黄芩素在正常及早期肝纤维化模型小鼠体外肝、肠微粒体中Ⅱ相代谢差异

Phase Ⅱ metabolism of biacalein in the liver and intestinal microsomes of normal and early hepatic fibrosis mice in vitro

目的考察肝纤维化模型小鼠肝、肠微粒体对黄芩素体外葡萄糖醛酸化代谢影响和酶促反应动力学研究。方法小鼠每周3次灌胃20%CCl4油溶液,持续6周,检测血清肝功能指标ALT和AST,ELISA试剂盒检测血清中HA,天狼猩红染色和免疫组化染色α-SMA评估肝纤维化模型。检测正常和肝纤维小鼠肠道内容物β-葡萄糖醛酸苷酶活性,同时建立小鼠肝、肠微粒体孵育体系对黄芩素葡萄糖醛酸化反应的酶促反应动力学进行研究,评估其代谢动力学类型,并对比在正常和肝纤维化模型中最大反应速率(Vmax)和米氏常数(Km)。结果经过6周CCl4诱导后,模型组的血清、病理和免疫组化检测结果显示肝纤维化模型复制成功。体外实验结果显示,黄芩素在小鼠肝微粒体中的代谢方式符合米氏方程,而在肠微粒体中属于底物抑制型。与正常组相比,黄芩素在肝纤维化后的肝微粒体中的代谢速率要显著提高(P<0.01),而在肠微粒体中则相反,但差异无统计学意义,同时发现肝纤维化后肠道菌群中的β-葡萄糖醛酸苷酶活性要显著高于正常组(P<0.01)。结论肝纤维化状态对葡萄糖醛酸苷转移酶等Ⅱ相代谢酶的活性带来显著的改变,这值得我们关注肝纤维化疾病状态下药物的代谢特征和临床疗效,促进安全合理用药。

Objective To determine the effect of liver and intestinal microsomes on the glucuronidation and enzyme kinetics of biacalein in normal and hepatic fibrosis mice, respectively. Methods Hepatic fibrosis mice models were induced by intragastric administration of 20% CC14 olive oil solution 3 times a week for 6 weeks. The serum level of ALT and AST were detected by automatic biochemistry equipment. The serum levels of hyaluronic acid （HA） was detected by ELISA kit. Sirius red staining was used to observe the pathological changes in the liver tissue. The content of a-smooth muscle contractile proteins （a-SMA） was detected by immunohistochemical method. The active of β-d-glucuronidase of intestinal contents were compared between the normal and the model mice. The liver and intestinal microsomes from the normal and the hepatic fibrosis mice were prepared for incubation of baicalein in vitro. The enzyme kinetics parameters Fm^x and Km of the normal and the hepatic fibrosis mice were compared. Results After 6 weeks, the hepatic fibrosis models were successfully rebuilt according to the serum, pathological and immunohistochemical changes. The glucuronidation behaviors of baicalein metabolized in intro in the liver and intestinal microsomes followed the typical Michaelis-Menten kinetics and the characteristics of substrate inhibition in both groups. The rate of glucuronidation of baicalein was significantly enhanced in the hepatic fibrosis liver microsomes as compard with that in the normal group （P 〈 0.01）, but quite the opposite in the intestinal microsomes. more active fl-d-glucuronidase of intestinal contents in the model group was significantly enhanced than in normal group （P 〈 0.01）. Conclusion Hepatic fibrosis can significantly change phase Ⅱ metabolizing enzymes such as glucuronosyl transferase （UGT） activity. We should pay more attention to the metabolic characteristics and clinical efficacy of drugs for hepatic fibrosis, and promote safety rational medicine use.