RNA interference(RNAi)is useful for selective gene silencing.Cytochrome P4503A4(CYP3A4),which metabolizes approximately 50% of drugs in clinical use,plays an important role in drug metabolism.In this study,we aimed to develop a short hairpin RNA(shRNA)to modulate CYP3A4 expression.Three new shRNAs(S1,S2 and S3)were designed to target the coding sequence(CDS)of CYP3A4,cloned into a shRNA expression vector,and tested in different cells.The mixture of three shRNAs produced optimal reduction(55%)in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells.Endogenous CYP3A4 expression in HepG2 cells was decreased about 50%at both mRNA and protein level after transfection of the mixture of three shRNAs.In contrast,CYP3A5 gene expression was not altered by the shRNAs,supporting the selectivity of CYP3A4 shRNAs.In addition,HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids,whose toxic metabolites are produced by CYP3A4.These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS,and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.
目的建立基于人孕烷X受体(human pregnane X receptor,hPXR)的UGT1A1的报告基因模型,研究中药提取物通过人孕烷X受体途径对UGT1A1的潜在诱导能力。方法以人基因组DNA为模板扩增UGT1A1的远端和近端启动子,连接到pGL3载体上,构建pGL3-PXRE重组质粒,并与人孕烷X受体表达质粒共转染HepG2细胞,从而建立报告基因模型。运用该模型考察了9种常见中药对人孕烷X受体的激活作用,即对UGT1A1的潜在诱导作用。结果将UGT1A1启动子片段克隆到pGL3载体上,构建了pGL3-PXRE重组质粒,成功构建了报告基因模型,并考察了多种常见中药的提取物通过人孕烷X受体途径对UGT1A1的诱导作用。结论成功地建立了基于人孕烷X受体的UGT1A1的报告基因模型,为人孕烷X受体激活剂的体外筛选提供了一种有效的方法。
Pharmacological activities and adverse side effects of ginkgolic acids(GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA(17:1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA(17:1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA(17:1) metabolism were human CYP1 A2, CYP3 A4, UGT1 A6, UGT1 A9, and UGT2 B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA(17:1) in HepG2 cells occurred in a time-and dose-dependent manner. Further investigation showed that GA(17:1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1 A-and CYP3 A-mediated metabolism.
YAO Qing-QingLI LiXU Ming-ChengHU Hai-HongZHOU HuiYU Lu-ShanZENG Su
Ginkgolic acids(GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA(15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA(15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA(15 : 1) on MDCK cells displayed a time-and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA(15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential(ΔΨm). In propidium iodide(PI) staining analysis, GA(15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA(15 : 1) induced renal toxicity.
YAO Qing-QingLIU Zhen-HuaXU Ming-ChengHU Hai-HongZHOU HuiJIANG Hui-DiYU Lu-ShanZENG Su
