Protection Effects Of Arbutin On Radiation-induced Apoptosis In Human Lymphoma U937 Cells And Its Mechanisms

Background: When cells are exposed to ionizing radiation,various reactive oxygen species(ROS)are produced.Excessive ROS has the potential to damage cellular macromolecules and that may lead to cell death.When the body is exposed to a certain dose of ionizing radiation for a long period of time,chronic radiation injury such as hematopoietic dysfunction can be caused.Hydroxyl radical,which play a pivotal role in cell deaths,is considered to be the most reactive and harmful of the so-called ROS.In this study,we screened eighty compounds for their intracellular hydroxyl radical scavenging ability in X-irradiated human lymphoma U937 cells and selected one compound,named arbutin for further detail analysis.Therefore,hydroxyl radical scavengers might be an effective radio-protection agent.Arbutin(4-hydroquinone-?-D-glucopyranoside),a naturally occurring glucoside of hydroquinone.It has been traditionally used to treat pigmentary disorders.However,there are no reports describing the effect of arbutin on IR-induced apoptosis.Objective: The aimfcd of our presg hent sthgudy hgwas to investigate the radioprotective effect of arbutin on X-radiation induced damage and to study if the radioprotective effect of arbutin is related to its cytoprotection and apoptosis suppression.And to explore its possible signaling pathways and molecular mechanism further.Methods: Divided U937 cells into four groups: the control group,the arbutin group(50?mol/L ? 100?mol/L ? 200?mol/L and 500?mol/L respsctively),the X-irradiation group(10Gy)and the combined group(arbutin+X-irradiation).The morphological changes in the U937 cells were examined by using Giemsa staining.After annexin V/ PI staining,the apoptosis detection of U937 cells were analysed by flow cytometry.Quantitative DNA fragmentation of U937 cells assay was carried out by spectrophotometer.Survival detection of U937 cells was performed by a Cell Counting Kit-8(CCK-8).The intracellular ROS generation was detected using HPF staining.Mitochondrial membrane potential of U937 cells was detected using the fluorescence of Tetramethylrhodamine methylester(TMRM).The apoptosis-related protein expressions of U937 cells after radiation were detected by western blot analysis.To measure caspase-8 activity,we used the FLICE/Caspase-8 Colorimetric Protease Assay Kit.FITC-labeled anti-Fas monoclonal antibody and then analyzed by flow cytometry.Statistical analysis: Data are expressed as the means ± SD.Statistical analysis was carried out using the Student's t-test.P-values < 0.05 were regarded as significant.All the experiments were performed in triplicate.Results: We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation in a concentration-dependent manner.The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss.It also could down-regulate the expression of phospho-JNK,phospho-p38 in whole cell lysate and activate Bax in mitochondria.Typical morphological changes associated with apoptosis were significantly less prominent.Arbutin also inhibits cytochrome C release from mitochondria to cytosol and can reduce the expression of caspase-3.To verify the role of JNK in X-irradiation-induced apoptosis,the cells were pretreated with a JNK inhibitor for 1 h,The results revealed that percentage of early apoptotic cells of the X-irradiation group,the X-irradiation group+the arbutin group,JNK inhibitor+the X-irradiation group and JNK inhibitor+the arbutin group were 17.30±3.47%,12.90±1.57%,12.67±1.78% and 9.30±1.31% respectively.Compared the combined group with the X-irradiation group of each group,there were significant differences between the two groups(P<0.05).The positive rate of Fas in the control group,the arbutin group,the X-irradiation group and the combined group were 3.15±0.07%,4.2±0.72%,8.91±1.0% and 7.07±1.18% respectively.Compared the combined group with the X-irradiation group,there were significant differences between the two groups(P<0.05).The activity of caspase-8 in the control group,the arbutin group,the X-irradiation group and the combined group were 0.05±0.01%,0.07±0.01%,0.26±0.02% and 0.17±0.13% respectively.Compared the combined group with the X-irradiation group,there were significant differences between the two groups(P<0.05).Conclusions: Arbutin exhibited no significant cytotoxicities on U937 cells,and could improve the viability of these cells after radiation.Arbutin might be the potential radio-protector.Arbutin exerted its protective effect on the proliferative activity of U937 cells as evidenced by decreased cytotoxicity after exposure to X-irradiation.It was possible that Arbutin achieved its radioprotective action,at least in part,by reducing DNA damage and scavenging ROS.Arbutin reduced the intracellular hydroxyl radical generation in irradiated U937 cells.Arbutin might be an effective intracellular hydroxyl radical scavenger.Arbutin exhibited its radioprotective activity via inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.