Effect Of The EphB2 On Aβ_25-35-induced Autophagy And Apoptosis In Mouse HT22 Cells

Objective: Alzheimer’s disease（AD）is a common central nervous system degenerative dementia disease,which is mainly manifested as progressive cognitive impairment and behavioral abnormalities.The main pathological features of AD are neurofibrillary tangles and senile plaques in brain tissue.Among them,the main component of age spots is β-amyloid peptide（β-amyloid peptide,Aβ）,but also the pathogenesis of AD pathology factor,which has a strong neurotoxicity,can induce cell damage and apoptosis,and ultimately lead to the occurrence of AD The Autophagy through the lysosomal degradation of part of its own components,in order to achieve their own organelle renewal and metabolism needs.Cells produce autophagy,can remove toxic and useless protein,maintain cell homeostasis.Relevant data show that autophagic abnormalities also play an important role in the pathology of AD.In this study,Aβ_25-35 was used to induce the transplanted mouse hippocampal neuronal cell line HT22 cells to establish the Alzheimer ’s disease model.When the Aβ_25-35 induced HT22 cells was damaged,the autophagy-related proteins AKT / p-AKT,mTOR / P-mTOR and LC3,the expression of Bax,Bcl-2 and Caspase-12,and the effect of EphB2 on autophagy and apoptosis.Methods: HT22 cells were divided into two groups: differentiated and undifferentiated group.MTT assay was used to detect the toxicity of EphB2 to HT22 cells.The degree of damage of Aβ_25-35 to different concentrations of HT22 cells,lactate dehydrogenase（LDH）The assay kit detects the LDH leakage of the corresponding HT22 cells to determine the optimal concentration of the AD model,the safe concentration range of EphB2,and the optimal concentration of EphB2.At the same time,the morphology of the two groups was observed before and after microscopic differentiation.HT22 cells were divided into control group,Aβ treatment group and Aβ + EphB2 low dose group,Aβ + EphB2 middle dose group,Aβ + EphB2 high dose group.Three concentrations of EphB2（25,50,100μmol / L）were applied to HT22 cells for 4 hours.The optimal concentration of Aβ_25-35 was incubated for 24 hours.The protein was extracted and the protein concentration was measured by BCA.The expression of AKT / p-AKT,mTOR / p-mTOR and LC3 in E22 cells induced by Aβ_25-35 was detected by Western Bloting,and the expression of apoptosis-related proteins Bax,Bcl-2 and Caspase-12 Level of change.Hoechst33258 staining,inverted fluorescence microscope to observe the changes in the nucleus of each group.Apoptosis of each group was detected by flow cytometry.Results:（1）The morphology and structure of HT22 cells were closer to that of hippocampal neurons in vivo,and the results of MTT assay showed that the survival rate of differentiated HT22 cells was negatively correlated with Aβ_25-35 concentration,while that of undifferentiated group Correlation.（2）With the increase of Aβ_25-35 concentration,LDH content in cell supernatant increased gradually,and the survival rate of HT22 cells decreased gradually.The survival rate of HT22 cells was about 63% when the concentration of Aβ_25-35 was 5μmol / L,and it could be determined as the best concentration of AD model.MTT assay showed that EphB2 at 400 ng / ml concentration,the significant increase in the value of HT22 cells.（3）Western blot results showed that the expression of p-AKT and p-mTOR in the Aβ-treated group and Aβ + EphB2 group were decreased gradually and the expression of LC3Ⅱ / Ⅰ was gradually increased compared with the control group.The expression of Bax and Caspase-12 in Aβ-treated group was significantly higher than that in control group,and the expression level of Bcl-2 in Aβ + EphB2 was lower than that in group Aβ On the contrary.（4）Hoechst33258 staining showed that the Aβ treatment group had irregular shape,deep staining and cleavage,and the Aβ + EphB2 low middle and high group was gradually relieved compared with the Aβ treatment group.（5）The results of flow cytometry showed that the apoptotic rate of Aβ treatment group increased significantly compared with that of control group,while that of Aβ + EphB2 low middle and high group was increased gradually than that of the cell viability of the β-treated group.Conclusion:（1）Aβ_25-35 can induce differentiated HT22 cells to establish AD model.（2）EphB2 has enhanced autophagy induced by Aβ_25-35 in HT22 cells.（3）EphB2 has the effect of attenuating the apoptosis induced by Aβ_25-35 in HT22 cells.