Effect Of RXRα Exporting Inhibition On The Apoptosis Of Neurons

Objective: To detect the changes of the expression and subcellular localization of RXRα and the excessive apoptosis of N2 a cells and neurons in C57BL/6 mice hippocampus on AD pathologic condition, and investigate the effect of RXRα exporting inhibition caused by 9-cis-RA which is ligand of RXRα on the apoptosis of neurons. Method: N2 a cells and mice hippocampus were treated with Aβ、Aβ together with 9-cis-RA respectively.Cell culture method was used to cultivate N2 a cells and collect and handle them including protein extraction、nucleoplasm separation 、cell climbing slice and so on according to the requirements and purpose of experiment. C57BL/6 mice were fed and their hippocampus were injected in groups according to the requirements and purpose of experiment.Protein extraction、nucleoplasm separation and brain slices were used to deal with the hippocampus.In regard to N2 a cells western blotting and RT-PCR were applied to detect the expression of RXRα and Nur77; nucleoplasm separation and confocal observations were performed to observe the translocation of RXRα and Nur77; cell apoptosis was detected by flow cytometry 、 DAPI staining 、MTT and the expression of Bcl-2 and Bax. In regard to C57BL/6 mice, western blotting were applied to detect the expression of RXRα and Nur77; nucleoplasm separation and confocal observations were performed to observe the translocation of RXRα and Nur77; cell apoptosis was detected by DAPI staining and the expression of Bcl-2 and Bax. Result: Comparing with control group, mRNA/protein expression levels of RXRα and Nur77 in N2 a cells treated with Aβ25-35 for 24 h remained almost the same, however, the protein ratio of RXRα and Nur77 in the cytoplasm increased from 5.26%、4.82%(in control group) to 42.2% 、35.5%(in Aβ group) respectively, and the ratio of cells apoptosis increased from4.36%(in control group) to 15.1%(in Aβ group) at the same time. Comparing with control group without 9-cis-RA treatment in advance, N2 a cells treated with Aβ25-35 for 24 h after 9-cis-RA treatment for 12 h had almost the same mRNA /protein expression levels of RXRα and Nur77, however, 9-cis-RA inhibited the shuttling of RXRα and Nur77 from the nucleus to the cytoplasm.The protein ratio of RXRα and Nur77 in the cytoplasm reduced from 42.2% 、35.5%(in control group) to 6.67%、5.44%(in 9-cis-RA group)respectively, and the ratio of cell apoptosis reduced from15.1%(in control group) to 5.31%(in Aβ+9-cis-RA group). Comparing with control group, mice treated with Aβ25-35 for 24 h remained almost the same protein expression levels of RXRα and Nur77, but some RXRα and Nur77 shuttled from the nucleus to the cytoplasm together with more cells apoptosis.The ratio of RXRα and Nur77 in the cytoplasm increased from 26.1%、11.1%(in control mice) to 59.2% 、73.7%(in Aβ treated mice) respectively. Comparing with control group without 9-cis-RA treatment in advance, mice treated with Aβ25-35 for 24 h after 9-cis-RA treatment for 12 h had almost the same protein expression levels of RXRα and Nur77, but the protein ratio of RXRα and Nur77 in the cytoplasm reduced from 59.2% 、73.7%(in Aβ treated mice) to 30.2%、19.8%(in Aβ+9-cis-RA treated mice) respectively together with less cell apoptosis. Conclusion:Aβ treatment resultes in the translocation of RXRα and Nur77 from the nucleus to the cytoplasm; Aβ treatment resultes in the apoptosis of neurons;RXRα exporting inhibition reduces the apoptosis of neurons caused by Aβ.