Water Soluble PM2.5 Extracts Induces Apoptosis Via P53-Noxa-mediated ROS Generation In HBE Cells

Objective:The aims of this study were to detect the effects of water soluble PM2.5 extracts(WSPE) on toxicity in HBE cell line, explore the possible mechanism of cytotoxic and pathogenesis induced by Effects of water soluble PM2.5 extracts(WSPE), providing theoretical and experimental bases for the respiratory system hazard assessment of PM2.5 in Henan region. Materials and Methods:1 PM2.5 was collected by a high volume PM2.5 sampler through quartz fiber filter in the Hi-Tech Zone, Zhengzhou(September 19-25, 2013). We measured the concentration and chemical characterization of atmospheric PM2.5. WSPE was prepared for the following experiments.2 The HBE cells were Exposured with different concentrations of WSPE(0, 50, 100, 200, 400, or 800μg/mL) for 12 h, 24 h and 48 h. MTT assays were performed as a measure of cell survival rate to observe WSPE influence on HBE cells proliferation. The infected dose of the following experiments is determined by the results.3 The HBE cells were Exposured with different concentrations of WSPE(0, 50, 100, 200, 400, or 800μg/mL) for 24 h.We carry on the micro nuclear test to observe the genetic toxicity of cells; the Hochst dyeing to observe the cell apoptosis; Flow cytometry experiments to observe the oxidative stress and apoptosis.4 Using 400 mg /L WSPE as Dyeing poison, HBE cells were divided into ten groups:(1) normal control group;(2)WSPE group;(3)WSPE+NAC group;(4) NAC group;(5)PFT-α+NAC group;(6)PFT-α group;(7)WSPE+Noxa group;(8)Noxa group;(9)WSPE+NC-RNA group;(10)NC-RNA group. N acetylcysteine(NAC) is the Inhibitor of the active oxygen. PFT-α is the Inhibitor of p53. Liposomes was used to transfect Noxa-RNA and NC-RNA of SiRNA into HBE cells.5 MTT growth experiment was used to evaluate the effects of cell proliferation in different conditions on HBE cells.6 Flow cytometry assays were used to evaluate the cellular oxidatie stress and apoptosis effect of WSPE expression in different conditions on HBE cells.7 Western blot method to detect the expression of p53 and Noxa target protein, as well as the caspase-3, caspase-9, Cyt.c protein expression levels. The target protein of p53 and Noxa of expression was detected by Western blot, and the proteins expression levels of caspase-3, caspase-9 and Cyt.c.8 The obtained experimental data are express by mean plus or minus standard deviation( x ± s), analysis by SPSS 12.0. The differences between the groups were compared using analysis of variance(One Way ANOVA), pairwise comparisons using LSD assay, the detection level of α = 0.05. Results:1 The daily average concentrations of PM2.5 was 74.95-164.87μg/m3, averages 122.76±30.01μg/m3 in the Hi-Tech Zone, Zhengzhou. Carbon and water soluble ion proportion is the largest all kinds of chemical composition.2 MTT growth experiments showed that the cell survival rate was obvious inhibited exposured with different concentrations of WSPE(200, 400, or 800μg/mL) for 12 h, 24 h and 48 h, and the difference was statistically significant(P < 0.05). Detected using NAC pretreatment cell, the cell survival rate increased significantly in the infected group and the difference was statistically significant(P < 0.05).After pretreatment with PFT-α, cell survival rate increased significantly in the infected group and the difference was statistically significant(P < 0.05).3 WSPE(200 mg/L) or higher making HBE cells micronucleus rate increased significantly, resulting cell chromosome damage, the difference was statistically significant(P <0.05).4 Exposured with different concentrations of WSPE for 24 h, intracellular reactive oxygen species(ROS) content increased with the increase of exposured concentration, the difference was statistically significant(P < 0.01) compared with the blank control group. Using NAC pretreatment cells, cell DCF positive rate increased significantly(P < 0.05). After pretreatment with PFT-α, cell DCF positive rate were obviously higher in the infected group and the difference was statistically significant(P < 0.05).5 Exposured with different concentrations of WSPE for 24 h, the cell apoptosis rate increased with the increase of exposured concentration, the difference was statistically significant(P < 0.01) compared with the blank control group. Using NAC pretreatment cells, the cell apoptosis rate increased significantly(P < 0.05).After pretreatment with PFT-α, the cell apoptosis rate were obviously higher in the infected group and the difference was statistically significant(P < 0.05).6 Western blot analysis results show that with prolonged WSPE exposured p53 expressed with time dependence and downgraded Noxa. After pretreatment with NAC and PFT-α, the apoptosis related proteins of caspase-3, caspase-9 and Cyt.c change significantly. Compared with NC-siRNA group, missing Noxa directly reduced caspase 3, caspase 9 and Cyt.c expression. ConclusionsWSPE could decrease HBE cells proliferation, and result in chromosome damage. Above all, PM2.5 induced apoptotic cell death via p53-Noxa-mediated ROS generations.