Establishment And Its Application Of A Cell Model Of Antioxidant Drugs Screening

Oxidative stress is closely related with the initiation and progression of many diseases, suchas aging, diabetes and cardiovascular disease. The establishment of a rapid and efficientscreening methods and screening model has important significance in discovering effectiveantioxidant drugs with low toxicity. This paper aims at establishing an efficient, simple andhighly sensitive cell model for the antioxidant drugs screening. And the cell model can be usedfor the preliminary screening of antioxidant drugs. Keap1-Nrf2-ARE signaling pathwayregulates the expressions of antioxidant enzyme genes and phase ΙΙ detoxification enzyme genesand plays an important role in oxidative stress. This study used antioxidant responseelement(ARE) to regulates the expression level of the luciferase reporter gene(Luc) to establish acell model for antioxidant drugs screening.The recombinant plasmid vector pARE-Luc-Neo was constructed to express reporter geneluciferase regulated by the ARE. MTT assay was used to evaluate the toxic effects of G418onHek293, and the suitable concentration of G418for selecting was800μg/mL. The recombinantplasmid vector pARE-Luc-Neo was transfected into Hek293cells(human embryonic kidneyepithelial cells) using FuGENE HD transfection reagent. After24hours transfection, positivecells were screened by G418, and26positive monoclonal cells were selected by using thedilution method. The monoclonal cell line Hek293-ARE with high activity of luciferase inresponse to phase ΙΙ detoxification enzymes inducer TBHQ was obtained by the detection ofluciferase activity. The screening effect of Hek293-ARE cells was evaluated by using TBHQ,resveratrol, curcumin and andrographolide. The results showed that the induced effect ofexpression of luciferase of Hek293-ARE cells with treatment of inducers were better, andbetween the expression level and the concentration of the inducer had a dose-responserelationship within a certain range. We tested the model of its genetic stability and expressionstability and the test results showed that this model is robust. After the above verificationindicated that the cell model for the antioxidant drugs screening was established successfully andthe cell model can be used for the preliminary screening of antioxidant drugs.The cell model was used to screen for18kinds of traditional Chinese medicine monmoners.The results showed that Chinese medicine monomer ursolic acid, cryptotanshinone, berberine,silymarin, apigenin, genistein, daidzein, breviscapine, baicalin, wogonin, emodin and rhein had abetter induced effect on expression of luciferase of Hek293-ARE cells and had a significantdose-response relationship within a certain range. These12kinds of raditional Chinese medicinemonomer could play a role of anti-inflammatory and antioxidant by activating Keap1-Nrf2-ARE signaling pathway. The results of screening crude polysaccharide from Cordyceps militarisshowed that the induced effect on expression of luciferase of Hek293-ARE cells were better andhad a significant dose-response relationship within a certain range, and its roles in antioxidantmay be achieved by the induction of phase ΙΙ detoxification enzymes. The results of screeningwater extract of Cordyceps Militaris showed that2#,3#,4#,7#,11#,12#and14#componentshad a better induced effect on expression of luciferase of Hek293-ARE cells, and indicated thatthese components can activate Keap1-Nrf2-ARE signaling pathway and have a antioxidantaction.The successful establishment of the cell model for antioxidant drugs screening provides anadvanced, rapid and efficient screening approach for discovering new antioxidant drugs andlarge-scale and rapid selecting active components which have antioxidant activity in naturalmedicine, and has a great significance in study of the mechanism of antioxidant drugs.