阿魏酸和异阿魏酸对HepG2细胞增殖及其细胞色素P450酶的影响

Effects of ferulic acid and isoferulic acid on proliferation of HepG2 cells and its cytochrome P450 enzymes

目的观察阿魏酸和异阿魏酸对HepG2细胞中细胞色素P450同工酶1A1、3A4(CYP1A1、CYP3A4)的作用特点。方法采用MTT比色法测定不同质量浓度阿魏酸和异阿魏酸对体外培养人肝癌HepG2细胞增殖的影响;采用流式细胞术测定阿魏酸和异阿魏酸对HepG2细胞周期的影响;实时定量PCR技术检测阿魏酸和异阿魏酸处理后CYP1A1、CYP3A4 mRNA的表达;Western blotting检测CYP3A4蛋白表达。结果作用48 h后,阿魏酸和异阿魏酸对HepG2细胞均有抑制作用,且有明显的剂量依赖关系;阿魏酸和异阿魏酸(50μg/mL)均阻滞HepG2细胞周期于G2/M期;阿魏酸和异阿魏酸在不同作用质量浓度下均是CYP1A1、CYP3A4 mRNA的抑制剂;50μg/mL阿魏酸和异阿魏酸处理细胞48 h后,CYP3A4蛋白表达均明显低于对照组,相比于对照组,阿魏酸和异阿魏酸的表达量分别为0.57、0.39。结论阿魏酸和异阿魏酸均能抑制HepG2细胞的增殖,其机制之一是影响细胞周期使其阻滞于G2/M期,能够抑制药物代谢酶CYP1A1、CYP3A4 mRNA的表达,同时抑制CYP3A4的蛋白表达,但作用程度差别较大,可能与其羟基和甲氧基异构有关。

Objective To observe the action characteristics of ferulic acid and isoferulic acid on cytochrome P450 isoenzymeslA1 and 3A4 （CYP1A1, CYP3A4） in HepG2 cells. Methods MTT colorimetry was used to investigate the effects of ferulic acid and isoferulic acid at different concentration on the proliferation of in vitro cultured HepG2 cells. Flow cytometry was used to determine the effects of ferulic acid and isoferulic acid on cell cycle of human liver HepG2 cells; The expression of CYP1A1 and CYP3A4 mRNA was tested by real time quantitative PCR technology after drug treatment and then CYP3A4 protein expression was detected by protein inlmunoblot method （Western blotting）. Results HepG2 cells were inhibited by ferulic acid and isoferulic acid after 48 h action, with a clear dose-response relationship; Two drugs （50 μg/mL） blocked HepG2 cell cycle in G2-M phase; All of the concentration （100, 50, and 25 μg/mL） of the two drugs were CYP1A1 and CYP3A4 mRNA inhibitors; After cells were disposed by the two kinds of drugs （50 μg/mL） with 48 h, protein expression was significantly lower than that in the control group; And compared with the control group, the expression amounts of ferulic acid and isoferulic acid were 0.57 and 0.39. Conclusion The proliferation of HepG2 cells is inhibited by the two drugs, and the cell cycle was arrested at GffM phase, which that may be one of its mechanisms, so as to inhibit the mRNA expression of CYP1A1 and CYP3A4. At the same time, both of them could inhibit the protein expression of CYP3A4, as well as the effect degree of difference is bigger, which may be associated with its hydroxyl and the methoxyl isomerisation.