The Oxidative Damage Of Atmospheric PM_2.5 On Human Alveolar Epithelial Cells A549 In Shijiazhuang

Objective:To study the main toxic effect of fine particles PM_2.5 on human alveolar epithelial,and the interactional effect between different concentration and time exposed to PM_2.5,and to provide a reliable scientific basis for control of haze and prevention of human respiratory diseases.Method:The particulate matters(PM_2.5)was collected in glass fiber membrane with EP-100 Intelligent PM_2.5 collection device;and the fiber membrane was eluted by the ultrasonic cleaning instrument;and the eluted fluid was desiccated through vacuum freeze-drying machine.The desiccated PM_2.5 were prepared into the following PM_2.5 concentration with PBS and F12 medium: 0,25,50,100,200 and 400 μ g/ml.A549 cells were cultured in different PM_2.5 concentrations respectively during a period of 6h,24 h and 48 h,and the supernatant fluid and cell lysis buffer were collected.Lactate dehydrogenase（LDH）leakage in the cell supernatant fluid was measured by trace enzyme method;the activity of catalase（CAT）by method of visible light;the malondialdehyde（MDA）production by TBA,and the activity of intracellular superoxide dismutase（SOD）by hydroxylamine method.The tables and charts for experimental data were made with Excel 2013.The data was analyzed by SPSS21.0.The one-way analysis of variance was used for analyzing the differences between the different concentrations and between different exposed times;the Dunnett-t test for analyzing differences between the control group and the various experimental groups.Results:1 The changing tendency of cytotoxic parameter for A549 cells: The cytotoxicity for A549 cells in different PM_2.5 concentrations were stronger by the increase of LDH leakage with the PM_2.5 concentration increasing,and the order from lowest to highest was 79.45、103.38、115.24、127.69、142.53 and 175.81U/gprot.The cytotoxicity for A549 cells were stronger by the increase of LDH leakage with different prolonged time,which showed 48h（167.28U/gprot）>24h（109.92U/gprot）>6h（94.86U/gprot）.The tendency of MDA production was consistent with that of LDH leakage in the different PM_2.5 concentration and time,however,the activity of CAT and SOD were decreased with the increase of the PM_2.5 concentration or the prolonged time.2 The effects of cytotoxicity in different PM_2.5 concentrations: the Dunnett-t test showed that there were significant differences for LDH、CAT、MDA and SOD between the 0μg/ml group and the other five concentration groups in the same exposed time.The variance analysis for the above 4 parameters also showed the significant differences between any two concentration groups in the same exposed time.And the above results indicated that the leakage of LDH and the production of MDA were increased along with the increase of PM_2.5 concentration,while the activity of CAT and SOD were decreased.3 The effects of cytotoxicity in different exposed times: the Dunnett-t test showed that there was the significant difference of above 4 cytotoxicity parameter between the exposed 6h and the other two time groups.There were also the significant differences between any two exposed time groups.The leakage of LDH and the production of MDA were increased along with the prolonged time.While CAT and SOD activities were decreased.4 The interactions between PM_2.5 concentration and the exposed time:Factorial analysis showed there was the interaction effect of PM_2.5 concentration and the exposed time,which meant the cytotoxicity for A549 cell was induced by the comprehensive effects of PM_2.5 concentration and the exposed time;the higher PM_2.5 concentration and the longer the exposed time was,the more severe oxidative damage was.Conclusions:1 Both of the different PM_2.5 concentrations and exposed time caused cytotoxic effects on A549 cell.With the increase of PM_2.5 concentration or the prolonged exposure time,the oxidative damage of A549 cell was more severe,and the antioxidant capacity would be weaker,which meant cytotoxicity of PM_2.5 was stronger.2 There was the interaction effects of PM_2.5 concentration and exposed time,the cytotoxic effects for A549 cells is resulted from the comprehensive effects of the PM_2.5 concentration and the exposed time,and both of them promoted the cytotoxicity of PM_2.5 for A549 cells.