,PP2 B,PP1 were detected by Western blotting(WB)method.Results:(1)Effect of TSG on learning and memory ability in 3×Tg-AD mice: compared with the normal control group,the escape latency in model group mice was significantly prolonged,the residence time in the original platform quadrant was shortened,and The times of crossing the platform was significantly reduced(P < 0.01);Compared with model group,the escape latency of mice in positive control group and TSG groups was significantly shortened,the residence time in the original platform quadrant was significantly prolonged,and The times of crossing the platform was significantly increased(P < 0.05 or P < 0.01).Compared with the positive control group,the residence time of the original platform quadrant in TSG medium and high dose groups was significantly prolonged,and The Times of crossing the platform was significantly increased(P < 0.05 or P < 0.01).(2)The effect of TSG on Nissl staining in 3×TgAD mice: Compared with the normal control group,the number of neuronal Nissl bodies in model group was significantly reduced and the staining was shallow;Compared with model group,the number of neuronal Nissosomes of mice in positive control group and TSG dose groups was significantly increased and the staining was darker.Compared with the positive control group,there were no significant changes in the number and staining of neuronal Nissosomes in TSG groups.(3)The effect of TSG on Tau protein phosphorylation in 3×Tg-AD mice: Western blotting results show that: phosphorylation Tau protein(Thr205 site)expression in model group mice brain tissue was significantly higher than normal control group(P<0.01);Compared with model group,the expression level of phosphorylated Tau protein(Thr205 site)in brain tissue of mice in positive control group and TSG dose groups was significantly decreased(P < 0.01);Compared with the positive control group,the expression level of phosphorylated Tau protein(Thr205 site)in brain tissue of mice in TSG medium and high dose groups was significantly decreased(P < 0.05 or P < 0.01).The results of immunofluorescence staining showed that compared with the normal control group,the average optical density of Tau protein and phosphorylated Tau protein(Ser404 site)in brain tissue of mice in model group were significantly increased(P < 0.01).Compared with model group,the average optical density values of Tau protein and phosphorylated Tau protein(Ser404 site)in positive control group and TSG dose groups were significantly decreased(P < 0.01).Compared with the positive control,the mean optical density of Tau protein and phosphorylated Tau protein(Ser404 site)in TSG groups had no significant difference.(4)The effect of TSG on Tau protein phosphokinase in 3×Tg-AD mice: Western blotting results show that the expression of protein CDK5,ERK1/2,JNK in model group mice brain tissue was significantly higher than normal control group(P<0.01);Compared with model group,the protein expressions of CDK5,ERK1/2 and JNK in brain tissue of mice in positive control group and TSG dose groups were significantly decreased(P < 0.05 or P < 0.01).Compared with the positive control group,the protein expression level of ERK1/2 in brain tissue of mice in TSG high-dose group was significantly decreased(P< 0.05),and the protein expression level of JNK in brain tissue of mice in TSG groups was significantly decreased(P<0.01).The q RT-PCR results showed that compared with the normal control group,the mRNA relative expressions of CDK5,ERK1/2and JNK in the brain tissues of mice in model group were significantly increased(P < 0.01).Compared with model group,TSG each dose group mice brain CDK5 mRNA expression of relative volume increased significantly(P < 0.05 or P < 0.01),positive control group and TSG each dose group mice brain ERK1/2 mRNA relative expression increased significantly(P <0.05 or P < 0.01),TSG high dose group mice brain JNK mRNA relative expression increased significantly(P < 0.05);Compared with the positive control group,the relative expression levels of CDK5 and JNK mRNA in brain tissue of mice in TSG high-dose group were significantly decreased(P < 0.05).(5)The ffect of TSG on Tau protein phosphatase in 3×Tg-AD mice:Western blotting results show that compared with normal control group,the expression of PP2 B and PP1 in brain tissue of mice in model group was significantly decreased(P<0.01);Compared with model group,the expression of PP2 B and PP1 protein in brain tissue of mice in positive control group and TSG dose groups were significantly increased(P<0.05 or P< 0.01).Compared with the positive control group,the expression of PP2 B and PP1 protein in brain tissue of mice in TSG medium and high dose groups were significantly increased(P < 0.05 or P<0.01).q RT-PCR results showed that compared with the normal group,the relative expression levels of PP2 B and PP1 mRNA in the brain tissues of mice in model group were significantly decreased(P<0.01).Compared with model group,the relative expression of PP2 B mRNA in brain tissue of mice in positive control group and TSG dose groups was significantly increased(P<0.05 or P<0.01),and the relative expression of PP1 mRNA in brain tissue of mice in TSG dose groups was significantly increased(P<0.05 or P<0.01).Compared with the positive control group,the relative expression levels of PP2 B and PP1 mRNA in brain tissue of mice in TSG high-dose group were significantly increased(P<0.05 or P< 0.01).(6)The effect of TSG on Tau protein phosphokinase regulator in 3×Tg-AD mice: compared with the normal control group,the average optical density of P39 and the Immunohistochemical positive neurons of P39 in model group mice cortex,hippocampus were significantly increased(P < 0.01);Compared with model group,the average optical density of P39 and the Immunohistochemical positive neurons of P39 in positive control group and TSG different dose group mice cortex,hippocampus were significantly decreased(P<0.05 or P<0.01);Compared with the positive control group,the average optical density of P39 and the Immunohistochemical positive neurons of P39 in TSG high dose group mice cortex,hippocampus was significantly decreased(P<0.05).Conclusion: TSG can repair the damaged neurons of 3×Tg-AD mice and improve the learning and memory ability of 3×Tg-AD mice.The mechanisms may be associated with downregulating the phosphokinase CDK5,ERK1/2,JNK and their regulatory factor P39,upregulating the phosphatase PP2 B,PP1,and inhibition of Tau protein phosphorylation.