目的 建立一種靈敏、快速的高細(xì)胞色素P450(CYPs)酶活性的大鼠原代肝細(xì)胞模型,用于評價經(jīng)CYPs酶代謝后產(chǎn)生肝毒性的藥物。方法 分別采用苯巴比妥和β-萘黃酮兩種CYPs酶廣譜誘導(dǎo)劑構(gòu)建大鼠原代肝細(xì)胞模型;應(yīng)用“cocktail”探針底物法考察兩種誘導(dǎo)劑對CYPs酶的影響;以基于CYPs酶代謝導(dǎo)致肝毒性的藥物他克林(TAC)、雙氯芬酸鈉(DIC)和對乙酰氨基酚(PAR)為模型藥物,評價所構(gòu)建的高CYPs酶活性大鼠原代肝細(xì)胞模型與普通大鼠原代肝細(xì)胞間靈敏性的差異;應(yīng)用所構(gòu)建的高CYPs酶活性大鼠原代肝細(xì)胞模型評價維拉帕米(VER)的肝毒性。結(jié)果 與普通大鼠原代肝細(xì)胞相比,3種模型藥物在高CYPs酶活性大鼠原代肝細(xì)胞中,在較低劑量時可使細(xì)胞上清液中乳酸脫氫酶(LDH)、細(xì)胞中活性氧(ROS)水平升高,線粒體膜電位(MMP)水平下降,或相同劑量下得到更嚴(yán)重的損傷結(jié)果,CYPs酶抑制劑1-aminobenzotriazole(ABT)和metyrapone(MET)能抑制這3種損傷的發(fā)生;100 μmol/L維拉帕米在普通大鼠原代肝細(xì)胞中并未引起LDH、ROS和MMP的改變,但在高CYPs活性大鼠原代肝細(xì)胞模型中,會引起細(xì)胞損傷,ABT和MET能抑制這種損傷。結(jié)論 經(jīng)誘導(dǎo)建立的兩種高CYPs酶活性大鼠原代肝細(xì)胞模型能更靈敏的評價基于CYPs酶代謝致毒藥物的肝毒性;兩種誘導(dǎo)劑誘導(dǎo)得到的高CYPs酶活性大鼠原代肝細(xì)胞模型對經(jīng)不同CYPs酶亞型代謝的藥物評價結(jié)果有各自優(yōu)勢。應(yīng)用高CYPs酶活性大鼠原代肝細(xì)胞模型證實維拉帕米的致毒機(jī)制是經(jīng)CYPs酶代謝產(chǎn)生肝毒性。

Objective To establish a rapid and sensitive primary rat hepatocytes model with high cytochrome P450 enzyme (CYPs) activity for evaluating the drug-induced liver injury based on CYPs metabolism. Methods The primary rat hepatocytes model was established with CYPs nonspecific inducer phenobarbital and β-naphthoflavone; The “cocktail” probe method was applied to inspect the effect of inducers on CYP450 enzyme. Tacrine (TAC), diclofenac sodium (DIC), and paracetamol (PAR) which induced liver injury based on the metabolism of CYPs were as the model drugs. The difference and sensibility between primary rat hepatocytes model with high CYPs activity and general primary rat hepatocytes model were evaluated; The liver injury of verapamil (VER) was evaluated with the primary rat hepatocytes model with high CYPs activity. Results After administration with three kinds of model drugs (100 μmol/L) to primary rat hepatocytes with high CYPs activity, the levels of lactate dehydrogenase (LDH) in hepatocytes supernatant and the reactive oxygen species (ROS) in hepatocytes were increased, and the mitochondrial membrane potential (MMP) was decreased. Nonspecific CYPs inhibitors, 1-aminobenzotriazole (ABT) and metyrapone (MET) could suppress the occurrence of this liver injury; VER (100 μmol/L) could not lead to LDH, ROS and MMP change in general primary rat hepatocytes model, however, it could cause cytotoxicity in primary rat hepatocytes model with high CYPs activity. The CYPs inhibitors ABT and MET suppressed this damage. Conclusion The primary rat hepatocytes model with high CYPs activity is more sensitive and suitable to evaluate the hepatotoxicity of drugs based on metabolism of CYPs. Two types of primary rat hepatocytes models with high CYPs activity have respective advantages in evaluation of different types of drug. VER was confirmed to induce liver injury based on metabolism of CYPs with the application of primary rat hepatocytes model with high CYPs activity.

國家自然科學(xué)基金(No.81373890);教育部高等學(xué)校博士學(xué)科點專項科研基金(No.20121210110011);教育部長江學(xué)者與創(chuàng)新團(tuán)隊發(fā)展計劃(No.IRT-14R41)