Regulation Of Stilbene Glycosides On Tau Phosphorylation In AD Rats Via PI3K/AKT/GSK-3β Signaling Pathway

Objective:Alzheimer’s disease rats induced by Aβ_25-35 were used as experimental subjects to analyze the effect of stilbene glycosides on the phosphorylation of related proteins in the brain tissue of Alzheimer’s disease rats via PI3K/AKT/GSK-3βsignaling pathway,so as to provide a new theoretical basis for the clinical treatment of Alzheimer’s disease.Methods:SD male rats at aged 24 months were used as AD model by injecting Aβ_25-35 in hippocampus.The rats were screened by passive avoidance test to meet the experimental requirements.Except the normal group and the sham operation group,the remaining rats were randomly divided into 5 groups,including model group,positive drug group,low-dose group,medium-dose group and high-dose group,with 10 rats in each group.Each group was given medicine once a day for continuous gavage for 28 days.The morphology and number of neuronal Nishi bodies in hippocampus and cortex were observed by Nissl staining;The apoptosis of neuronal cells in hippocampus and cortex were detected by TUNEL apoptosis assay;The expression of PI3K,AKT,GSK-3βand NF-κB m RNA in the brain tissue of rats in each group were detected by time-fluorescence quantitative polymerase chain reaction;The expression of p-Tau（Thr205）protein in hippocampal and cortical regions of rat brain tissue in each group were detected by immunohistochemical staining;The expression of GSK-3βprotein in hippocampal region of rat brain tissue in each group were detected by immunofluorescence method;And the expression of p-PI3K（Y607）,p-AKT（Ser473）and p-Tau（Ser396）proteins in rat brain tissues of each group was detected by protein immunoblotting.Results:（1）In the Nissl staining experiment,compared with normal group,the number,morphology and arrangement of Nishi bodies in the hippocampus and cortex were not significantly changed in the sham operation group and the positive drug group,while the number of Nishi bodies was significantly reduced in the model group,the cell morphology was broken and the arrangement was irregular.Compared with model group,the number of Nishi bodies in positive drug group and TSG dose groups increased to different degrees,and the cell rupture situation was gradually improved.（2）In the TUNEL apoptosis experiment,compared with the normal group,there was no significant change in the number of apoptotic neurons in hippocampus and cortex in sham operation group and positive drug group,but the number of apoptotic neurons in model group increased significantly（P<0.01）;compared with the model group,the number of neuronal cell apoptosis was decreased in positive group,TSG medium and high dose groups（P<0.01）.（3）In the real-time fluorescence quantitative polymerase chain reaction experiment,compared with the normal group,the relative expression levels of genes in the sham operation group were not significantly changed,the m RNA relative expression levels of PI3K,AKT and GSK-3βwere not significantly changed in the positive drug group,and the m RNA relative expression levels of NF-κB was decreased（P<0.01）,and the relative expression of PI3K and AKT m RNA in the model group were significantly decreased（P<0.01）and GSK-3βand NF-κB m RNA relative expression were significantly increased（P<0.01）;Compared with the sham-operated group,PI3K and AKT m RNA relative expression in the model group were significantly decreased（P<0.01）and GSK-3βand NF-κB m RNA relative expression were significantly increased（P<0.01）;Compared with the model group,the relative expression levels of PI3K and AKT m RNA in positive drug group and TSG dose groups were significantly increased（P<0.05）,the m RNA relative expression levels of GSK-3βand NF-κB were significantly decreased（P<0.05）;（4）In immunohistochemical staining,the expression of p-Tau（Thr205）in hippocampus and cortex was not significantly changed in sham operation group and positive drug group,but significantly increased in model group compared with normal group（P<0.01）;Compared with model group,the expression level of p-Tau（Thr205）protein in positive drug group and TSG dose groups decreased to different degrees.（5）In the immunofluorescence experiments,there was no significant difference in GSK-3βprotein expression between sham operation group and positive drug group,while GSK-3βprotein expression in model group was significantly increased（P<0.01）;Compared with model group,GSK-3βprotein expression was decreased in positive drug group and TSG high-dose group（P<0.01）.（6）In Western Blot,compared with the normal group,there was no significant difference in the expression of p-Tau（Ser396）,p-PI3K（Y607）and p-AKT（Ser473）between the sham group and the positive group,while the expression of p-Tau（Ser396）in the model group was significantly increased（P<0.01）,the expressions of p-PI3K（Y607）and p-AKT（Ser473）were significantly decreased（P<0.01）;Compared with model group,the expression level of p-Tau（Ser396）protein was decreased in positive drug group and TSG dose groups（P<0.01）,the expressions of p-PI3K（Y607）and p-AKT（Ser473）were significantly increased（P<0.05）.Conclusion:TSG can reduces the apoptosis of neuronal cells in hippocampus and cortex of AD rats,and the mechanism may be by increasing the activity of PI3K/AKT signaling pathway and inhibiting the expression of NF-κB;Or by inhibiting GSK-3β,thereby reduceing Tau protein phosphorylation.