The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1
The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1
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DOI：
10.1042/bj20080918
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发表时间：
2009-01-01
影响因子：
4.1
通讯作者：
Tiribelli, Claudio
中科院分区：
文献类型：
--
作者：
Corich, Lucia;Aranda, Alejandro;Tiribelli, Claudio
体外和体内研究表明，UCB（未结合胆红素）具有神经毒性。虽然以前的研究表明，MRP 1（多药耐药相关蛋白1）和MDR 1（多药耐药蛋白1）可以保护细胞免受UCB的积累，但从未进行过直接比较它们在UCB转运中的作用。为此，我们采用可诱导的siRNA（small interfering RNA）表达系统沉默人神经母细胞瘤SH-SY 5 Y细胞中MRP 1和MDR 1的表达。在未沉默（对照）细胞和MRP 1或MDR 1表达降低相似的细胞之间比较了体外暴露于临床相关水平的未结合UCB的影响，通过RT-PCR（逆转录-PCR）（mRNA）、免疫印迹（蛋白质）和MDR 1（特定荧光底物的净摄取增强）记录。通过MTT [3-（4，5-二甲基噻唑-2-基）-2，5-二苯基-2H-溴化四唑]试验评估细胞毒性。与对照组相比，MRP 1缺陷细胞积累了更多的UCB，并遭受了更大的细胞毒性。相比之下，MDR 1缺陷细胞表现出与对照相当的UCB摄取和细胞毒性。在中等水平的沉默，UCB毒性的敏感性增加密切相关的MRP 1的表达，但不是MDR 1的减少。这些数据支持这样一个概念。细胞UCB积聚的限制，由MRP 1而不是MDR 1介导的UCB输出的线索在预防新生儿胆红素脑病中起重要作用。
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 ill 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 ill vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions ill 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, Clue to UCB export mediated by MRP1, but not MDR1, plays all important role in preventing bilirubin encephalopathy in the newborn.