Study On The NADPH Oxidase Related Mechanisms Of Pro-oxidant Activity Of Xanthohumol

Xanthohumol?XN?is a natural antioxidant polyphenolic substance with content up to 1%of the dry weight of hops.It has been reported a wide range of chemopreventive effects,such as inhibiting the initiation,promotion and metastasis of malignant tumors.Some recent studys indicate that in addition to the potential of antioxidant,xanthohumol may exert pro-oxidant properties,and thus may selectively form an imbalanced redox state by inducing the formation of excess reactive oxygen species?ROS?.As a result,some cells are induced into apoptosis.Comparing the effect of ROS generated from NADPH oxidase and mitochondria,the two main ROS sources of cells,ROS from NADPH oxidase usually exhibited cell signal function,while exhibited damage effect when from mitochondria.It is reported that XN induced ROS generation through mitochondria,and also induced mitochondria damage.In view of XN is the important natural product of hops,it is hard to understand that its ability to damage mitochondria.It is reasonable to postulate that there may be other sources of ROS generation except mitochondria.In our previous study,it was suggested that NOX might be the ROS generation site of xanthohumol treatment.To further evaluate our earlier discovery,molecular biology methods and human red blood cells were used in the present study.Material and mathods:Human acute myeloid leukemia HL-60 cells and human red blood cells were used as cell models.?1?Fluorescent probe DCFH-DA was used to detect intracellular ROS,and the results were analyzed by Flow cytometer and Flowjo software.?2?Immunofluorescence study was used to study the NOX activation after XN treatment and was observed under a confocal microscopy.?3?siRNA technique was used to knock down NOX subunit gp91^phox.?4?CRISPR-Cas9technique was used to further knock out gp91^phoxhox and establish HL-60^gp91p91 cell model.?5?Human red blood cells were used as a special cell model to eliminate the influence of mitochondria,and performed the ROS generation study too.Results:?1?XN induced ROS generation.ROS production peaked at a concentration of 10??XN treatment,thus 10?M was chosen as the optimal concentration.?2?Treatment of cells with both XN and NADPH oxidase inhibitor DPI showed a significant decrease in ROS compared with XN alone treatment group,indicated that XN induced ROS generation was associated with NADPH oxidase.?3?The results of confocal microscopy showed that the cytoplasmic subunits had a tendency to aggregate to the cell membrane,suggesting that NOX may be stimulated by XN.?4?Results of gp91^phoxhox knock down study showed that ROS production induced by XN decreased compared to the wild HL-60 cells,indicated the involvement of NOX in XN induced ROS generation.?5?Result of ROS detected on HL-60^gp-91p-91 knockout cell model showed a sharp decreased of ROS generation,which also indicated the involvement of NOX in XN induced ROS generation.?6?The results of human red blood cells experiments also showed that XN induced ROS burst could be inhibited by DPI potently;these results further confirmed that XN induced ROS generation through NOX.Conclusion:In conclusion,our study proved that XN induced ROS generation through NADPH oxidase,and the related evidence were as follows:1)DPI,a NOX inhibitor,inhibited the pro-oxidant peroperty of XN.2)Immunofluorescence results showed that XN could activate NOX.3)The XN induced ROS production decreased in the gp91^phoxknock down cells compared to the control cells.4)The XN induced ROS production decreased in the gp91^phoxhox knocked out cells compared to the wild type cells.5)Human red blood cells,without mitochondria but with NOX,exhibited ROS generation and DPI inhibition effects when treated with XN.