Effect Of1,25（OH）₂D₃on Mediating Aβ Across The Blood-Brain Barrier

Objectives:To study the effect of1,25(OH)2D3on mediating Abeta across the blood-brain barrier and its mechanism, we set up the model of blood-brain barrier by mouse brain micro vascular endothelial cells (bEnd.3). Methods:(1) MTT assay was used to find the non-cytotoxicity dosage of1,25(OH)2D3;the dosage at which the survival rate of cells is above90%was considered as the highest non-cytotoxicity dosage.(2)Using hypoxia to induce the abnormal expression of LRP1、BCRP and RAGE in the brain micro vascular endothelial cells, which would happen with pathological characteristics of Alzheimer’s disease.The expression of LRP1, BCRP, RAGE were analysed by real-time PCR and Western blot after giving1,25(OH)2D3(3) VDR mRNA and protein expression using real-time PCR and Western blot analysis.(4)Establish in vitro model to study the effect of1,25(OH)2D3on Abeta across the blood-brain barrier.Results:(1)We choose1nM,10nM,100nM for the next experiments by MTT assay.(2)The expression of LRP1and BCRP mRNA are decreased significantly, while the RAGE mRNA increases after hypoxia (P<0.05); After giving1,25(OH)2D3, the expression of LRP1mRNA was increased while RAGE mRNA was decreased in all groups (P<0.01).100nM1,25(OH)2D3could up regulate the level of BCRP mRNA (P<0.05). Hypoxia down-regulated the protein expression of LRP1and BCRP while increased RAGE expression (P<0.01) Given1,25(OH)2D3,medium and high dose groups (10nM and100nM) could up-regulate LRP1expression (P<0.01). There is no significant differences between1nM and hypoxia control. All1,25(OH)2D3groups could down-regulate the expression of RAGE (P<0.01); only100nM could increased BCRP expression (P<0.05).(3)1,25(OH)2D3could increased the expression of VDR at10nM and100nM significantly (P<0.05).(4)The result of A(3bidirectional transport assay has shown that the permeability was similar between the low dosage and the control (proximately4.3%), and ER was1.85with insignificant effect. While the medium and high dose groups could make the transport rate from BL to AP increased significantly (5.6%and6.2%respectively), and the efflux rates ER are2.62and3.23respectively. Conclusions:1,25(OH)2D3could increased the expression of Aβ efflux transporters BCRP and LRP1and decreased the influx receptor RAGE expression on blood brain barrier, which may increase the Abeta transport rate from BL-AP remarkably. The effect that1,25(OH)2D3affects on BCRP、LRP1and RAGE may be realized via Vit D-VDR signaling pathway.