Corich Lucia, Aranda Alejandro, Carrassa Laura, Bellarosa Cristina, Ostrow J Donald, Tiribelli Claudio
Centro Studi Fegato, Bld Q, AREA Science Park, Basovizza Campus, Ss 12 km 163.5, c 3412 Trieste, Italy.
Biochem J. 2009 Jan 1;417(1):305-12. doi: 10.1042/BJ20080918.
In vitro and in vivo studies have demonstrated that UCB (unconjugated bilirubin) is neurotoxic. Although previous studies suggested that both MRP1 (multidrug resistance-associated protein 1) and MDR1 (multidrug resistance protein 1) may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA (small interfering RNA) expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (reverse transcription-PCR) (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] test. MRP1-deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable with controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease in the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.
体外和体内研究均表明，未结合胆红素（UCB）具有神经毒性。尽管先前的研究表明，多药耐药相关蛋白1（MRP1）和多药耐药蛋白1（MDR1）都可能保护细胞免受UCB蓄积的影响，但从未对它们在UCB转运中的作用进行过直接比较。为此，我们使用了一种可诱导的小干扰RNA（siRNA）表达系统来沉默人神经母细胞瘤SH-SY5Y细胞中MRP1和MDR1的表达。通过逆转录聚合酶链反应（RT-PCR）（mRNA）、免疫印迹（蛋白质）以及对于MDR1，通过特定荧光底物的净摄取增强来记录，比较了未沉默（对照）细胞与MRP1或MDR1表达有类似降低的细胞在体外暴露于临床相关水平的游离UCB时的效果。通过MTT [3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐]试验评估细胞毒性。与对照相比，MRP1缺陷细胞积累的UCB明显更多，且细胞毒性更大。相比之下，MDR1缺陷细胞表现出与对照相当的UCB摄取和细胞毒性。在中等沉默水平下，对UCB毒性的易感性增加与MRP1表达的降低密切相关，而与MDR1的表达无关。这些数据支持了这样一种观点，即由MRP1介导而非MDR1介导的UCB输出导致的细胞内UCB蓄积受限，在预防新生儿胆红素脑病中起重要作用。
