| ObjectiveIn vivo, our study focuses on the effects of hyperglycemia and hyperlipidemia on cognition, neural degeneration and synaptic plasticity in hippocampus. In vitro, our experiments study the changes of proliferation and autophagy on PC12 cell with high glucose or high oleic acid, respectively.Methods1. C57B/L mice were injected with STZ (30mg/kg, i.p) once a day for four days consecutively. The control mice were injected with the equal volume of sterile citric acid/sodium citrate buffer. After one week, blood plasma was harvest from tail vein, and the concentration of glucose was determined, and≥250mg/dL was used. Plasma glucose was measured by oxidase-methods and plasma cholesterol was test by Enzyme assay every month. Learning and memory was test using the moires water maze. And neural degeneration in hippocampus was valued through Fluoro-Jade C-staining.β- amyloid deposition in hippocampus was measured by ELISA and immunohistochemistry. Golgi-Cox staining together with semi-quantitative stereological method was used to detect the density of dendritic spine of pyramidal cells in CA 1 of hippocampus. Additionally, autophagosome of pyramidal cells in hippocampus were observed by transmission electron microscopy.2. PC12 cells were cultured at different condition to mimicry high glucose or high lipid by adding the different concentration of glucose or oleic acid to medium. Cell viability was valuated by MTT assay. Cell morphology were observed by microscope and cell proliferation by immunofluorescence microscope with high glucose; Also cell cycle was determined by flow cytometry. Under high lipid condition, presenting of 3-MA, autophagy inhibitor, or rapamycin, autophagy inducer, respectively, the expression level of LC3-Ⅱwere detected by western blotting, cell viability were valuated by MTT.Results1. In vivo, our results indicates that serum insulin levels were lower and plasma insulin sensitivity were decreased in diabetic mouse compared with control group at 4 weeks after induced. The ability of learning and memory declined compared to the age-matched group after 2 mouth (p<0.05). Neural degeneration in hippocampus aggregated at 4 month compared with that of control. More deposition ofβamyloid in hippocampus of DM-mouse exits than control. The density of dendritic spine in hippocampus is lowed in DM-mouse compared to the control group (p< 0.01). Autophagysome were observed in cytoplasma of pyramidal cells in hippocampus in diabetic mouse by transmission electron microscopy.2. In vitro, the viability of PC 12 cell was obviously inhibited with different concentrations of glucose in time- and dose-dependent manners, half of inhibition concentration(IC50) value was 150mM. The proliferation of PC 12 cells significantly were inhibited with high glucose by incorperation of Brdu. Most of cells were arrested at G1 phase, and S phase synthesis of were reduced after 48h with high glucose. Autophagy were induced in PC 12 cells with oleic acid, and the expression levels of autopha related protein LC3-Ⅱwere significantly increased and autophagy vesica was observed using fluorescent microscope. Giving autophagy inhibitors. MTT show the cell vibility and the expression of LC3-Ⅱlevels decreased significantly compared with single oleic acid, while the opposite effect of rapamycin.Conclusion1. Our study suggested persistant high glucose and lipid induce the neurodegeneration and synapse-loss,enhancement of beta amyloid deposition in hippocampus, which impaired the cognition in type 2 diabetes model.2. PC 12 cells proliferation were inhibited by high glucose, autophagy was induced by oleic acid, also inhibit autophaby influence cell viability, but has protective effect of rapamycin. |
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