雷公藤甲素诱导正常人肝细胞L02的毒性及甘草酸二铵的保护作用

目的: 研究雷公藤甲素(triptolide, TP)诱导的正常人肝细胞L02的毒性及甘草酸二铵(diammonium glycyrrhizinate, DG)保护作用。方法: 首先利用CCK-8法测定不同浓度TP对L02的毒性,然后研究DG预处理48 h后对TP诱导L02毒性的影响。利用活性氧(ROS)探针结合高内涵,研究不同浓度DG预处理对TP诱导L02 ROS的影响。进一步利用CYP3A4苯妥因诱导剂和酮康唑抑制剂阐明肝药酶在DG解毒中的作用。结果: 经细胞计数试剂盒(cell counting kit, CCK-8)法测定,L02经TP孵育1、2和4 h时,IC₅₀分别为26、14和8 μmol·L^-1。当正常人肝细胞经DG(10、100和1 000 μmol·L^-1)预处理48 h后,再与TP共同孵育1 h,DG 10和100 μmol·L^-1组的IC₅₀分别升高至58、79 μmol·L^-1,而DG 1 000 μmol·L^-1组的IC₅₀则降低至18 μmol·L^-1。TP诱导L02产生ROS呈现明显的剂量相关性。DG(10和100 μmol·L^-1)预处理48 h后可明显降低TP诱导ROS的能力。CYP3A4的诱导剂与DG共同预处理后,可显著性降低TP诱导ROS的能力。CYP3A4的抑制剂和DG共同预处理,反证诱导CYP3A4可能是DG保护作用的机制之一。结论: 本研究提示TP诱导L02的活性氧(reactive oxygen species, ROS)水平增加,产生毒性作用;低浓度和中浓度DG可以显著性降低TP对L02的毒性,而高浓度DG反而增强TP对L02的毒性。DG诱导CYP3A4上调和降低ROS水平对减轻TP诱导的正常肝细胞毒性起到重要作用。

Objective: To study the cytotoxicity influence of triptolide (TP) in normal human hepatic cell L02 and the protective effects of diammonium glycyrrhizinate (DG). Methods: The cytotoxicity of TP and the protective effect of DG on L02 were investigated by CCK-8. TP induced reactive oxygen species (ROS) in L02 and the protective effect of DG were also investigated using high content analysis (HCA). Ketoconazole (CYP3A4 inhibitor) and phenytoin (CYP3A4 inducer) were used to verify whether DG upregulated CYP3A4 activity to protect L02 cells from TP induced cytotoxicity. Results: The IC₅₀ on L02 after 1, 2 and 4 h TP treatments were 26, 14 and 8 μmol·L^-1, respectively.After 48 h pretreatment of DG (10, 100 μmol·L^-1), the IC₅₀ on L02 (1 h TP treatment) were increased to 58 μmol·L^-1 and 79 μmol·L^-1 groups, respectively. However, 1 000 μmol·L^-1 DG pretreatment increased the IC TP IC₅₀ on L02 (1 h TP treatment) to 18 μmol·L^-1. After DG pretreatment, the ability of TP induced ROS was significantly reduced in the low and middle dose groups.Reactive oxygen species (ROS)-induction on L02 of TP can be inhibited after 48 h CYP3A4 induction or DG pretreatment. Conclusion: DG showed significantly protective effects on the TP induced L02 cytotoxicity in both low and middle dose groups.However, high dose of DG increased the TP induced liver toxicity. DG protects L02 cells from TP induced liver toxicity by up-regulating CYP3A4 and reducing ROS levels.