Chafuroside B, an Oolong tea polyphenol, ameliorates UVB-induced DNA damage and generation of photo-immunosuppression related mediators in human keratinocytes

Abstract

Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK). Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD), in NHEK exposed to UVB (20 mJ/cm2). In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE2, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.

Figure 1. Chafuroside B attenuated DNA damage, cell damage, and apoptosis in UVB-exposed human keratinocytes.

A Chemical structure of chafuroside B. B NHEK were irradiated with UVB (20 mJ/cm²), and then treated with chafuroside B (0.3 and 1 µM). Alamar blue assay was used to evaluate cell viability at 48 h after treatment. C, D NHEK were irradiated with UVB (20 mJ/cm²), and then treated with chafuroside B (1 µM). After 6 h, apoptotic cells were detected with CellEvent Caspase-3/7 Green Detection Reagent, which produces bright green fluorescence. Nuclei were stained using Hoechst 33342, exhibiting blue fluorescence. Numbers below the panel indicate percent of caspase-3/7-active cells detected in each population (100 cells, at least, were counted in each of the plates). E NHEK were irradiated with UVB (20 mJ/cm²), and then treated with chafuroside B (1 µM). After 24 h, CPD in genomic DNA was detected by an immunofluorescence method using FITC-labeled CPD specific antibodies. Cha B  =  Chafuroside B. All data are expressed as the mean ± sd (n = 3 or 4). *P<0.05, **P<0.01 and ***P<0.001 compared with UVB (+).

Figure 2. Chafuroside B decreased the UVB-induced production of TNF-α, PGE₂, and IL-10 in human keratinocytes.

NHEK were irradiated with UVB (20 mJ/cm²), and then treated with chafuroside B (0.3 and 1 µM). After 48 h, supernatants were collected and the levels of TNF-α, PGE₂, and IL-10 were evaluated by ELISA. (A) TNF-α. (B) PGE₂. (C) IL-10. Cha B  =  Chafuroside B. All data are expressed as the mean ± sd (n = 4). *P<0.05, **P<0.01 and ***P<0.001 compared with UVB (+).

Figure 3. Chafuroside B enhanced IL-12 mRNA expression and production in human keratinocytes.

A NHEK were treated with chafuroside B (0.3 and 1 µM). The cells were harvested after 3 h and IL-12 mRNA was quantitated by means of RT-PCR. B NHEK were treated with chafuroside B (0.3 and 1 µM). After 72 h, supernatants were collected and IL-12 was evaluated by means of ELISA. Cha B  =  Chafuroside B. All data are expressed as the mean ± sd (n = 3 or 4). *P<0.05 compared with the control.

Figure 4. Chafuroside B decreased the mRNA expression of RANKL induced by UVB radiation in human keratinocytes.

NHEK were irradiated with UVB (20 mJ/cm²), and then treated with chafuroside B (0.3 and 1 µM). The cells were harvested after 24 h and RANKL mRNA was evaluated by means of RT-PCR. Cha B  =  Chafuroside B. All data are expressed as the mean ± sd (n = 3 or 4). *P<0.05 compared with UVB (+).