Establishment of a drug screening model based on anti-oxidative response element / 中草药

ABSTRACT

Objective: To establish a cell model for anti-oxidative drug screening by using anti-oxidative response element (ARE) to regulate the expression level of luciferase (Luc) reporter gene. Methods: The recombinant plasmid vector pARE-Luc-Neo, expressed by ARE-regulated Luc reporter gene, was constructed. The recombinant plasmid vector was transfected into human embryonic kidney epithelial cells (Hek293 cells) using FuGENE® HD transfection reagent. The positive cells were screened by G418, and the positive monoclonal cells were selected using the dilution method. The monoclonal cell lines with high activity of Luc reporter gene in response to anti-oxidative protein inducer TBHQ were obtained using the Luc activity detection. The screening effect of the positive clones was evaluated by resveratrol and curcumin. The effects of andrographolide, cryptotanshinone, silymarin, matrine, and polydatin at different concentration (0, 3.125, 6.25, 12.5, 25, 50, and 100 μmol/L) on Luc expression level were observed. Results: The Hek293-ARE cells were obtained by detecting the Luc reporter gene activity, the Luc reporter gene expression was regulated by ARE, meanwhile there exists a dose-response relationship between the expression level and the concentration of the inducer within a certain range. The andrographolide, cryptotanshinone, and silymarin have significant induction effects. Conclusion: The cell model for anti-oxidative drug screening is established successfully and could be effectively and high-throughput for the preliminary screening of anti-oxidative drugs.