The Effect Of Aβ Mixture On Transport Receptor Of Aβ Across Blood Brain Barrier And The Protective Effect Of 1,25（OH）₂D₃

Objective:To investigate the effect of Aβmixture of Aβ_1-42 oligomer and Aβ_1-40monomer on transport receptor of Aβacross blood-brain barrier and the protective effect of1,25（OH）₂D₃,we treated endothelial cells（hCMEC/D3）with Aβmixture.Methods:（1）hCMEC/D₃ were cultured in vitro and identified by immunocytochemistry.（2）The preparation of Aβ_1-40-40 and Aβ_1-42 peptide solutions,and identification of Aβ_1-42oligomers by Western blotting.（3）To find the subtoxic concentration of Aβpreparation and the best drug concentration of 1,25（OH）₂D₃,MTT cytotoxicity assay was performed to measure toxicity of different concentrations of Aβ_1-40-40 monomer、Aβ_1-42-42 oligomers、Aβmixture and 1,25（OH）₂D₃.（4）experimental grouping:（1）control group（normal hCMEC/D₃ cells group）;（2）Aβmixture group(subtoxic concentrations of A_β1-40 and Aβ_1-42);（3）Aβmixture and 1,25（OH）₂D₃ intervention group:Aβmixture and low dose of 1,25（OH）₂D₃;Aβmixture and middle dose of 1,25（OH）₂D₃;Aβmixture and high dose of 1,25（OH）₂D₃.（5）The transcription level of low-density lipoprotein receptor-related protein-1（LRPl）,P-glycoprotein（P-gp）and receptor for advanced glycation end product（RAGE）was detected by RT-PCR,while the expression level of LRPl,P-gp and RAGE was detected by Western-Blot.Results:（1）The results of Western-Blot showed that the Aβ_1-42-42 oligomers were prepared,and its molecular weight was 8-12kDa.（2）According to the MTT experiment,we selected the sub-toxic concentration of Aβpreparation with Aβ_1-42 at 25nM and Aβ_1-40 at 100nM with the cell viability above 90%and the best experimental drug concentration of1,25（OH）₂D₃ at lnM,10nM and 50nM.（3）Aβmixture group showed m RNA and protein expression levels of RAGE were significantly higher than the control group（P<0.01）,and1,25（OH）₂D₃ low,middle and high dose groups showed mRNA and protein expression level of RAGE were lower than Aβmixture group（P<0.01）;Compared to the control group,mRNA and protein expression level of LRP1 were no difference（P>0.05）,however 1,25（OH）₂D₃ low,middle and high dose groups showed that mRNA and protein expression level of LRP1 were higher than Aβmixture group（P<0.01）;Aβmixture group showed mRNA and protein expression level of P-gp were lower than the control group（P<0.01）,and compared with the Aβmixture group,1,25（OH）₂D₃ low,middle and high dose group showed m RNA and protein expression level of P-gp were no difference（P>0.05）.Conclusion:（1）The mixture of soluble Aβspecies itself can regulate its own transporter expression of RAGE and P-gp on the blood brain barrier endothelial cells,which may cause further Aβaccumulation.（2）1,25（OH）₂D₃ can play a protective role by regulating the expression of RAGE and LRP1 and reducing Aβaccumulation,which makes it a potential tool treat and prevent AD.