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目的:初步考察尿毒清颗粒(NDQ)对重组人CYP450(细胞色素P450)和P-gp(P糖蛋白)的体外影响。方法:①使用Promega P450-GloTMScreening System,通过荧光发光原理来检测NDQ组、抑制剂组对重组CYP450的IC50值,比较NDQ组与抑制剂组IC50来评价NDQ对重组人CYP1A2,CYP2D6,CYP3A4,CYP2C19,CYP2C9的抑制作用。②使用BD ATPase Assay Kit,通过发光法检测NDQ组、空白对照组P-gp ATP酶活性,比较NDQ组、空白对照组ATP酶活性来评价NDQ是否为重组人P-gp的底物或抑制剂。结果:①NDQ组与抑制剂组IC50值(mg/ml)如下,CYP1A2:17.47、3.37×10-6;CYP3A4:33、4.1813×10-5;CYP2C9:10.39、1.056×10-3;CYP2D6:13.98、2.2459×10-6;CYP2C19:9.251、1.442×10-3。②NDQ组的P-GP ATP酶的活性为16.39nmol/mg protein/min,空白对照组的ATP酶活性为4.72 nmol/mg protein/min。结论:①NDQ组与抑制剂组IC50值差异较大,不在一个数量级,NDQ对重组人CYP 1A2、CYP 2D6、CYP 3A4、CYP2C19、CYP2C9基本没有抑制作用。②NDQ组ATP酶活性高于空白组并具有显著性差异,因此,NDQ可能为重组人P-GP的抑制剂或底物。
OBJECTIVE: To investigate in vitro the effects of niuduqing granule (NDQ) on the recombinant human CYP450 (cytochrome P450) and P-gp (P-glycoprotein) in vitro. Methods: ① Promega P450-GloTMScreening System was used to detect the IC50 value of NDQ group and inhibitor group against recombinant CYP450 by fluorescence emission principle. NDQ group and IC50 of inhibitor group were compared to evaluate the effects of NDQ on recombinant human CYP1A2, CYP2D6, CYP3A4, CYP2C19 , CYP2C9 inhibition. ② BD ATPase Assay Kit was used to detect the activity of P-gp ATPase in NDQ group and blank control group by luminescence method. The activities of ATPase in NDQ group and blank control group were compared to evaluate whether NDQ was the substrate or inhibitor of recombinant human P-gp . Results: ① The IC50 values (mg / ml) of NDQ group and inhibitor group were as follows: CYP1A2: 17.47, 3.37 × 10-6; CYP3A4: 33, 4.1813 × 10-5; CYP2C9: 10.39, 1.056 × 10-3; CYP2D6: 13.98 , 2.2459 × 10-6; CYP2C19: 9.251, 1.442 × 10-3. ② The activity of P-GP ATPase in NDQ group was 16.39nmol / mg protein / min, and the ATPase activity in blank control group was 4.72nmol / mg protein / min. Conclusion: ①The IC50 values of NDQ group and inhibitor group are quite different, which is not an order of magnitude. NDQ has no inhibitory effect on recombinant human CYP 1A2, CYP 2D6, CYP 3A4, CYP2C19 and CYP2C9. (2) The ATPase activity in NDQ group was higher than that in blank group and had significant difference. Therefore, NDQ might be the inhibitor or substrate of recombinant human P-GP.