Ozone-induced Atherosclerotic Alterations Of Co-cultured Human Endothelial Cells And Underlying Mechanisms

Background:With the rapid development of society and industrialization,air pollution has become a global public hazard problem,especially in China.At present,the control of haze pollution with particulates as the main component has achieved initial results.At the same time,as a widespread secondary pollutant,ozone has gradually developed into one of the main pollutants causing health hazards.Thus far,a large number of in vitro and in vivo studies have demonstrated that ozone exposure can directly affect human respiratory system to cause health hazards.However,documented evidence of the effects of ozone on human cardiovascular system damage is mainly from epidemiological studies,and the specific action of ozone and underlying mechanisms are still unclear.Objective:To investigate whether ozone can induce atherosclerotic changes in endothelial cells? What are the specific pathways and mechanisms responsible for this alteration?Methods:In this study,trans-well chamber was used to construct a co-culture model of epithelial cells and endothelial cells in vitro.Meanwhile,single culture of epithelial cells or endothelial cells was also performed under the same concentrations of ozone exposure for 4h.The expression levels of inflammatory cytokines IL-6,IL-8 and ET-1 were detected by ELISA.The levels of m RNA expression of IL-6,IL-8,ICAM-1,VCAM-1 and other biomarkers was detected by RT-PCR pretreatment of co-culture medium with neutralizing antibody against IL-6 or IL-8 prior to ozone exposure,the protein expression level of ET-1 was determined by ELISA,and the m RNA expression level of ICAM-1 was measured by RT-PCR.Results:1.Ozone exposure shows a direct damage to endothelial cells,which is more severe in the co-cultured endothelial cells.The LDH level of individual epithelial cells and endothelial cells increased significantly after high concentration of ozone exposure(P<0.05).LDH levels were significantly increased at all ozone concentrations in the co-culture exposure group(A549+HCAEC),with statistically significant differences(P<0.05).Under the same concentration of ozone exposure,the LDH level of the co-culture exposure group was significantly higher than that of the separately exposed epithelial cells and the separately exposed endothelial cells,with statistical difference(P<0.05).2.Ozone exposure induces inflammatory responsesThe expression level of IL-6 protein in the endothelial cells alone and the co-cultured group was significantly increased after high concentration of ozone exposure,and the difference was statistically significant(P<0.05).With high concentration of ozone exposure,the expression level of IL-6 gene in endothelial cells was significantly higher in the group exposed to isolated endothelial cells and the group exposed to co-culture than in the control group and the group exposed to medium and low concentration of ozone,and the difference was statistically significant(P<0.05).In the case of medium-high concentration of ozone exposure,the IL-6 gene expression level of the co-culture exposure group was significantly higher than that of the endothelial cells alone,and the difference was statistically significant(P<0.05).In the acute ozone exposure group,IL-8 protein expression levels were significantly increased in the single epithelial cells,the single endothelial cells and the co-culture exposure group,and the difference was statistically significant(P<0.05).In the case of acute ozone exposure,the expression level of IL-8 protein in the co-culture exposure group was higher than that in the single epithelial cells and the single endothelial cells,with statistical difference(P<0.05).The IL-8 gene expression level of single epithelial cells and single endothelial cells was significantly increased after high concentration of ozone exposure,and the difference was statistically significant(P<0.05).The expression level of IL-8 gene in endothelial cells was significantly increased in the co-culture exposure group with medium-high concentration of ozone exposure,and the difference was statistically significant(P<0.05).At the same ozone concentration exposure,the expression level of IL-8 gene in A549 cells cultured alone was higher than that in the co-culture model and the endothelial cells cultured alone,and the difference was statistically significant(P<0.05).3.Ozone exposure up-regulates the expression of adhesion molecules and endothelin-1.VCAM-1 gene expression level was significantly increased in the endothelial cells alone and in the co-culture group after high concentration of ozone exposure,and the difference was statistically significant(P<0.05).The expression level of ICAM-1 gene in endothelial cells was significantly increased with medium-high concentration of ozone exposure,and the difference was statistically significant(P<0.05).In the acute ozone exposure group,the expression level of ICAM-1 gene was significantly increased in the co-culture exposure group,and the difference was statistically significant(P<0.05).In the case of high concentration of ozone exposure,gene expression levels of VCAM-1 and ICAM-1 in the co-culture exposure group were significantly higher than those in the individual endothelial cells,with statistically significant differences(P<0.05).In the acute ozone exposure group,the expression levels of ET-1 Protein were significantly increased in both the endothelial cells alone and the co-culture exposure group,with statistically significant differences(P<0.05),and the expression levels of ET-1 Protein in the co-culture exposure group were higher than that in the endothelial cells alone,with statistically significant differences(P<0.05).4.Blockage of ozone-induced atherosclerotic alteration in co-cultured endothelial cellsPretreatment of co-culture medium with neutralizing antibodies against IL-6 or IL-8 significantly reduced the expression levels of adhesion factors and endothelin-1.This effect was more pronounced in co-cultured endothelial cells exposed to 400 ppb ozone Similarly,at the same exposure conditions the neutralizing antibody against IL-6 blocked ozone-induced atherosclerotic alterations of endothelial cells exposed to 400 ppb ozone(P<0.05).Conclusions:1.Ozone exposure induces cell damage and pro-inflammatory response of co-cultured cells.2.Ozone exposure induces up-regulation of cell adhesion molecules and endothelin-1,the biomarkers of atherosclerotic changes in endothelial cells.3.The results from the neutralization assay demonstrate that IL-6 and IL-8 play an important role in ozone-induced atherosclerotic changes in endothelial cells.