Our final results demonstrate for the first time that digitoflavone is in a position to attenuate oxidative injury in colonic cells by up regulate the expression in the antioxidant defense enzymes via a mechanism that in volved p38 MAPKs activation and Nrf2 translocation and additional confirmed chemopreventive effect by absolutely free radical scavenging and inhibition of inflammation. Outcome Digitoflavone induced high levels of ARE driven luciferase activities in Caco two, HT 29, HepG2 and HEK 293 cells A DNA fragment containing 8 copies of your ARE se quence were subcloned into the pGL3 vector. Soon after transient transfection with the expres sion plasmid, diverse concentrations of digitoflavone had been added to the cell culture and incubated for eight hours and 24 hours respectively.
Parallel cell viability assays re vealed no of course cytotoxic effects for the digitoflavone treatment when the concentration of digitoflavone is reduced NVP-BGJ398 BGJ398 than 10 uM in Caco two, HepG2, HEK 293 cells and five uM in HT 29 cells. ten uM digitoflavone induced the highest degree of luciferase activity right after 8 hours exposure, about 5 fold increases of manage. A different human epithelial colorectal adenocarcinoma cell line HT 29 also showed that low concentrations of digitoflavone can boost the ARE luciferase activity with no obviously cytotoxic effects. To evaluate the ARE driven luciferase activity of digitoflavone in other cell lines, HepG2 and HEK 293 cell lines were transient transfected using the pGL3 ARE luciferase plasmid respectively and tested with 1 20 uM digitoflavone for 8 hours.
All tested cell lines selleck inhibitor showed over two fold increases of your luciferase ac tivity at 1 10 uM concentrations of digitoflavone. These outcome recommended that digitoflavone, at low concentrations, is usually a potent activator from the Nrf2 ARE antioxi dant pathway. Digitoflavone stimulated the expression from the Nrf2 ARE mediated antioxidant defense proteins in Caco 2 cells To verity whether or not activation of luciferase activity by digi toflavone in Caco two cells reflected the expression of your endogenous ARE driven genes, the mRNA levels of GR, TR, HO 1, GCSc, GCSm, NQO1, and MRP2 have been examined within the presence or absence of digitoflavone. In Caco 2 cells treated with 10 uM digitoflavone for eight hours, the mRNA levels of GR, TR, HO 1, GCSc, GCSm, UGT1A1 and UGT1A10 increased 1. 2, six. 0, 1. 5, 1. 7, 1. 8, 1. 5, 1. eight fold, respectively.
Simi larly, evaluation of your Nrf2 mediated antioxidant en zymes, including GCSc and TR by Western blotting showed that exposure of Caco two cells to 1 15 uM digi toflavone strongly induced GCSc, GCSm and TR protein expression within a dose and time dependent man ner. Digitoflavone induced Nrf2 protein expression and nuclear translocation Prior research described that under standard conditions, Keap1 sequestered Nrf2 within the cytoplasm and that trans location of Nrf2 into the nucleus is essential for the transactivation of a variety of targeted genes.