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.
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-1αand IL-33(all P<0.01). Pearson correlation analysis showed that the supernatant levels of the proinflammatory cytokines TSLP, IL-1αand 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-1αand IL-33, which may lead to impaired skin barrier function.