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目的:建立一个快速、准确的测定大鼠肝微粒体中CYP3A4酶活性的HPLC,研究雷公藤醇提物对肝损伤大鼠肝微粒体CYP3A4酶活性的影响,进而探讨归脾汤对其保护机制。方法:50只大鼠随机分为正常组、模型组、归脾汤组、P450诱导组、P450抑制组。正常组、模型组灌服生理盐水,归脾汤组灌服归脾汤(9g·kg-1),P450诱导组、P450抑制组分别腹腔注射地塞米松(100mg·kg-1)和酮康唑(80mg·kg-1),连续ig 4d后,再以雷公藤醇提物(3.25mg·kg-1)ig 3d造模。选用HPLC以咪达唑仑为探针药物,检测各组大鼠肝微粒体中CYP3A4酶活性。结果:在所建立的HPLC条件下,咪达唑仑的出峰时间在8.960min,能够完全分离,且无其他生物基质峰干扰;线性范围是0.25~20μmol·L-1(r=0.9999),符合检测要求,方法可靠。结果显示,与正常组比较,模型组大鼠CYP3A4活性明显降低,差异有统计学意义(P<0.05);与模型组比较,归脾汤组、CYP450诱导组大鼠CYP3A4活性亦明显升高,差异有统计学意义(P<0.05);归脾汤组与CYP450诱导组之间比较,大鼠CYP3A4活性差异不明显,无统计学意义。结论:本方法能够对CYP3A4活性进行准确评价,雷公藤对大鼠肝微粒体CYP3A4酶活性有一定的抑制作用,归脾汤可能通过诱导CYP3A4活性而降低雷公藤的毒性从而发挥对肝脏的保护作用。
OBJECTIVE: To establish a rapid and accurate HPLC method for the determination of CYP3A4 activity in rat liver microsomes and to study the effect of alcohol extract of Tripterygium wilfordii on the activity of CYP3A4 in liver microsomes of rats with liver injury, and to explore the protective mechanism of Guipi Decoction . Methods: Fifty rats were randomly divided into normal group, model group, Guipi Decoction group, P450 induction group and P450 inhibition group. Normal group and model group were fed with normal saline, Guipi Decoction group was treated with Guipi Decoction (9g · kg -1), P450 group and P450 group by intraperitoneal injection of dexamethasone (100mg · kg -1) (80mg · kg-1). After continuous ig for 4 days, the animals were treated with ig3d (3.25mg · kg-1) alcohol extract. Midazolam was used as a probe to detect CYP3A4 activity in liver microsomes of rats in each group. Results: Under the established HPLC conditions, the peak time of midazolam was 8.960min, which could be completely separated without any other peak of biological matrix. The linear range was 0.25 ~ 20μmol·L-1 (r = 0.9999) Meet the testing requirements, the method is reliable. The results showed that compared with the normal group, the activity of CYP3A4 in the model group was significantly lower (P <0.05). Compared with the model group, the activity of CYP3A4 in the Guipi Decoction group and the CYP450-induced group was also significantly increased, (P <0.05). There was no significant difference in CYP3A4 activity between Guipi Decoction group and CYP450-induced group, and there was no statistical significance. Conclusion: This method can accurately evaluate the activity of CYP3A4. Tripterygium on the liver microsomes CYP3A4 enzyme activity has a certain inhibitory effect, Guipi Decoction may induce CYP3A4 activity and reduce the toxicity of Tripterygium so as to play a protective effect on the liver .