何首乌水提物及其主要成分对人肝细胞L02中CYP1A2、CYP2C9和CYP2E1 mRNA表达的影响

Effects of aqueous extract of Polygoni Multiflori Radix and its main constituents on expression of mRNA of CYP1A2,CYP2C9,and CYP2E1 in human liver L02 cells

目的分析何首乌水提物中主要化学成分及其量,阐明何首乌水提物及其主要成分对人正常肝实质细胞L02中CYP1A2、CYP2C9和CYP2E1 mRNA表达的影响。方法 HPLC测定何首乌水提物中主要化学成分及其质量分数;MTT法确定何首乌水提物及其主要成分对L02细胞活力的影响;实时荧光定量PCR测定L02细胞中CYP1A2、CYP2C9和CYP2E1mRNA表达量。结果何首乌水提物主要含有二苯乙烯苷、大黄素-8-O-β-D-葡萄糖苷、大黄素和大黄素甲醚,各成分在何首乌水提物中的质量分数分别为(1.14±0.03)%、(0.106 9±0.001 6)%、(0.010 8±0.000 9)%、(0.003 55±0.000 19)%;何首乌水提物及其主要成分作用L02细胞24 h,何首乌水提物和大黄素对细胞活力的影响随着药物浓度的增加抑制作用增强,IC50值分别为7.290 mg/m L和0.082 mmol/L,二苯乙烯苷、大黄素-8-O-β-D-葡萄糖苷和大黄素甲醚在实验浓度范围内对细胞活力的抑制作用不明显;何首乌水提物、大黄素均可明显抑制L02细胞中CYP1A2、CYP2C9、CYP2E1 mRNA的表达;大黄素-8-O-β-D-葡萄糖苷抑制CYP1A2和CYP2C9的mRNA表达;二苯乙烯苷抑制CYP1A2 mRNA表达,但激活CYP2C9mRNA表达;大黄素甲醚浓度依赖性抑制CYP1A2、CYP2C9 mRNA表达,却表现低浓度抑制、高浓度激活CYP2E1 mRNA表达。结论何首乌水提物抑制L02细胞中CYP1A2、CYP2C9和CYP2E1 mRNA表达是其所含各种成分综合作用的结果,何首乌4种主要成分均可抑制CYP1A2 mRNA表达,蒽醌成分是抑制CYP2C9 mRNA表达的主要成分,游离蒽醌是抑制CYP2E1 mRNA表达的主要成分。

Objective To analyze the main chemical constituents and their contents in aqueous extract of Polygoni Multiflori Radix（PMR,root of Polygonum multiflorum）,and to elucidate the effects of aqueous extract of PMR and its main constituents on the expression of the mRNA of CYP1A2,CYP2C9,and CYP2E1 in human liver L02 cells.Methods The main chemical constituents and their content in aqueous extract of PMR were determined by HPLC.The cytotoxicity of aqueous extract of PMR and its main constituents on L02 cells was determined by MTT assay.The mRNA expression of CYP1A2,CYP2C9,and CYP2E1 in L02 cells were detected by quantitative real-time PCR.Results There were four main well-separated chromatographic peaks standing for tetrahydroxy stilbene glucoside,emodin-8-O-β-D-glucoside,emodin and physcion in aqueous extract of PMR.The content of these components in aqueous extract of PMR was（1.14 ± 0.03）%,（0.106 9 ± 0.001 6）%,（0.010 8 ± 0.000 9）%,（0.003 55 ± 0.000 19）%,respectively.The cytotoxicity of aqueous extract of PMR and emodin on L02 cells at 24 h was dose-dependent,and the concentration of 50% inhibition was 7.290 mg/m L and 0.082 mmol/L respectively.Tetrahydroxy stilbene glucoside,emodin-8-O-β-D-glucoside and physcion did not show significant cytotoxicity on L02 cells in the experimental concentrations.Aqueous extract of PMR and emodin significantly inhibited the expression of mRNA of CYP1A2,CYP2C9,and CYP2E1 in L02 cells.Emodin-8-O-β-D-glucoside inhibited the expression of mRNA of CYP1A2 and CYP2C9.Tetrahydroxy stilbene glucoside inhibited the expression of mRNA of CYP1A2 but activated the expression of mRNA of CYP2C9.Physcion inhibited the expression of mRNA of CYP1A2 and CYP2C9 in a dose-dependent manner,but inhibited the expression of mRNA of CYP2E1 in low concentration and activitated the expression of mRNA of CYP2E1 in high concentration.Conclusion The inhibition of aqueous extract of PMR on the expression of mRNA of CYP1A2,CYP2C9,and CYP2E1 in L02 cells is the combined effect of all components in it.The main four components all inhibit the expression of mRNA of CYP1A2.The anthraquinone is the main component inhibiting the expression of mRNA of CYP2C9.The free anthraquinone is the main component inhibiting the expression of mRNA of CYP2E1.