The Toxicity And Mechanisms Of Human Bronchial Epithelial Cells Exposed To Cigarette Smoking Condensate And Fie Particulate Matter

ObjectiveUsing cultured human bronchial epithelial cells?BEAS-2B?,to observe and compare the toxic effect of the two kinds of typical air particles of cigarette smoke condensate?cigarette smoke condensate,CSC?and fine particles(fine particulate matter,PM_2.5)on BEAS-2B cells,revealed that the related toxicity mechanism,provide scientific basis to prevent and control respiratory system diseasen induced by two typical air particles.Methods1.The preparation and treatment of two kinds of particles: CSC: a sample cigarette from Zhengzhou tobacco research institute.According to the GB/T5606.1-2004 sampling method,collecting tobacco smoke condensate and adjust the concentration of CSC to 10mg/ml as mother liquor,and stored at-80? for later use.PM_2.5: using the Laoshan Type 2030 TSP sampler collecting particles and make dry powder,adjust the concentration to 100mg/ml as fine particulate matter mother liquor,stored at-80? for later use..2.Detection the BEAS-2B cell viability induced by different concentrations of particulate matter by CCK-8 method,explicited the half inhibitory concentration of CSC and PM_2.5 and the later experiments intervention dose;using inverted microscope to observe the morphology change induced by two kinds of particles on BEAS-2B cells;Through the Annexin V-FITC/PI assay to observe the effect in apoptosis induced by two kinds of particles on BEAS-2B cells.3.The MDA,GSH,8-OHdG and EC-SOD were detected by ELISA Kit to research whether CSC and PM_2.5 induce BEAS-2B cells damage through oxidative stress mechanism.The IL-1?,TNF-?,IL-6 and IL-8 were detected by ELISA Kit to research whether CSC and PM_2.5 induce BEAS-2B cells damage through inflammatory response.4.Using RayBio? Human Apoptosis Antibody Array G1 to screening proteins with high throughput involved in apoptosis induced by CSC and PM_2.5on BEAS-2B cell,validated the discrepant proteins and their pathway proteins by RT-PCR and Western blotting methods.5.Using RayBio? Human Inflammation Antibody Array G Series III to screening inflammatory factors induced by PM_2.5 on BEAS-2B cells,and validated the differences of inflammatory factors and their pathways by ELISA method.Results1.CSC and PM_2.5 component analysis.CSC detected a total of 13 kinds of polycyclic aromatic hydrocarbons,content from large to small is as follows: ANT?18.99%?,BAa?11.57%?,BkF?8.52%?,BbF?8.29%?,CHR?8.17%?,PHE?6.97%?,BPE?6.83%?,IPY?5.13%?,PYR?3.57%?,DBA?3.53%?,FLT?3.23%?,FLU?2.13%?,BaP?0.81%?;and thedetection of 5 kinds of heavy metal compounds,the content from large to small is as follows: Zn?8.86%?,Cu?2.65%?,Cd?0.44%?,Mn?0.22%?,Hg?0.09%?.PM_2.5 detected a total of 11 kinds of polycyclic aromatic hydrocarbons,content from more to less as follows: BbF?6.50%?,BkF?5.62%?,CHR?4.71%?,Ba P?4.53%?,BPE?4.05%?,BaA?3.92%?,FLT?3.87%?,IPY?3.21%?,PYR?2.82%?,PHE?0.69%?,ANT?0.26%?;also the detection of 8 kinds of heavy metal compounds,the content from large to small is as follows: Zn?28.95%?,Pb?11.47%?,Mn?9.80%?,Cu?9.29%?,Cr?0.13%?,Ni?0.13%?,Cd?0.06%?,Hg?0.002%?.2.CSC and PM_2.5 toxicity effect on BEAS-2B cells?1?According to the CCK-8 experimental results show that the IC50 of CSC exposed to BEAS-2B cell is about 80?g/ml and then we respectively set the infected dose 0?10?20?40 ?g/ml;the IC50 of PM_2.5 exposed to BEAS-2B cell is about 165?g/ml and then we respectively set the infected dose 0?17.5?35?70 ?g/ml.?2?BEAS-2B cells were exposed to 24 h by CSC,and the morphological changes of the cells were observed.Compared with the control group,the cytoplasm became loose,the antenna became narrow,cells became thinner,and the transparency decreased.After PM_2.5 exposure,the morphological change of BEAS-2B cells was increased,the density of cells was decreased,and the number of floating cells increased.With the increase of the concentration of PM_2.5,the floating cells increased,and the granular material was observed in BEAS-2B cells.?3?BEAS-2B cells was treated by CSC for 24 h,in Annexin V-FITC /PI scatter map visible with the increase of CSC concentration,located in the lower right and upper right quadrant of the early and late stage apoptotic cellsgradually increased,the apoptosis rate of BEAS-2B cells was significantly higher than the control group;and BEAS-2B cells was treated by PM_2.5 for 24 h,in Annexin V-FITC /PI scatter map visible with the increase of PM_2.5concentration,the damage of cell membrane is located on the left upper quadrant and right upper quadrant of late apoptotic cells gradually increased,the apoptosis rate of BEAS-2B cells was significantly higher than the control group.?4?The BEAS-2B cells under the stimulation of CSC,the level of EC-SOD,MDA,8-OHdG were higher than the normal control group,GSH levels decrease with the increase of dose,the results showed that CSC can induce cytotoxicity in BEAS-2B cells through oxidative stress;BEAS-2B cells under stimulation of PM_2.5 in low dose group,EC-SOD MDA and 8-OHd G levels increased slowly,but in high dose group was significantly higher than the control group,while the level of GSH was significantly lower than the control group,the results showed that PM_2.5 can induce not obviously oxidative stress reaction on BEAS-2B cells.?5?BEAS-2B cells under the stimulation of CSC,the level of TNF-?,IL-1?,IL-6,IL-8 were higher than the control group,the results showed that CSC can induce inflammatory reaction;while BEAS-2B cells stimulated by PM_2.5,the level of TNF-?,IL-1?,IL-6,IL-8 were significantly higher than the control group,the results showed that PM_2.5 can lead to the increasing levels of inflammatory reaction,releasing inflammatory mediators,causing cell damage.3.The apoptosis mechanism in BEAS-2B cells induced by CSC and PM_2.5?1?The results of antibody array and Western blotting showed that the BEAS-2B cells under the stimulus of CSC,it induced the expression increased of TNF-R1,p21,IGF-1R,PI3 K,and AKT;Fas L expression level decreased.The western blotting experimental results show that the expression increased ofIGF-1R,PI3 K,and Akt,thus we speculated that IGF-1R/PI3K/Akt pathway may be activated and it may caused BEAS-2B cell to undergo malignant transformation.?2?Antibody array,RT-PCR and Western blotting experimental results showed that BEAS-2B cells stimulated by PM_2.5,the expression level of apoptosis related proteins TNF-R1,Fas,HTRA,BID,Caspase8 were significantly increased,the expression level of IGF-2 and TNF-R2 were significantly decreased.4.The inflammatory mechanism on BEAS-2B cells induced by PM_2.5The results showed that the expression of IL-8,IL-13,TNF-,GM-CSF,IL-2,IL-17,TGF-b1,IP-10,I-309,EOTAXIN,IL-4,IL-3,EOTAXIN-2,and the expression of inflammatory factors TIMP-2 and IL-10 were decreased in PM_2.5microarray.The use of KEGG database and the ELISA method validation results showed that the expression of inflammatory cytokines IL-4,IL-13,Eotaxin,TNF-and IL-3 increased alpha activation of asthma related pathways resulting in BEAS-2B cell inflammation caused by bronchial injury.Conclusion1.The material composition of CSC and PM_2.5 are different.The polycyclic aromatic hydrocarbons: mainly the detection of 13 kinds of polycyclic aromatic hydrocarbons in CSC,the content of the top three were: ANT,BaA and BkF,PM_2.5 was mainly detected 11 kinds of polycyclic aromatic hydrocarbons,the content of the top three were: BbF,BkF and CHR.In the heavy metal material:mainly for the detection of 5 kinds of heavy metal compounds in CSC,the content of the top three were: Zn,Cu,Cd and PM_2.5 in detection of 8 kinds of heavy metal compounds,the content of the top three were: Zn,Pb,Mn.2.The toxic effects of CSC and PM_2.5 on BEAS-2B cells were different.The IC50 of CSC was about 80?g/ml,the IC50)of PM_2.5 was about 165?g/ml,so CSC toxicity was higher than PM_2.5;CSC cause BEAS-2B cells become narrow and thin,but PM_2.5 cauce BEAS-2B cells shrinkage and deformation,the granular material can be observed in the cells,thus there were difference morphological changes induce by CSC and PM_2.5 in BEAS-2B cells;oxidative stress and inflammatory reaction were be observed in BEAS-2B induced by CSC and PM_2.5,while CSC induced oxidative stress level was significantly higher than PM_2.5,PM_2.5 is mainly cause inflammatory reaction in BEAS-2B cells.3.The apoptosis mechanism was different in BEAS-2B cells caused by CSC and PM_2.5.The expression level of TNF-R1,p21 and IGF-1R were increased in BEAS-2B cells caused by CSC,and the increasing expression of IGF-1R can promoting the expression of PI3 K and AKT protein.And PM_2.5through the increasing of TNF-R1,Fas,BID,Caspase8,HTRA to induced BEAS-2B cell apoptosis.4.PM_2.5 can increasing the expression of IL-4,IL-13,Eotaxin,TNF-alpha,IL-3 and other related inflammatory factors to activate the asthma related pathway,which eventually leads to the inflammatory reaction of BEAS-2B cells to cause bronchial injury.