Abstract: 【Abstract】 Objective To evaluate the effect of atmospheric fine particulate matter （PM2.5） on the expression of skin barrier-associated proteins and proinflammatory cytokines in human keratinocytes. Methods Atmospheric PM2.5 samples were given by Professor Yufeng Zhou in Children′s Hospital of Fudan University. Human primary keratinocytes were isolated from circumcised foreskins of 5 males, and subjected to culture. These human primary keratinocytes were divided into several groups to be stimulated with PM2.5 at different concentrations of 0 （control group）, 10, 50, 100, 200 mg/L for 24 hours, and cell counting kit （CCK）-8 assay was performed to determine the survival rates of keratinocytes. Fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of filaggrin, keratin-14 and claudin-1 in these keratinocytes respectively, and enzyme-linked immunosorbent assay （ELISA） was performed to detect levels of interleukin （IL）-1α, thymic stromal lymphopoietin （TSLP） and IL-33 in the culture supernatant of these keratinocytes. Statistical analysis was carried out with SPSS13.0 software by using one-way analysis of variance, least significant difference （LSD）-t test and Pearson correlation analysis. Results After 24-hour treatment with different concentrations of PM2.5, there was no significant difference in the survival rate of keratinocytes between the 10-mg/L PM2.5 group and control group （P > 0.05）, while the survival rates of keratinocytes were significantly lower in the 50-, 100-, 200-mg/L PM2.5 groups than in the control group （all P < 0.05）. After the treatment with 50 mg/L PM2.5, the cell survival rate gradually decreased and then remained stable along with the increase of PM2.5 treatment duration, and the cell survival rate at 24 hours was 72.37% ± 3.12%. After 24-hour treatment with PM2.5, the mRNA expression of filaggrin and keratin-14 was significantly higher in both the 10- and 50-mg/L PM2.5 groups （filaggrin: 1.27 ± 0.15, 1.32 ± 0.09 respectively; keratin-14: 1.15 ± 0.13, 1.08 ± 0.16 respectively） than in the control group （all P < 0.05）, but lower in both the 100- and 200-mg/L PM2.5 groups （filaggrin: 0.84 ± 0.11, 0.42 ± 0.12 respectively; keratin-14: 0.67 ± 0.09, 0.74 ± 0.11 respectively） than in the control group （all P < 0.05）. The 10-, 50-, 100-and 200-mg/L PM2.5 groups showed significantly decreased mRNA expression of claudin-1 compared with the control group （all P < 0.05）. Western blot analysis showed that filaggrin expression was significantly higher in the 50- and 100-mg/L PM2.5 groups than in the control group （both P < 0.05）, while no significant difference was observed between the 10-, 200-mg/L PM2.5 groups and the control group （both P > 0.05）. The 10-, 50-, 100- and 200-mg/L PM2.5 groups showed significantly increased keratin-14 expression compared with the control group （all P < 0.05）. The claudin-1 expression did not differ between the 10-mg/L PM2.5 group and control group （P = 0.87）, but significantly higher in the 50-, 100- and 200-mg/L PM2.5 groups than in the control group （all P < 0.05）. The stimulation with PM2.5 at 10, 50, 100 and 200 mg/L could induce an increase in the supernatant levels of TSLP, IL-1α and IL-33 （all P < 0.01）. Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1α and IL-33 were positively correlated with the concentration of PM2.5 （r = 0.57, 0.67, 0.91 respectively, all P < 0.05）. Conclusion The exposure to PM2.5 can induce decreased survival rate of keratinocytes, aberrant expression of filaggrin, keratin-14 and claudin-1, and elevated secretion of the proinflammatory cytokines TSLP, IL-1α and IL-33, which may lead to impaired skin barrier function.

Key words: Keratinocytes, Particulate matter, Cell survival, Keratin-14, Claudins, Interleukin-1alpha, PM2.5, Barrier-associated proteins, Filaggrin, Interleukin-33, Thymic stromal lymphopoietin