基于UGT1A1酶介导的胆红素代谢考察大黄素在肝微粒体体系中的肝毒性

以胆红素代谢过程中UGT1A1酶介导的胆红素葡萄糖醛酸结合环节为切入点,通过考察待测物大黄素对该酶的抑制作用预测其肝毒性。以胆红素为UGT1A1酶底物,于人肝微粒体、大鼠肝微粒体及人重组UGT1A1酶中加入不同浓度胆红素及大黄素,分别以胆红素的总代谢产物生成量对胆红素底物浓度作图,以米氏方程双倒数法绘图,并以不同曲线的斜率对应胆红素底物浓度绘制slop图计算表观抑制常数Ki,考察其对胆红素葡萄糖醛酸结合的抑制作用,预测其肝毒性有无及大小。结果显示大黄素在3个体系中对UGT1A1酶均有中强抑制作用,且抑制类型均为竞争型抑制。HLM,RLM,r UGT1A1体系的Ki分别为(5.400±0.956),(10.020±0.611),(4.850±0.528),P均0.05。同时发现,大黄素对于UGT1A1酶的抑制在大鼠及人之间无明显种属差异。大黄素可通过抑制UGT1A1酶活性导致胆红素代谢异常,从而存在引发肝毒性的潜在危险。该试验所建立的体外研究方法为中药肝毒性药物的筛选提供了新思路和新方法,对中药安全性评价具有借鉴意义。

Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes

To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant K_i was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with K_i=5.400±0.956(P<0.05) in HLM system, K_i=10.020±0.611(P<0.05) in RLM system, K_i=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines.