细胞色素P450介导的补骨脂酚代谢减毒

目的:分析补骨脂酚代谢的细胞色素P450(cytochrome P450,CYP)表型,并在体外研究人肝微粒体(hu-man liver microsomes,HLM)对补骨脂酚代谢减毒的机制。方法:用HLM与化学抑制剂合用法以及人源重组CYP酶法分析代谢补骨脂酚的CYP酶亚型。用HPLC-MS法分析CYP亚型酶特异底物的代谢产物生成量,研究CYP酶广谱抑制剂1-氨基苯并三唑(1-aminobenzotriazole,ABT)对HLM中CYP亚型酶活性的抑制作用。用HPLC法分析补骨脂酚在HLM孵育液中的浓度,研究ABT对其代谢的影响。应用MTT法检测人肾近曲小管上皮细胞(human kid-ney-2,HK-2)的存活率来评价补骨脂酚对HK-2的毒性作用以及HLM中CYP酶和ABT对其毒性的影响。结果:HLM中的CYP1A2、CYP2C9、CYP2C19和CYP3A4参与了补骨脂酚的代谢,其中以CYP2C19的代谢转化率最高;2.5 mmol/L ABT能明显抑制0.5 g/L HLM中上述4种CYP同工酶活性,抑制率达到90%以上;2.5 mmol/L ABT对补骨脂酚在HLM的代谢抑制率为83.24%±2.13%。在HK-2细胞存活率实验中,ABT能显著降低HLM对30～90μmol/L补骨脂酚的代谢减毒作用(P0.05)。结论:HLM减低补骨脂酚HK-2细胞毒性的作用与HLM中CYP酶将补骨脂酚代谢转化为无毒或毒性较小的代谢产物有关,应用CYP酶广谱抑制剂能逆转HLM对补骨脂酚的代谢解毒作用。

Metabolic detoxification of bakuchiol is mediated by cytochrome P450 enzymes in human liver microsomes

Objective:To analyze cytochrome P450(CYP) phenotyping for bakuchiol metabolism and study the mechanism of detoxification of bakuchiol by human liver microsomes(HLM) in vitro.Methods: The CYP phenotyping for bakuchiol metabolism was determined using HLM combined with CYP specific inhibitors and recombinant human CYP isoforms.The relative activities of CYP isoforms were determined by analyzing the formation of the substrate metabolites using HPLC-MS/MS,in presence or absence of 1-aminobenzotriazole(ABT) which was CYP enzymes’ broad spectrum inhibitor.The residual concentrations of bakuchiol in microsomal incubates were determined using HPLC to investigate ABT’s effect on the metabolism of bakuchiol.The effects of CYP enzymes on the nephrotoxicity of bakuchiol were investigated using human kidney-2(HK-2) by MTT assay,in presence or absence of ABT.Results: CYP1A2,CYP2C9,CYP2C19 and CYP3A4 in HLM were involved in bakuchiol metabolism,among which CYP2C19 showed the highest metabolic rate.Co-incubation with ABT(2.5 mmol/L) could inhibit more than 90% of the enzyme activities for CYP1A2,CYP2C9,CYP2C19 and CYP3A4.ABT(2.5 mmol/L) could inhibit the HLM metabolism of bakuchiol with inhibition ratio 83.24% ±2.13%.When preincubated with ABT,the metabolic detoxification of bakuchiol by HLM was significantly reduced(P<0.05).Conclusion: The mechanism of metabolic detoxification of bakuchiol by HLM is associated with bakuchiol metabolism by CYP enzymes to form non toxic or lower toxic metabolites.The broad spectrum inhibitor of CYP could inverse the detoxification of HLM.