Our final results demonstrate for the very first time that digitoflavone is able to attenuate oxidative injury in colonic cells by up regulate the expression with the antioxidant defense enzymes via a mechanism that in volved p38 MAPKs activation and Nrf2 translocation and additional confirmed chemopreventive impact by free of charge radical scavenging and inhibition of inflammation. Outcome Digitoflavone induced high levels of ARE driven luciferase activities in Caco 2, HT 29, HepG2 and HEK 293 cells A DNA fragment containing 8 copies in the ARE se Quence have been subcloned into the pGL3 vector. Immediately after transient transfection with all the expres sion plasmid, distinct concentrations of digitoflavone have been added to the cell culture and incubated for 8 hours and 24 hours respectively.
Parallel cell viability assays re vealed no definitely cytotoxic effects for the digitoflavone therapy when the concentration of digitoflavone is reduced than 10 uM in Caco 2, HepG2, HEK 293 cells and five uM in inhibitor OSI-906 HT 29 cells. ten uM digitoflavone induced the highest level of luciferase activity soon after eight hours exposure, about five fold increases of handle. An additional human epithelial colorectal adenocarcinoma cell line HT 29 also showed that low concentrations of digitoflavone can boost the ARE luciferase activity with no certainly cytotoxic effects. To evaluate the ARE driven luciferase activity of digitoflavone in other cell lines, HepG2 and HEK 293 cell lines had been transient transfected with all the pGL3 ARE luciferase plasmid respectively and tested with 1 20 uM digitoflavone for eight hours.
All tested cell lines showed over 2 fold increases with the luciferase ac tivity at 1 10 uM concentrations of digitoflavone. These result suggested inhibitor MK-1775 that digitoflavone, at low concentrations, is actually a potent activator on the Nrf2 ARE antioxi dant pathway. Digitoflavone stimulated the expression in the Nrf2 ARE mediated antioxidant defense proteins in Caco two cells To verity whether activation of luciferase activity by digi toflavone in Caco two cells reflected the expression on the endogenous ARE driven genes, the mRNA levels of GR, TR, HO 1, GCSc, GCSm, NQO1, and MRP2 were examined within the presence or absence of digitoflavone. In Caco two cells treated with 10 uM digitoflavone for 8 hours, the mRNA levels of and UGT1A10 improved fold, respectively.
Simi larly, evaluation on the Nrf2 mediated antioxidant en zymes, like 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 in a dose and time dependent man ner. Digitoflavone induced Nrf2 protein expression and nuclear translocation Previous research described that beneath standard circumstances, Keap1 sequestered Nrf2 inside the cytoplasm and that trans location of Nrf2 in to the nucleus is essential for the transactivation of several targeted genes.