CYP1A2低表达增强何首乌水提物及其相关单体成分肝细胞毒性的研究

目的建立CYP1A2低表达人正常肝细胞株,探究何首乌水提物(PMR)及其相关单体成分的肝细胞毒性与CYP1A2低表达的关系。方法运用RNA干扰技术构建稳定转染shCYP1A2的2种人正常肝细胞系L02、HepaRG,RT-qPCR验证干扰效果;PMR及其相关单体成分:二苯乙烯苷(2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside,TSG)、大黄素-8-O-β-D-葡萄糖苷(emodin-8-O-β-D-glucopyranoside,EG)、没食子酸(gallic acid,GA)、大黄素(emodin,EM)、大黄素甲醚(physcion,PH)、芦荟大黄素(aloe-emodin,AE)、大黄酸(rhein,RH)作用CYP1A2敲低的L02及HepaRG细胞48 h,MTT检测细胞活力。结果成功构建了稳定转染shCYP1A2的2种人正常肝细胞系,L02-shCYP1A2、HepaRG-shCYP1A2与相应空质粒对照组相比,CYP1A2 mRNA表达分别下降了59.81%(L02-shCYP1A2)和66.60%(HepaRG-shCYP1A2)。1.5~3 mg/mL PMR作用CYP1A2敲低的L02-shCYP1A2细胞48 h,细胞毒性显著增加(P<0.01);2.5~3 mg/mL PMR作用CYP1A2敲低的HepaRG-shCYP1A2细胞48 h,细胞毒性显著增加(P<0.05、P<0.01);40~120μmol/L GA作用CYP1A2敲低的L02-shCYP1A2及HepaRGshCYP1A2细胞48 h,细胞毒性显著增加(P<0.01、P<0.01);AE作用CYP1A2敲低的HepaRG-shCYP1A2细胞48 h,细胞毒性显著增加(P<0.01)。结论CYP1A2低表达显著增加PMR的肝细胞毒性,推测GA和AE可能为相关的增毒成分。

CYP1A2 Depletion by shRNA Enhances Hepatotoxicity of Extract of Polygoni Multiflori Radix and Related Monomer Compositions

Objective To construct the CYP1A2 depletion human liver cell lines by shRNA,and to explore the relationship between depletion of CYP1A2 and hepatotoxicity of extract of Polygoni Multiflori Radix(PMR)and related monomer compositions.Method We constructed stably transfected L02-shCYP1A2 and HepaRG-shCYP1A2 cells with depletion of CYP1A2 by shRNA,verified by RT-qPCR.CYP1A2 depletion L02 and HepaRG cells were treated for 48 h with extract of PMR,and related monomer compositions included 2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-glucoside(TSG),emodin(EM),aloe-emodin(AE),emodin-8-O-β-D-glucopyranoside(EG),gallic acid(GA),physcion(PH),rhein(RH).Cell viability was detected by MTT assay.Result Successfully constructed two stably transfected cell lines,named L02-shCYP1A2 and HepaRGshCYP1A2 cells,and the mRNA expression of CYP1A2 decreased by 59.81%in L02-shCYP1A2 and 66.60%in HepaRG-shCYP1A2,respectively.After L02-shCYP1A2 were treated with PMR for 48 h,cytotoxicity were significantly increased(P<0.01).After HepaRG-shCYP1A2 were treated with PMR for 48 h,cytotoxicity were significantly increased(P<0.05,P<0.01).The cytotoxicity of GA was significantly increased in L02-shCYP1A2 cells and HepaRG-shCYP1A2 cells(P<0.01,P<0.01).The cytotoxicity of AE was also significantly increased in HepaRG-shCYP1A2 cells(P<0.01).Conclusion The depletion of CYP1A2 significantly increased the hepatotoxicity of PMR,of which GA and AE were suggested to be the related monomer compositions.