Effects Of Long-term E-cigarette Aerosol Exposure On Lung Bronchial Epithelial Cells And Its Mechanism

Objective:To establish an animal and cell model of e-cigarette aerosol exposure,to study the long-term effects of e-cigarette aerosol exposure on mouse lung tissue and human bronchial epithelial cells and to explore its molecular mechanismMethods:(1)Establish a long-term passive e-cigarette aerosol/mist model in mice.Twenty-four male clean-grade C57BL/6J mice were randomly divided into a control group(air group),an electronic cigarette aerosol group,and a cigarette smoke group.The weight changes of mice in each group were recorded every week(2)Lung function measurement:set breathing parameters,and measure the forced vital capacity(FVC),50th millisecond breathing volume(FEV0.05),airway resistance(Rn),tissue damping(G)and tissue elasticity(H)(3)Collect mouse left lungs for HE,Masson staining,and immunohistochemistry(4)ELISA method was used to detect inflammation indicators(IL-6,TNF-α,IL-1β,IFN-y,and IL-8)in mouse serum,alveolar lavage fluid,and lung tissue homogenate(5)The xanthine oxidase method and thiobarbituric acid method were used to detect the levels of superoxide dismutase and malondialdehyde in serum,alveolar lavage fluid,and lung tissue homogenate(6)Establishment of cell poisoning model:Beas-2B cells were divided into a control group,an electronic cigarette IQOS group,and a CSE group,with three samples in each group The IQOS group was treated with complete medium containing 1%IQOS aerosol extract;the CSE group was treated with complete medium containing 1%CSE;the control group was treated with 0%IQOS aerosol extract/CSE complete medium.Total processing for March.(7)The high-throughput sequencing technology was used to detect the gene changes in the e-cigarette IQOS-treated group,CSE-treated group and control group,to screen out differentially expressed genes,and to conduct GO pathway and KEGG pathway enrichment analysis.Results:(1)General conditions and body weight changes in mice:Compared with the control group,mice in the IQOS and cigarette groups showed signs of dry hair,decreased activity,decreased appetite,and shortness of breath.There was no death in each group of mice during the experiment.The body weight of mice in the IQOS and cigarette groups were lower than those in the control group at the same time point(p<0.05).There was no significant difference in body weight between the IQOS group and the cigarette group(p>0.05).(2)Pulmonary function results:tissue damping(G),tissue elasticity(H),FEV0.05/FVC of IQOS and cigarette groups were lower than those of control group(p<0.05);Road resistance(Rn)was significantly increased compared with the control group(p<0.05).There was no significant difference in airway resistance(Rn),tissue damping(G),tissue elasticity(H),forced vital capacity,and FEV0.05/FVC in the IQOS group compared with the cigarette group(p>0.05).(3)IL-6 and TNF-a in serum,alveolar lavage fluid and lung tissue homogenate of mice in IQOS and cigarette groups were significantly higher than those in control group(p<0.05),but there was no statistical difference between IQOS and cigarette groups.Significance(p>0.05).(4)SOD in serum,alveolar lavage fluid,and lung tissue homogenate of mice in the IQOS and cigarette groups were significantly lower than those in the control group(p<0.05),and MDA were higher than those in the control group(p<0.05).There was no significant difference between the cigarette and cigarette groups(p>0.05).In addition,SOD levels in lung tissue of mice in IQOS group were higher than those in cigarette group(p<0.05);MDA levels in serum and lung of mice in IQOS group were lower than those in cigarette group(p<0.05).(5)HE staining of lung tissue:Compared with the control group,mice in the IQOS and cigarette groups showed enlarged alveolar space,local rupture of the alveolar space,alveolar cavity fusion and emphysema formation.Mean linear intercept(MLI)showed:μm in the IQOS group(41.6 ± 1.6)μm,cigarette(44.6 ± 1.8)μm,and control group(31.5 ± 1.6)μm.Compared with the control group,both the IQOS group and the cigarette group were enlarged(p<0.05),while there was no significant difference between the IQOS group and the cigarette group(p>0.05).(6)Masson staining of lung tissue:The airway walls of mice in the IQOS and cigarette groups were thicker than those in the control group(p<0.05,p<0.01),but there was no significant difference between the IQOS group and the cigarette group(p>0.05);Collagen deposition in the airway wall of mice in the IQOS and cigarette groups increased compared with the control group(p<0.01),however,there was no significant difference between the IQOS group and the cigarette group(p>0.05).(7)Immunohistochemical results:The expression of E-cadherin in bronchial epithelium of mice in the IQOS and cigarette groups decreased compared with the control group(p<0.01),but there was no significant difference between the IQOS group and the cigarette group(p>0.05);The expression of α-SMA in bronchial epithelium of mice in the IQOS and cigarette groups increased compared with the control group(p<0.05),but there was no significant difference between the IQOS group and the cigarette group(p>0.05).(8)High-throughput sequencing:Compared with the control group,there were 2,572 differentially expressed genes in the IQOS group,and the others included 1,592 up-regulated genes and 980 down-regulated genes.There were 2092 differentially expressed genes in the CSE group,including 1171 up-regulated genes and 921 down-regulated genes.In addition,1530 overlapping genes were found in the IQOS and CSE groups.The GO enrichment showed that 16 of the first 20 comments in the IQOS group were the same as the CSE group.these enriched GO terms are mainly classified as cellular components.KEGG pathway enrichment analysis.There are 13 differential pathways in the IQOS group,and 7 differential pathways in the CSE group.It mainly focuses on cell adhesion and intercellular interaction factor pathways.Conclusion:Long-term e-cigarette aerosol exposure can lead to growth restriction,reduced lung function,oxidative and antioxidant imbalance,increased inflammatory factors,airway remodeling,and EMT in mice;it can also cause changes in the transcriptome of human bronchial epithelial cells,which in turn affects epithelium Cell function.Therefore,the use of electronic cigarettes is not safe and is not a good substitute for traditional cigarettes.