The Effect Of A? Mixture On Transport Receptor Of A? Across Blood Brain Barrier And The Protective Effect Of 1,25?OH?₂D₃

Objective:To investigate the effect of A?mixture of A?_1-42 oligomer and A?_1-40monomer on transport receptor of A?across blood-brain barrier and the protective effect of1,25?OH?₂D₃,we treated endothelial cells?hCMEC/D3?with A?mixture.Methods:?1?hCMEC/D₃ were cultured in vitro and identified by immunocytochemistry.?2?The preparation of A?_1-40-40 and A?_1-42 peptide solutions,and identification of A?_1-42oligomers by Western blotting.?3?To find the subtoxic concentration of A?preparation and the best drug concentration of 1,25?OH?₂D₃,MTT cytotoxicity assay was performed to measure toxicity of different concentrations of A?_1-40-40 monomer?A?_1-42-42 oligomers?A?mixture and 1,25?OH?₂D₃.?4?experimental grouping:?1?control group?normal hCMEC/D₃ cells group?;?2?A?mixture group(subtoxic concentrations of A_?1-40 and A?_1-42);?3?A?mixture and 1,25?OH?₂D₃ intervention group:A?mixture and low dose of 1,25?OH?₂D₃;A?mixture and middle dose of 1,25?OH?₂D₃;A?mixture 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 A?_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 A?preparation with A?_1-42 at 25nM and A?_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?A?mixture 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 A?mixture 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 A?mixture group?P<0.01?;A?mixture group showed mRNA and protein expression level of P-gp were lower than the control group?P<0.01?,and compared with the A?mixture 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 A?species itself can regulate its own transporter expression of RAGE and P-gp on the blood brain barrier endothelial cells,which may cause further A?accumulation.?2?1,25?OH?₂D₃ can play a protective role by regulating the expression of RAGE and LRP1 and reducing A?accumulation,which makes it a potential tool treat and prevent AD.