冬凌草甲素诱导小鼠肝癌细胞醌还原酶活性及其机理研究

Oridonin-Induced Quinone Reductase Activity and Its Mechanism in Mouse Hepatoma Hepa1c1c7 Cells

为筛选鉴定出溪黄草中抗癌活性组分,本研究以溪黄草为材料得到乙酸乙酯提取物,通过硅胶柱层析、半制备色谱分离纯化,利用小鼠肝癌细胞Hepa1c1c7体外模型测定分离产物对细胞存活率和醌还原酶(quinone reductase,QR/NAD(P)H:reductase 1,NQO1)诱导活性。通过核磁共振、质谱分析鉴定组分结构;采用细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)、磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)、丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)p38、蛋白激酶C(protein kinase C,PKC)和c-Jun N-末端激酶(c-Jun N-terminal kinase,JNK)5种蛋白激酶的特异性抑制剂,确定冬凌草甲素诱导QR活性的信号转导通路,采用实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)和Western blot法分别检测NQO1的m RNA和蛋白表达情况。结果:1)从溪黄草中筛选鉴定出一较强诱导QR活性组分为冬凌草甲素;2)冬凌草甲素质量浓度为0.94μg/m L时,即可诱导QR活性倍增;3)PI3K抑制剂处理显著抑制冬凌草甲素诱导的QR活性,而ERK抑制剂处理显著促进冬凌草甲素诱导的QR活性,但PKC、p38和JNK抑制剂处理对冬凌草甲素诱导的QR活性无明显影响;4)冬凌草甲素显著提高细胞内NQO1的m RNA和蛋白表达水平。结论:溪黄草中的冬凌草甲素具有抗癌活性,其抗癌活性通过提高Hepa1c1c7细胞中NQO1基因的转录及翻译水平以增加QR活性实现,并且其抗癌功能主要通过激活PI3K通路、抑制ERK通路进行信号传导。研究结果为深入开展溪黄草和冬凌草甲素的癌症化学预防研究奠定前期理论基础。

To screen and identify the anticancer components of Rabdosia serra(Maxim)Hara,the effects of the crude ethyl acetate extract of Rabdosia serra(Maxim)Hara and its purified fractions obtained by silica gel column chromatography and semi-preparative chromatography on cell viability and quinone reductase(QR/NQO1)activity were evaluated using mouse hepatoma Hepa1c1c7cells.The purified fractions were identified by mass spectrometry(MS),1H-nuclear magnetic renonsance(1H-NMR)and13C-NMR.Furthermore,mouse hepatoma cells were pretreated with specific protein kinase inhibitors including phosphatidylinositol3-kinase(PI3K)inhibitor,extracellular signal-regulated kinase(ERK)inhibitor,mitogen activated protein kinases(MAPK)p38inhibitor,protein kinase C(PKC)inhibitor and c-Jun N-terminal kinase(JNK)inhibitor to determine the signal transduction pathways leading to the induction of QR activity.The mRNA and protein expression levels of NQO1were measured by quantitative PCR and Western blot,respectively.Our results suggested as follows:1)oridonin was identified as a potential anticancer component of this extract;2)oridonin at a concentration of0.94μg/mL exhibited a remarkable potency in inducing QR;3)PI3K inhibitor significantly inhibited the induction of QR activity by oridonin,while ERK inhibitor showed an opposite effect.Moreover,p38,PKC and JNK inhibitors did not significantly affect the induction effect of oridonin on QR;4)oridonin markedly increased the expression of both mRNA and protein of NQO1.In conclusion,oridonin from Rabdosia serra(Maxim)Hara can induce QR activity in cultured Hepa1c1c7cells by activating the PI3K signal transduction pathway and inhibiting the ERK signal transduction pathway,and it also can significantly induce the transcription and translation of NQO1.These results would provid a preliminary theoretical basis for cancer chemoprevention with Rabdosia serra(Maxim)Hara and oridonin.