吡咯里西啶生物碱(PAs)是国际公认的植物性肝毒成分。研究表明：PAs需经肝CYP酶催化生成活性代谢物而致肝毒性。CYP酶存在于肝细胞内，故肝细胞对PAs的摄取及转运对其毒性至关重要，然而该机制尚未阐明。肝细胞基底侧表达OCT1转运体，胆管侧表达MATE1，两者分别介导有机阳离子由血液入肝细胞，及由肝细胞外排至胆汁。我们已发现，OCT1介导肝细胞对PAs代表化合物野百合碱、倒千里光碱的摄取，OCT1抑制剂可显著减少其摄取并减弱其毒性，推测OCT1/MATE1可能介导PAs的肝脏摄取和转运。本项目拟应用稳定表达人OCT1、MATE1及CYP酶的单转、双转及三重转染的转基因细胞模型、人原代肝细胞、基因敲除小鼠，阐明PAs的肝脏摄取和转运机制，揭示OCT1/MATE1在PAs致肝毒中的重要性；为PAs的肝毒防治提供新思路；获得PAs结构-体外肝毒关系，为预测PAs体内肝毒性提供依据。

Pyrrolizidine alkaloids(PAs) are well known hepatic toxin widely distributed in many plant species. It has been demonstrated that the PAs have minimal toxicity in their original form, and will be metabolized by cytochrome CYP in liver (especially by CYP3A4), to form reactive metabolites which react with nucleophiles (such as SH, OH, NH groups on nucleotides, proteins, glutathione) to form adducts, finally lead to the acute and chronic liver diseases. Since CYP located in hepatocyte, the uptake and transport of PAs is one of the key factor on hepatic toxicity, however this mechanism has not been clarified yet. The basolateral localization of organic cation transporter 1(OCT1) and the apical localization of multidrug and toxin extrusion protein 1 (MATE1) will mediate the uptake of organic cation from blood into hepatocyte and excretion to bile, respectively. Our previous results revealed that OCT1 mediated hepatocyte uptake of monocrotaline and retrorsine (typical PAs), the OCT1 inhibitors reduced the uptake of monocrotaline and attenuate the hepatocyte toxicity, which can be deduced that OCT1(MATE1) mediated monocrotaline and retrorsine uptake and transport in liver. The present study will establish and employ a few of cell models stable expression of human OCT1(hOCT1), human MATE1(hMATE1) and human CYP(especially CYP3A4), the primary cultured human hepatocytes, and OCT1 transporter knockout mice, to elucidate the effect of OCT1 and MATE1 in the uptake and extrusion of PAs in liver, to study the effect of OCT1 inhibitors on accumulation of PAs in liver and toxicity induced by PAs, and to explore the structure-toxicity relationship. The results will be helpful for discovering of a new strategy to reduce the liver toxicity caused by PAs and predicting the liver toxicity of PAs in vivo.

吡咯里西啶生物碱(PAs)是国际公认的植物性肝毒成分，目前对PAs 的肝脏毒性，尚无理想的治疗手段，因此阐明 PAs 致毒机制，寻找具有针对性的治疗方法仍然是国际关注的热点。基于PAs的结构特征及文献报道，我们推测肝细胞基底侧表达有机阳离子转运体1（OCT1）、胆管侧表达毒素及外排转运蛋白（MATEs）以及肝脏CYP酶分别在PAs的肝脏积聚及代谢激活中发挥至关重要的作用。本项目首先构建了稳定表达人OCT1、CYP3A4、OCT1/CYP3A4，OCT1/MATE1的转基因细胞模型，并应用上述细胞模型、原代肝细胞，考察了OCT1及CYP3A4 在PAs致肝毒中的作用。研究发现，野百合碱（MCT）、倒千里光碱（RTS）等PAs为OCT1底物，但仅是MATE1及P-gp等外排转运体的弱底物。进一步研究发现，MCT及RTS等在mock 及MDCK-hOCT1 细胞未显示明显的毒性，而对MDCK-CYP3A4细胞，显示出时间、浓度依赖性的细胞损伤，提示CYP3A4介导的代谢激活是RTS发挥毒性的关键步骤；RTS等对MDCK-hOCT1/CYP3A4细胞的毒性显著大于对MDCK-CYP3A4及MDCK-hOCT1的毒性，提示OCT1介导的肝细胞摄取及CYP3A4介导的代谢激活，对RTS等PAs的细胞毒性起了非常重要作用。上述研究为寻找RTS等PAs的减毒方法提供新的策略。在完成上述研究的同时，我们构建了几乎所有重要的阳离子类转运体的转基因细胞模型，研究了原小檗碱及类似生物碱的细胞及组织转运，特别是阐明了OCT1介导的氯化两面针碱（NC）的肝脏转运、CYP3A4代谢解毒，OCT2介导的NC肾脏积聚及致肾毒性机制，为NC的成药性评价提供了重要依据。我们还阐明了抗精神病药物舒必利、抗乙肝药物恩替卡韦的肾脏及胎盘转运机制；筛选获得了减少顺铂肾积聚，降低顺铂肾毒性的候选化合物。