Protective Effect Of Antioxidants On Alveolar Macrophage Injury Induced By Uranium Dust

ObjectiveIn order to reduce the lung injury induced by uranium dust and provideexperimental basis for protective agent, the objective is to explore the macrophagesfunction and the protection of four kinds of antioxidants (NAC, SOD, CAT andmannitol) on macrophages injury induced by uranium dust.MethodsThe experiment was divided into the control group (0μg/cm2) and experimentalgroups (25,50,100,200,400μg/cm2uranium dust). Cell viability, apoptosis andapoptosis morphology of THP-1macrophages were examined by MTT method tests,Annexin V/PI cell apoptosis assay and Hoechst33258fluorescence labeling method.Reactive oxygen species (ROS) levels of THP-1macrophages stained with2,7-dichloride fluorescent yellow double acetate (DCFH-DA) were examined byfluorescence microscopy and chemical/fluorescent light meter. The intracellularglutathione (GSH), Ca2+levels and mitochondrial membrane potential were observedby chemical/fluorescent light meter after respectively stained with CMFDA,FLUO-8AM and JC-10. FITC labeled Escherichia coli incubation with THP-1macrophages which exposed to different concentration of uranium ore dust for2hours, then used the Chemical/fluorescence luminous fluorescence detection to assaythe phagocytosis function of THP-1macrophages. The changing of cytokines (IL-1β,GM-CSF, TNF-α and IL-8, etc.) were detected by Liquid suspension chip system.After advanced incubation by antioxidants (NAC, SOD, CAT, mannitol)，detected the results of apoptosis，related indicators of apoptosis，phagocytosis andcytokines which induced by uranium dust. Comparatively study on the protection ofdifferent antioxidants on macrophage function mediated by uranium dust. ResultsThe survival rate of macrophages was significantly reduced in a concentration-and time-dependent manner after exposed to uranium dust. Intracellular ROS levelsand apoptosis rate were changed correspondly, GSH depletion was accelerated by therising levels of ROS. The membrane potential of mitochondrial was reduced and theintracellular Ca2+levels were increased at the same time. The phagocytosis functionwas gradually inhibited with the increasing concentration of uranium dust. Lowdoses of uranium dust increase the expression of cytokines such as IL-1β, GM-CSF,TNF-α and IL-8, accelerates the response of inflammatory.After incubated by four kinds of antioxidants (SOD, CAT, mannitol, NAC), thesurvival rate, apoptosis rate and the oxidative damage index (ROS and GSH levels)of THP-1macrophages are increased significantly. The effects of NAC and mannitolgroup were most obvious among them. The decreased trend of macrophagephagocytosis and the increased trend of cell factor can be obviously inhibited afterincubated by NAC. The production of intracellular ROS (H2O2and O2-·,· OH, etc.)was positively correlated with macrophages’ function injury cause by uranium dust,·OH is probably one of the main active oxygen free radicals during the process of cellinjury, which needs further research about its specific mechanism.Conclusion1. Uranium dust can induce apoptosis of THP-1macrophage, reduce the cellsurvival rate. Early cell damage caused by uranium dust is mainly displays inapoptosis.2. The phagocytosis function of THP-1macrophage can be inhibited gradually,with the relative rising intracellular ROS and reducing GSH levels. The low doses ofuranium dust can also increase the amount of cytokines such as IL-1β, GM-CSF,TNF-α and IL-8etc.3. Four kinds of antioxidants (NAC, CAT, SOD and mannitol) can protectmacrophage oxidative damage caused by uranium dust, the protective effects ofNAC is most obvious. NAC can also increase the phagocytosis of macrophage after stimulated by uranium dust, along with the decreasing ROS levels and increasingGSH levels, the secretion of cytokines such as IL-8, TNF-α, GM-CSF etc. haveincreased.