Ecklonia cava Extract and Dieckol Attenuate Cellular Lipid Peroxidation in Keratinocytes Exposed to PM10

Abstract

Airborne particulate matter can cause oxidative stress, inflammation, and premature skin aging. Marine plants such as Ecklonia cava Kjellman contain high amounts of polyphenolic antioxidants. The purpose of this study was to examine the antioxidative effects of E. cava extract in cultured keratinocytes exposed to airborne particulate matter with a diameter of <10 μm (PM10). After the exposure of cultured HaCaT keratinocytes to PM10 in the absence and presence of E. cava extract and its constituents, cell viability and cellular lipid peroxidation were assessed. The effects of eckol and dieckol on cellular lipid peroxidation and cytokine expression were examined in human epidermal keratinocytes exposed to PM10. The total phenolic content of E. cava extract was the highest among the 50 marine plant extracts examined. The exposure of HaCaT cells to PM10 decreased cell viability and increased lipid peroxidation. The PM10-induced cellular lipid peroxidation was attenuated by E. cava extract and its ethyl acetate fraction. Dieckol more effectively attenuated cellular lipid peroxidation than eckol in both HaCaT cells and human epidermal keratinocytes. Dieckol and eckol attenuated the expression of inflammatory cytokines such as tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, and IL-8 in human epidermal keratinocytes stimulated with PM10. This study suggested that the polyphenolic constituents of E. cava, such as dieckol, attenuated the oxidative and inflammatory reactions in skin cells exposed to airborne particulate matter.

Figure 1

The effects of PM10 on viability of and lipid peroxidation in cultured HaCaT keratinocytes. The cells were treated with PM10 at the indicated concentrations for 48 h, followed by the determination of the cell viability (a) and cellular lipid peroxidation (b). The data are presented as a percentage of the control values (mean ± SE, n = 3). ^∗p < 0.05 versus control.

Figure 2

The effects of E. cava extract on viability of and lipid peroxidation in HaCaT keratinocytes exposed to PM10. In (a), the cells were treated with E. cava extract at the indicated concentrations for 48 h. In (b) and (c), the cells were exposed to PM10 (100 μg mL⁻¹) for 48 h in the absence and presence of E. cava extract at the indicated concentrations. The cell viability (a, b) and cellular lipid peroxidation (c) were determined. The data are presented as the percentage of the control value (mean ± SE, n = 3). ^∗p < 0.05 versus control and ^#p < 0.05 versus PM10 control.

Figure 3

The effects of various solvent fractions of E. cava extract on viability of and lipid peroxidation in HaCaT keratinocytes exposed to PM10. In (a), E. cava extract was fractionated into the methylene chloride (MC), ethyl acetate (EA), n-butyl alcohol (BA), and water (WT) fractions. In (b) and (c), the cells were exposed to PM10 (100 μg mL⁻¹) for 48 h in the absence and presence of each fraction (100 μg mL⁻¹). The cell viability (b) and cellular lipid peroxidation (c) were determined. The data are presented as the percentage of the control values (mean ± SE, n = 3). ^∗p < 0.05 versus control and ^#p < 0.05 versus PM10 control.

Figure 4

HPLC of Ecklonia cava extract and the chemical structures of eckol and dieckol. Typical HPLC patterns of E. cava extract (a) and its ethyl acetate fraction (b). The peaks of eckol and dieckol were identified by the comparison of the retention times with those of standard compounds. The chemical structures of eckol and dieckol are shown in (c) and (d).

Figure 5

The effects of eckol and dieckol on viability of and lipid peroxidation in HaCaT keratinocytes exposed to PM10. The cells were exposed to PM10 (100 μg mL⁻¹) for 48 h in the absence and presence of eckol or dieckol at different concentrations (1–10 μg mL⁻¹). The cell viability (a, b) and cellular lipid peroxidation (c, d) were determined. The data are presented as a percentage of the control values (mean ± SE, n = 3). ^#p < 0.05 versus PM10 control.

Figure 6

The effects of eckol and dieckol on viability of and lipid peroxidation in human epidermal keratinocytes exposed to PM10. The cells were exposed to PM10 (100 μg mL⁻¹) for 48 h in the absence and presence of eckol or dieckol (10 μg mL⁻¹). The cell viability (a) and cellular lipid peroxidation (b) were determined. The data are presented as the percentage of the control values (mean ± SE, n = 3). ^#p < 0.05 versus PM10 control.

Figure 7

The effects of eckol and dieckol on the expression of inflammatory cytokines in human epidermal keratinocytes exposed to PM10. The cells were exposed to PM10 (100 μg mL⁻¹) for 24 h in the absence and presence of eckol or dieckol (10 μg mL⁻¹). The mRNA levels of TNF-α (a), IL-1β (b), IL-6 (c), and IL-8 (d) were analyzed by qRT-PCR and normalized to those of GAPDH, a housekeeping gene. The data are presented as the percentage of the control value (mean ± SE, n = 3). ^#p < 0.05 versus PM10 control.

Figure 8

The effects of eckol and dieckol on the expression of inflammatory cytokines in human epidermal keratinocytes exposed to PM10. The cells were exposed to PM10 (100 μg mL⁻¹) for 48 h in the absence and presence of eckol or dieckol (10 μg mL⁻¹). The protein levels of TNF-α, IL-1β, IL-6, and IL-8 in the culture medium were analyzed by ELISA and normalized to the total protein content of the cells. Data are presented as fold changes compared to the control value (mean ± SE, n = 3). ^#p < 0.05 versus PM10 control.