黄连素对HepG2细胞CYP3A4和P-gp的影响和作用机制研究

摘要/Abstract

摘要： 目的: 阐明黄连素即盐酸小檗碱(berberine hydrochloride,BBR)对人肝癌细胞HepG2中细胞色素P450 3A4(CYP3A4)、多药耐药基因(MDR1)在mRNA和蛋白水平表达的影响,初步探讨黄连素对CYP3A4和P-糖蛋白(P-gp)的影响及其作用机制。方法: 采用MTT法检测不同浓度的BBR(1~100 μg/mL)对HepG2细胞活力的影响;并在BBR对HepG2细胞毒性较小的浓度范围进行实验,实验分为空白对照组、不同浓度BBR组、诱导剂利福平(Rif)组以及不同浓度BBR和Rif共同给药组,与对数期HepG2细胞孵育48 h后,提取细胞RNA和总蛋白,分别采用荧光定量PCR法(QRT-PCR)、Western blot法检测HepG2细胞中CYP3A4、MDR1、孕烷X受体(pregnane X receptor,PXR)、视黄醛X受体(retinoid X receptor,RXR)在mRNA水平的表达和CYP3A4、P-gp在蛋白水平的表达。结果: BBR在1~40 μg/mL 浓度范围内对HepG2细胞毒性小,细胞活力大于80%,超过 40 μg/mL 时对细胞毒性大,细胞存活率低。Western blot结果表明,与空白对照组比较, 0.1、0.5 和 2 μg/mL BBR对HepG2细胞CYP3A4和P-gp蛋白的表达有诱导作用(P<0.05),但10、40 μg/mL BBR对HepG2细胞CYP3A4和P-gp蛋白的表达有显著性抑制作用(P<0.01)。QRT-PCR结果表明,与空白对照组比较,10、20和 40 μg/mL BBR均对HepG2细胞PXR、CYP3A4、MDR1 mRNA的表达有显著抑制作用(P<0.05);但 10 μg/mL BBR对RXR mRNA无显著性影响(P>0.05),20、40 μg/mL BBR对RXR mRNA有显著的抑制作用(P<0.05)。结论: 黄连素对CYP3A4和P-gp有双向作用,低剂量时诱导,高剂量时抑制,并且其作用很有可能通过PXR信号通路实现。

关键词: 黄连素, CYP3A4, P-糖蛋白, 孕烷X受体, 视黄醛X受体, 药物相互作用

Abstract: AIM: To study the effects of berberine hydrochloride(BBR) on the mRNA and protein expression of cytochrome P450 3A4(CYP3A4) and multidrug resistance 1(MDR1) in HepG2 cells, then to investigate the mechanisms of regulation of CYP3A4 and P-glycoprotein(P-gp) by berberine. METHODS: HepG2 cells were treated with different concentrations of BBR(1-100 μg/mL)for 48 h, then effects of BBR on the cell proliferation were detected by MTT assay. The experiments were taken when the cytotoxicity of BBR on HepG2 cells was low.The experiments were divided into the following groups: control group, groups administrated with different concentrations of BBR(0.1-40 μg/mL), 10 μmol/L Rifampicin(Rif)group and groups administrated with different concentrations of BBR (0.1-40 μg/mL) and together with 10 μmol/L Rif, incubated with the logarithmic phase of HepG2 cells for 48 h. Total proteins and RNA were extracted. The mRNA levels of CYP3A4, MDR1, pregnane X receptor (PXR) and retinoid X receptor (RXR) in HepG2 cells were assayed with QRT-PCR, and the protein levels of CYP3A4,P-gp in HepG2 cells were assayed with Western blot. RESULTS: When the concentration of BBR was during 1- 40 μg/mL, the cytotoxicity of BBR on HepG2 cells was low, besides, the living rates of HepG2 cells were all above 80%.Compared with the control group,Western blot analysis showed that groups administrated with 0.1,0.5 and 2 μg/mL BBR significantly induced the expression of CYP3A4 and P-gp proteins in HepG2 cells(P<0.05). However, the 10 and 40 μg/mL BBR groups could markedly inhibit the expression of CYP3A4 and P-gp proteins in HepG2 cells(P<0.01).QRT-PCR analysis indicated that groups administrated with 10, 20 and 40 μg/mL BBR could strongly down-regulated the CYP3A4, PXR, and MDR1 mRNA expression in HepG2 cells(P<0.05) but 10 μg/mL BBR group had no significant inhibitory effects on the RXR mRNA expression(P>0.05),20 and 40 μg/mL BBR groups markedly inhibited the RXR mRNA expression(P<0.05). CONCLUSION: Berberine had a dual role on the expression of CYP3A4 and P-gp. Induction was leaded by low doses berberine, inhibition was leaded by high doses berberine. The mechanisms of regulation of CYP3A4 and P-gp by berberine was probably through PXR pathways.

Key words: berberine hydrochloride, CYP3A4, P-gp, PXR, RXR, drug interactions

中图分类号:

R965.2

唐霞, 辛华雯, 李维亮, 欧阳萌. 黄连素对HepG2细胞CYP3A4和P-gp的影响和作用机制研究[J]. 中国临床药理学与治疗学, 2015, 20(1): 7-13.

TANG Xia, XIN Hua-wen, LI Wei-liang, OUYANG Meng. Study of the effects of Berberine on CYP3A4 and P-gp in HepG2 cells and its mechanism in vitro[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2015, 20(1): 7-13.

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