NADPH Oxidase Is Involved In Xanthohumol-Induced ROS Increase In HL-60 Cells

Xanthohumol is a prenylated flavonoid that occurs only in the hop plant. Scavenging of ROS represents one of the mechanisms by which flavonoids exert their antioxidant activities. The production of ROS is the unavoidable consequence of aerobic life. ROS production can be triggered by increases or decreases in ROS-related enzymes such as NADPH oxidase. NADPH oxidase is a membrane-bound enzyme complex. ROS generation is the primary and sole function of NADPH oxidase. Preliminary work in our laboratory had found that xanthohumol can also induce ROS. Also this process can be inhibited by DPI. The purpose of this reaserch is to find out whether NADPH oxidase is involved in xanthohumol-induced ROS burst. The human promyelocytic leukemia cell line was treated with doses ranging from 0.1 to 40μM of xanthohumol in order to assess the effects on cell proliferation and oxidative stress. Trypan blue was used to measure cell viability and reactive oxygen species production was determined by 2’,7’-dichlorofluorescein diacetate. Cells were observed by scanning electron microscope and confocal. Protein expression of gp91phox and p47phox were done by Western blot. The results show:1、 Treatments XN led to a decrease in cell proliferation. The decrease in xanthohumol-induced cell proliferation was inhibited by 1mM antioxidant NAC 2、 Treatments XN 0.1 to 10μM led to an increase of ROS production, however, treatments XN 20 to 40μM led to a decrease. We found the influence of serum on cell survival.3、DPI, inhibitor of NADPH oxidase, decreased XN-induced ROS production. Otherwise L-NMMA, inhibitor of nitric oxide synthase, had no influence. 4、After treatment with xanthohumol, gp91phox and p47phox expression were increased and more gp91phox and p47phox combined to become active NADPH oxidase. Taking all the results into account, we make sure that NADPH oxidase is involved in xanthohumol-induced ROS increase. Our study also found that it’s important to select proper conditions to do research on XN-induced ROS mechanism.