扶正化瘀方对原代大鼠肝细胞中CYP450的影响

Effect of Fuzheng Huayu Recipe on CYP450 in Primary Rat Hepatocytes

目的：采用原代大鼠肝细胞（PRH）研究扶正化瘀方（FZHY）对大鼠细胞色素P450酶（CYP450）的影响。方法：采用两步灌流法分离大鼠原代肝细胞,形成肝板结构后加入FZHY共培养24 h,换成含有4种CYP450同工酶（CYP1A2,CYP2C9,CYP2D6,CYP3A4）对应的特异性探针药物（非那西丁、甲苯磺丁脲、氢溴酸右美沙芬、睾酮）的培养基,培养2 h后吸取上清液,利用UPLC-MS/MS对探针药物的代谢产物进行检测,考察FZHY对探针药物代谢产物生成量的影响,评价FZHY对PRH中CYP450代谢活力的影响。结果：PRH中CYP1A2,CYP2C9,CYP2D6与CYP3A4具有良好的代谢活力。4种探针药物在0-500μmol·L（-1）内对PRH没有毒性,在0.5-500μg·L（-1）时,FZHY对PRH没有毒性。FZHY在5μg·L（-1）时对PRH具有轻微的保护作用,并能使甲苯磺丁脲的代谢活化值——米氏常数（Km）降低20.8%;氢溴酸右美沙芬的Km降低39.2%;非那西丁的Km降低17.4%;并使睾酮的Km显著降低。结论：FZHY能抑制原代大鼠肝细胞中CYP1A2,CYP2C9,CYP2D6和CYP3A4的代谢活性。

Objective：Taking primary rat hepatocytes（PRH） model,to explore the effect of Fuzheng Huayu recipe（FZHY） on rat cytochrome P450 enzyme（CYP450）.Method：PRH were isolated and cultured by two-step perfusion method.With the formation of the hepatic plate structure,FZHY was added to culture for 24 h,then replaced with culture medium containing specific probe substrates of CYP450（phenacetin-CYP1A2,tolbutamide-CYP2C9,dextromethorphan hydrobromide-CYP2D6,testosterone-CYP3A4）,the supernatant was aspirated after 2 h,the corresponding products of the probe drugs were detected by UPLC-MS/MS.Result：CYP1A2,CYP2C9,CYP2D6 and CYP3A4 in PRH had good metabolic activity.The probe drugs had no toxicity on PRH in the range of 0-500 μmol·L^（-1）,FZHY had no toxicity on PRH in the range of 0.5-500 μg·L（-1）.FZHY could slightly protect PRH with concentration of 5 μg·L（-1）.After PRH was cultured with 5 μg·L（-1） FZHY for 24 h,Michaelis constant（Km） of tolbutamide decreased by 20.8%,Kmof dextromethorphan hydrobromide decreased by39.2%,Kmof phenacetin decreased by 17.4%,Kmof testosterone decreased significantly.Conclusion：FZHY can inhibit the activity of CYP1A2,CYP2C9,CYP2D6 and CYP3A4 in PRH.