The Radioprotective Mechanism Of Gen Against X-rays Induced Oxidative Damage In HUVEC Cells

In recent years, with the widespread use of nuclear power andnuclear technology, opportunities that people exposured toradiation are increasing, more attentions are paid to the harm ofradiation damage induced by radiation. One mechanism of radiationdamage is that excess free radicals are produced after ionizingradiation, it can induce oxidative stress when scavenging andproducing free radicals are out of banlance, which could cause theinjury of cell membranes、DNA and proteins and other biologicalmacromolecules, so that morphology and function of the cells、tussues and the organs would change, causing damage to the body,eventhe cancer.Genistein is flavonoid compound,and widely exist in vegetables、fruits and tea,with a variety of biological activity,theantioxidant activity of genistein has been proved.Studies haveshown that genistein also has anti-radiation effect due to thestructure of phenolic hydroxyl.Genistein protects cells fromoxidative damage by quenching free radicals, inhibiting lipidperoxidation and enhancing the activity of antioxidant enzymesystem.Vascular endothelial cells are more sensitive to ionizingradiation, and one of the important targets after radiation damage,the dysfunction of VECs are a great significance in hypertension, heart failure,atherosclerosis(AS) and other cardiovasculardiseases. Based on the present studies on antioxidant of genistein,in this research, we select HUVEC cells as the model cells ofradiation damage in the cardiovascular system. Focus on the damagemechanism of free radicals, investigate the radiation damage effectof single dose of X-rays, and explore the protective effect ofgenistein and the possible mechanisms to provide experimentalevidences for radiation damage research in cardiovascular system.The details are as follows:(一)X-rays induced HUVEC cell injury modelLow doses of ionizing radiation generated excitatory effect,the stimulation on the immune system can enhance the body immunity,and beget the adaptability of high-dose radiation after exposureto small pre-dose radiation. Larger doses X rays are easy to makea large number of cells death, reduce the immune system and inducetumors. Therefore, this part of the experiment took human umbilicalvein endothelial cells (HUVEC) as experimental subjects,established a cell model of radiation damage, and observed thesurvival rate of HUVEC cells after different doses of X-rayirradiation at different time points, in order to select theappropriate experimental irradiation dose.HUVEC cells were irradiated with75mGy、100mGy、200mGy、400mGy of middle-low dose X-rays,with1.2Gy、2.4Gy、3.6Gy、4.8Gy、6.0Gy of middle-high dose X-rays, respectively, after irradiationgo on culturing for24h、48h、72h respeetively, detected the cellviability by MTT assay. From the experiment results, campared withthe control group, the viability of HUVEC cells were all above100% in middle-low dose X-ray groups at24h.48h after Irradiation, theviability of400、1200mGy groups were decreased(<100%,p<0.05).72hafter Irradiation, the viability of low-dose groups were all below100%, and with the addition of radiation doses, the viability weredecreased.The viability of HUVEC cells of high doses groups wereall below100%after24h,while the injury severity of3.6Gy doseX-ray had statistical difference(p<0.05).Irradiation after48h and72h, the viability of high doses X-ray groups were decreasing whenthe radiation doses were increasing, and the injury severitybetween groups were statistically significant(p<0.05), and while3.6Gy,4.8Gy6.0Gy groups were significantly defferent(p<0.01).Theresults suggested that middle-high dose irradiation could induceoxidative damage in HUVEC cells, there were a certain amount ofactivity relationship between the degree of damage and theradiation doses while the incubation time.(二)Protective Effect of Genistein on High Dose X-radiation InducedOxidative Stress Damage in HUVECsHUVECs cells were incubated with the concentrations of0、5、20、50μmol/L respectively for2h before being irradiated to3.6Gy、6.0Gy X-rays respectively. Detected the levels of superoxidedismutase(SOD)、 reactive oxygen species(ROS) and glutathioneperoxidase(GSH-Px) after24h and48h respectively. From theexperiment results, compared with control group, the level of ROSwere apparently increased(p<0.01), SOD and GSH-Px weresignificantly decreased(p<0.01) in HUVECs after irradiation, thestatistical difference was much obvious when the radiation doseswere increasing; Compared with pure irradiated group, we did observe a significant decrease in the level of ROS, while increaseof SOD(p<0.01) in HUVECs treated with genistein, the protectiveeffects and concentrations were positively correlated. Theprotective effect of genistein to3.6Gy X-ray irradiation wassuperior to6.0Gy X-ray irradiation. But there was no obvious changeon the level of GSH-Px in HUVECs treated with genistein after24h and48h (p>0.05). The results suggested that genistein couldameliorates X-radiation induced oxidative damage in HUVECs, and ata certain range of concentration, the radioprotective efficacy werewell correlated.(三)The mechanism of Genistein against high-dose X-rays radiationinduced damge in HUVECsThe signal transduction of cell damage response mechanisminvolved with cycle arrest、apoptosis or DNA repair depends on thephosphatidylinositol3-kinase(PI3K) related protein kinase family,which plays a regulatory role on cell cycle、DNA repair and apoptosis;AKT is the direct downstream target of PI3K, the formation ofPI3K/AKT pathway is an important intracellular surivival signalingpathway, it can mediate the transcription of factors and cytokinesdirectly or indirectly, regulate cell proliferation、differentation、apoptosis and tumor angiogenesis. This part of theexperiment will detect the cell cycle changes in irradiated HUVECcells and the possible mechanism of PI3K/AKT signaling pathway inthe process in gene and protein level.HUVECs cells were incubated with different concentrations(0、5、20、50μmol/L) for2h before being irradiated to3.6Gy、6.0Gy X-rays respectively. Collected cells at the24h、48h and detected the cell cycle by flow cytometry;collected cells of3.6Gy dose groupat48h to do PCR and Western Blot, detected the expression of PI3K、AKT、CyclinB mRNA and protein. From the experiment results, camparedwith control group,24h and48h after irradiated with3.6Gy、6.0Gydoses X-ray, there wre abvious G2/M phase arrest in HUVEC cells.After treated with genistein, the G2phase arrest had eased bothat24h and48h, showing that the cell number reduced in G2phase,increased in S、G1phase,the differences were that the optimalconcentration of genistein to relieve G2/M phase arrest was20μmol/L at24h, while5μmol/L at48h.The PCR results showed that, compared with the normal controlgroup cells,48h after irradiated with3.6Gy doses X-ray,expression of PI3K mRNA was increased slightly, the expression ofAKT mRNA was significantly increased (p <0.01), cyclin B (cyclinB1)mRNA expression increased slightly; after treated with genistein,compared with the model group (M),the expression of PI3K mRNA wasincreased, but no statistically differences (p>0.05), theexpression of AKT mRNA was decreased, there were statisticallydifferences among the concentration groups (p <0.05), theexpression of CyclinB1mRNA was increased, but no differences (p>0.05).The Western Blot results indicated that, campared with controlgroup,48h after irradiated with3.6Gy doses X-ray, expression ofPI3K protein was reduced(p<0.05)，the expression of AKT proteindecreased(p<0.05), p-AKT was extraordinarily increased(p<0.01),the expression of cyclinB was abnormally elevated. After treatedwith genistein, the expression of PI3K protein added compared withmodle group, but there were no statistical differences among concentration groups(p>0.05);the expression of AKT protein reducedcompared with modle group(p<0.05); p-AKT protein added comparedwith modle group(p<0.05); and increasing expression in cyclinBprotein. The results suggested that genistein could effectivelyinhibit the radiation damage-induced by high-dose X-ray in HUVECcells, and could relieve G2/M phase arrest induced by ionizingradiation, the mechanism may be associated with the activation ofthe AKT signaling pathway.