| Objective:1.To observe the effect of Cornel Iridoid Glycoside(CIG)on the activity and protein expression of glycogen synthase kinase-3?(GSK-3?),and its protective effect of nerve function on Alzheimer's disease.2.To elucidate the mechanism of CIG inhibiting tau phosphorylation by increasing PP2 A activity.Methods:1.The first part: effect of CIG on WT/GFX AD model rats,(1)Wistar rats were divided into six groups randomly: sham operation group,sham operation group + CIG(60 mg/kg),WT/GFX model group,low dose of CIG(60 mg/kg),high dose of CIG(120 mg/kg),positive drug group(Memantine 3 mg/kg).(2)Model preparation and administration:Wistar rats were given CIG for 7 days,and used Morris water maze to observe the learning and memory ability of these rats.On the 7th day,the escape latency was recorded.On the8 th day,the rats were injected with WT/GFX 200 ?M on the unilateral lateral ventricle and the tau hyperphosphorylated AD model was prepared.On the 9th day,the Morris water maze was tested to record escape latency,the animals' brain tissue were took at last.The morphological changes of hippocampal neurons were observed by immunofluorescence.The protein expression of PI3K/AKT signal pathway and synaptic related protein in the hippocampus and cortex were detected by Western blotting.2.The second part: effect of CIG on transfection of GSK-3?into N2 a cells,the wild-type GSK-3?(Wt GSK-3?)plasmid was transfected transiently into the mouse neuro blastoma cells(Neuro-2A cell,N2 a cells),and the tau hyperphosphorylation model was established.The experiment is divided into blank group,empty vector group,empty vector group+CIG(200 ?g/m L),Wt GSK-3 ?,Wt GSK-3 ? + CIG(50 ?g/m L),Wt GSK-3 ? + CIG(100 ?g/m L),Wt GSK-3 ? + CIG(200 ?g/m L).Western blotting was used to observe the phosphorylation level of tau protein at multiple sites,the expression of GSK-3?,p-GSK-3?-ser9 and multiple proteins related in autophagy pathway.3.The third part:effect of CIG on N2 a cells transfected with Src,(1)To determine optimal transfection conditions of Src: Transfecting Src plasmid DNA(0.2,0.4,0.6,0.8 ?g)into mouse Neuro-2A cell(N2a cells)was performed to observe the effects of different quantity Src on phosphorylation of PP2 A catalytic subunit C and tau phosphorylation.(2)After transfecting 0.6 ?g Src plasmid DNA into N2 a cells 24 h,the cells incubated with CIG(50,100,200 ?g/ml)24h,then observed the effects of CIG on Src,PTP1 B,p-PP2 Ac and tau phosphorylation.Results:1.The first part: Morris water maze experiment: Compared with the sham operation group,the escape latency was increased obviously in the model group,and the escape latency was decreased significantly by CIG administered orally in the model group.Immunofluorescence assay: The number of nerve axon in the model group decreased,the axons was ruptured significantly,and the arranged mussily,after given CIG,ruptured axon were reduced significantly and it arranged neatly.Western blotting test: model group compared to sham operation group,phosphorylation of tau protein at Thr217 and PHF-1(identified Ser396/404)increased significantly,CIG can reduced the phosphorylation of these sites obviously;the expression of p-GSK-3?-ser9 in the hippocampus and cortex of model group decreased,that is GSK-3?was activated,CIG administered orally can inhibit GSK-3?activity.The expression of PI3K/AKT/GSK-3?signal path in the model group was inhibited by the expression of p-IGF-IR,p-PI3 K,PI3K-110?,p-PDK1 and p-AKT were decreased,that is,the activity of IGF-IR,PI3 K,PDK1 and AKT was inhibited,and the expression of p-IRS1 was increased and IRS1 activity was inhibited,CIG administration orally can increase the activity of PI3 K,IRS1,PDK1,AKT significantly,and thus inhibite the activity of GSK-3?.In the model group,the expression of p-synapsin,Synaptophysin and PSD95 were decreased,and these protein expression increased by CIG.2.The second part:N2a cells overexpressed GSK-3?,the expression of p-GSK-3?-ser9 and GSK-3? in the N2 a cells increased,and the phosphorylation of tau protein at 199/202 and PHF-1 also increased significantly.After administration of CIG,the expression of p-GSK-3?-ser9 was increased obviously,that is activity of GSK-3?was inhibited,while CIG can decrease GSK-3?protein expression,and phosphorylation level of tau protein at199/202?PHF-1 site was reduced significantly.In N2 a cells,overexpressed of GSK-3?,the protein expression level of Beclin1,LC3?,ATG12 was increased after given CIG(50,100,200 ?g/m L),that is,the level of autophagy was increased.3.The third part:(1)Expression of Src protein was increased significantly,the expression of p-PP2 Ac was up-regulated and the expression of PP2 A was not changed when Src(0.2,0.4,0.6 ?g)plasmid transfected into N2 a cells,and the tau phosphorylation at the sites of Ser199/202,Ser396 increased significantly;In the N2 a cells transfected with 0.8 ug Src,the expression of p-PP2 Ac was increased apparently,the expression of PP2 A was not changed,and the phosphorylation of tau at Ser199/202 and Ser396 sites was decreased.(2)In the N2 a cells transfected with 0.6 ?g Src,the expression of Src was significantly increased,the expression of p-PP2 Ac was significantly increased,and the tau phosphorylation at the sites of Ser199/202,Thr205,Thr217 and Ser396 sitesincreased significantly;CIG can inhibited the expression of p-PP2 Ac,the expression of tau phosphorylation at the sites of Ser199/202,Thr205,Thr217,and Ser396.In addition,CIG can up-regulate protein expression of PTP1 B.Conclusion:1 CIG can reduce tau hyperphosphorylation,protect neuronal morphology,increase synaptic function,and improve learning and memory disorders of AD;2 CIG can degrade GSK-3?protein by activating cell autophagy;3 CIG can inhibit the activity of GSK-3 ? by activating PI3K/AKT/GSK-3 ? signaling pathway;4 CIG can decrease the phosphorylation of PP2 A catalytic subunit C by increasing the expression of PTP1 B,and then increase the activity of PP2 A,and further reduce the level of tauhyperphosphorylation;5 CIG has no obvious regulation effect on Src. |
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