Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia.
Pharm Res. 2021 Jan;38(1):97-111. doi: 10.1007/s11095-021-03006-y. Epub 2021 Feb 2.
P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of β-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown.
P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 μM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay.
FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation.
These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain β-amyloid clearance.
血脑屏障（BBB）上的 P-糖蛋白（P-gp）可阻止许多外源性物质进入大脑，并介导β-淀粉样蛋白从大脑向血液的清除，而β-淀粉样蛋白在阿尔茨海默病（AD）大脑中会积累。据报道，锌和铜可以调节 BBB 上 P-gp 的表达和功能；然而，外源性铁（在 AD 中积累）对 P-gp 动力学的影响尚不清楚。
通过 Western blot 和 RT-qPCR 分别检测用柠檬酸铁铵（FAC；250 μM，72 h）处理的永生人脑血管内皮细胞（hCMEC/D3）中 P-gp 蛋白和 MDR1 转录本的水平。使用罗丹明 123 和 [H]-地高辛积累来评估 P-gp 功能。使用 2'，7'-二氯荧光素二乙酸酯测定细胞内活性氧（ROS）水平，并使用铁嗪测定法定量细胞内铁水平。
FAC 处理显著降低了 P-gp 蛋白（36%）和 MDR1 mRNA（16%）水平，但罗丹明 123 或 [H]-地高辛积累没有显著变化。虽然 P-gp/MDR1 下调与 ROS 和细胞内铁升高有关，但抗氧化剂 N-乙酰半胱氨酸或铁螯合剂去铁胺和地拉罗司不能减轻 MDR1 下调，这表明涉及 ROS 独立机制或不完全铁螯合。
这些研究表明，铁在 BBB 上负调节 P-gp 的表达，可能影响 CNS 药物输送和大脑β-淀粉样蛋白清除。
