Effects of fine particulate matter PM2.5 in ambient air on the proliferation, cell cycle and apoptosis of a human keratinocyte cell line HaCaT / 中华皮肤科杂志

ABSTRACT

Objective To evaluate effects of fine particulate matter PM2.5 in ambient air on the proliferation,cell cycle and apoptosis of a human keratinocyte cell line HaCaT.Methods PM2.5 in hazefog episodes during the heating season was collected in Beijing from 2015 to 2016,and processed into PM2.5 suspensions.HaCaT cells were divided into several groups to be treated with culture medium alone (control group),PM2.5 suspensions at different concentrations of 100-400 mg/L (experiment groups,50-800 mg/L for observation of cellular morphology and analysis of cell proliferation) for 24 hours,or cell culture medium without cells or PM2.5 suspensions (blank group).Cellular morphological changes were observed under an inverted microscope.Cell counting kit-8 (CCK-8) assay was performed to determine cell survival rate,flow cytometry to determine the cell cycle distribution and detect cell apoptosis,and Western blot analysis to determine the protein expression of cyclin A2 and cyclin-dependent kinase1 (CDK1).Results Along with the increase of PM2.5 concentration,HaCaT cells lost their normal shape gradually,and the number of viable cells gradually decreased.Compared with the control group (100％ ± 4.95％),the 50-mg/L PM2.5 group showed no changes in cell survival rates (P ＞ 0.05),while the 100-,200-,400-and 800-mg/L PM2.5 group showed significantly lower survival rates (91.77％ ± 2.04％,80.01％ ± 1.57％,57.80％ ± 1.56％,21.98％ ± 0.86％,respectively,all P ＜ 0.05).Flow cytometry revealed that the 100-,200-and 400-mg/L PM2.5 groups showed gradually increased proportion of cells at S phase,but gradually decreased proportion of cells at G2/M phase compared with the control group (all P ＜ 0.05).As Western blot analysis showed,the protein expression of cyclin A2 and CDK1 significantly decreased in the 100-,200-and 400-mg/L PM2.5 groups compared with the control group,which was lowest in the 200-mg/L PM2.5 group(all P ＜ 0.05).In addition,the 100-,200-and 400-mg/L PM2.5 groups showed significantly higher total apoptosis rates (9.98％ ± 0.21％,12.56％ ± 0.74％,16.74％ ± 1.48％,respectively) compared with the control group (6.24％ ± 0.17％,all P ＜ 0.05).Conclusion PM2.5 can inhibit cell proliferation and promote apoptosis of HaCaT cells,likely by downregulating the expression of cyclin A2 and CDK1 and arresting HaCaT cells at S phase.