The Effect Of FOXO3a On Oxidative Damage Induced By Organic Extracts Of Diesel Exhaust In 16HBE Cells

Objective: Diesel engine exhaust is a product during diesel engine working.With the extensive application of diesel engine,the relationship between diesel exhaust exposure and human health has been concerned increasingly.In recent years,it has been reported that diesel exhaust might induce oxidative damage,DNA damage,inflammatory response,cell toxicity,and other serious damage to human health.FOXO3a is an important antioxidant stress factor which plays a very important role in the process of oxidative damage.Therefore,it is important to study the effect of FOXO3a on the affections caused by organic extracts of diesel exhaust.In this study,we detected the oxidative damages of 16 HBE cells after organic extracts of diesel exhaust treatment and found FOXO3a is the key point of the cytotoxic effects induced by organic extracts of diesel exhaust.Methods:1 Collection and extraction of diesel engine exhaustThe polytetrafluoroethylene(PTFE)membrane was used to collect the diesel engine exhaust.Ultrasound radiation in a dichloromethane bath was used for the extraction of samples.The supernatants were mixed and concentrated by rotary evaporator.And then the concentrated extracts were transferred into small glass tubes to dry by nitrogen.The dry residue was dissolved in DMSO.2 Cell culture and cell model establishmentThe 16 HBE cells were cultured in Minimum essential medium(MEM)with 10% fetal bovine serum(FBS)at 37°C with a 5% CO2 saturated humidity.The 16 HBE cells in logarithmic growth phase were exposed to 0,5,10,20?g/mL organic extracts of diesel exhaust for 24 h.The cell lines of FOXO3a over expression and silence were created bytransfection of plasmid FOXO3a TM and targeted silencing siRNA FOXO3a in 16 HBE cells.The levels of FOXO3a mRNA expression were detected by RT-PCR.The 16 HBE cells with FOXO3a over expression and silence were exposed to 0 and 10?g/mL organic extracts of diesel exhaust.3 Morphology observation of 16 HBE cellsThe morphology changes of 16 HBE cells were observed by inverted microscope.4 Determination of cellular oxidative damage indexThe cells were digested and incubated with DCFH-DA fluorescent probe,and the flow cytometry was used to detect the reactive oxygen species(ROS)contents of 16 HBE cells after treatment with different doses of organic extracts of diesel exhaust.SOD and GSH-Px activities as well as MDA content were detected by commercial biochemical kits to evaluate the oxidative damages in 16 HBE cells after organic extracts of diesel exhaust exposure.5 DNA damage in 16 HBE cellsDNA damage was detected by the SCGE(comet assay)after organic extracts of diesel exhaust treatment.6 Host cell reactivation assay(HCR)The 16 HBE cells were transiently co-transfected with CdCl2(2?mol/L)damaged pGL3-Control plasmid and pRL-TK reporter plasmid.The cells after transfection were treated with organic extracts of diesel exhaust for 24 h.Protein concentration and the activities of firefly-luciferase and renilla-luciferase in the specimen were determined.The ratios of firefly-luciferase and renilla-luciferase activities were calculated to evaluate the repairabilities of 16 HBE cells.7 Western blotThe FOXO3a and pFOXO3a protein expression was detected by Western blot.Results:1 The changes of 16 HBE cells after exposed to organic extracts of dieselexhaustMorphological observation found that normal 16 HBE cells were elongated,loosead,herent compact growth,high refractive index.After organic extracts of diesel exhaust exposure,some cells shrinked into circular,the refractive index decreased,the adherent ability decreased and some of cells floated in the culture medium.This shows that OEDE has a certain effect on cell morphology.2 Identification of FOXO3a low and high expression cell linesCompared with the siRNA control group,the expression levels of FOXO3a mRNA in siRNA FOXO3a group were significantly decreased(P<0.05);compared with the pcDNA 3.1 group,the expression levels of FOXO3a mRNA in FOXO3a TM group were significantly increased(P<0.05).This shows that the FOXO3a low,high expression of the cell line is successfully established.3 The effect of OEDE on oxidative damages in 16 HBE cellsWith the increase of concentration of the organic extracts of diesel exhaust,the SOD and GSH-PX activities gradually reduced,compared with the control(P<0.05);MDA contents increased,compared with the control(P<0.05);the levels of ROS increased,compared with the control(P<0.05).This shows that OEDE can induce oxidative damage of 16 HBE cells,which makes the cells appear oxidative stress.4 DNA damage induced by organic extracts of diesel exhaustWith the increase of concentration of organic extracts of diesel exhaust,the Olive tail moment significantly increased,compared with the control(P<0.05).This shows that OEDE can induce DNA damage in cells.5 Changes in cell repair capacityWith the increase of concentration of organic extracts of diesel exhaust,the DNA repair capacities significantly decreased,compared with the control(P<0.05).This shows the ability of OEDE to inhibit the repair of DNA cells.6 FOXO3a and pFOXO3a protein expression in 16 HBE cells after exposed to organic extracts of diesel exhaustCompared with the control group,the expression levels of FOXO3a and pFOXO3a protein expression in 5?g/mL and 10?g/mL exposed groups increased significantly(P<0.05);while compared with all the other groups,the20?g/mL treatment group decreased significantly(P<0.05).The ratios of pFOXO3a/FOXO3a were increased significantly(P<0.05).This shows that OEDE affects the expression of FOXO3a and pFOXO3a.7 Effect of FOXO3a on cell oxidative damagesAfter the treatment of organic extracts of diesel exhaust,the SOD and GSH-PX activities gradually reduced,while MDA,ROS and OTM levels increased in FOXO3a knock down cells(P<0.05);the SOD and GSH-PX activities gradually increased,while MDA,ROS and OTM levels decreased significantly in FOXO3a high expressed cells(P<0.05).This shows that FOXO3a play an antioxidant role in cells.Conclusions:1 Organic extracts of diesel exhaust can induce oxidative stress and oxidative damages in 16 HBE cells.2 Organic extracts of diesel exhaust can induce DNA damages and affect the DNA repairabilities in 16 HBE cells.3 The mechanism of oxidative damages induced by OEDE might relate with the expression of FOXO3a.