离子载体和生物金属对人脑血管内皮细胞 P 糖蛋白表达和功能的调节作用。
Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.
机构信息
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,, Monash University,, 381 Royal Parade, Parkville, VIC,, 3052, Australia.
The Florey Institute of Neuroscience and Mental Health,, Parkville, VIC, Australia.
出版信息
Pharm Res. 2018 Mar 5;35(4):83. doi: 10.1007/s11095-018-2377-6.
PURPOSE
Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown.
METHODS
Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking.
RESULTS
A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold).
CONCLUSIONS
Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.
目的
锌和铜等生物金属已被证明会影响紧密连接的表达，进而影响血脑屏障 (BBB) 的完整性。然而，这些生物金属是否也会影响 BBB 转运体（如 P-糖蛋白 (P-gp)）的表达和功能目前尚不清楚。
方法
使用永生化人脑血管内皮细胞 (hCMEC/D3) 细胞系，通过细胞内 Western 测定法（结合 Western 印迹）评估金属载体化合物氯碘羟喹以及生物金属锌和铜处理后相对 P-gp 表达情况。采用荧光 P-gp 底物罗丹明 123 观察功能调节，电感耦合等离子体质谱法 (ICP-MS) 提供生物金属转运信息。
结果
24 小时用氯碘羟喹、锌和铜（0.5、0.5 和 0.1 μM）处理可诱导 P-gp 的显著上调（细胞内 Western 测定法为 1.7 倍），Western 印迹法证实为 1.8 倍增加）。相同的处理导致罗丹明 123 在 1 小时孵育期间的积累减少 23%。ICP-MS 表明，尽管这种联合处理对细胞内锌浓度没有影响，但处理显著增加了生物可利用的铜（4.6 倍）。
结论
向人脑血管内皮细胞中输送更多的铜与重要外排泵 P-gp 的表达和功能增强有关，这可能为 P-gp 调节提供治疗机会。
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