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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8μmol·L-1。当正常人肝细胞经DG(10、100和1 000μmol·L-1)预处理48 h后,再与TP共同孵育1 h,DG 10和100μmol·L-1组的IC50分别升高至58、79μmol·L-1,而DG 1 000μmol·L-1组的IC50则降低至18μmol·L-1。TP诱导L02产生ROS呈现明显的剂量相关性。DG(10和100μmol·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 toxicity of triptolide (TP) induced L02 in normal human hepatocytes and the protective effect of diammonium glycyrrhizinate (DG). Methods: CCK-8 method was used to determine the toxicity of different concentrations of TP to L02. Then the effect of DG preconditioning on the TP-induced L02 toxicity was studied. Using reactive oxygen species (ROS) probe combined with high content, the effects of different concentrations of DG pretreatment on L02 ROS induced by TP were studied. Further use of CYP3A4 phenytoin inducer and ketoconazole inhibitors clarify the role of hepatic enzymes in the detoxification of DG. Results: The IC50 of L02 was 26, 14 and 8 μmol·L-1 at 1, 2 and 4 h after incubation with TP by CCK-8 assay. When the normal human hepatocytes were pretreated with DG (10, 100 and 1 000 μmol·L-1) for 48 h and then incubated with TP for 1 h, the IC50 of DG 10 and 100 μmol·L-1 groups increased to 58, 79μmol·L-1, while the IC50 of DG 1 000μmol·L-1 group decreased to 18μmol·L-1. TP-induced L02 production of ROS showed a significant dose-related. The pretreatment of DG (10 and 100μmol·L-1) for 48 h significantly reduced the ability of TP to induce ROS. The pretreatment of CYP3A4 with DG could significantly reduce the ability of TP to induce ROS. CYP3A4 inhibitors and DG co-pretreatment, reverse evidence-induced CYP3A4 may be one of the mechanisms of DG protection. Conclusions: This study suggests that TP induces an increase in reactive oxygen species (ROS) levels in L02 and produces toxic effects; low and medium concentrations of DG can significantly reduce the toxicity of TP to L02, whereas high concentrations of DG enhance the effect of TP on L02 Toxicity. DG-induced CYP3A4 upregulation and decreased ROS levels play an important role in reducing TP-induced normal hepatotoxicity.