Amentoflavone-Enriched Selaginella rossii Protects against Ultraviolet- and Oxidative Stress-Induced Aging in Skin Cells

Abstract

Selaginellaceae plants are used in cosmetics to limit skin aging. This study is the first to investigate the anti-aging effects of Selaginella rossii (SR) on ultraviolet B (UVB)- and oxidative stress-induced skin cells. The 95% ethanol extract of Selaginella rossii (SR95E) contained much higher amounts of amentoflavone (AMF), an active compound, than other Selaginellaceae plants and was more effective in inhibiting matrix metalloproteinase (MMP)-1 expression in CCD-986sk fibroblasts. SR95E significantly decreased UVB-induced MMP-1, MMP-2, MMP-3 and MMP-9 expression and enhanced procollagen type I C-peptide content and mRNA expression of collagen type I alpha (COL1A)1 and COL1A2 in CCD-986sk fibroblasts. In HaCaT keratinocytes, SR95E treatment also dose-dependently decreased UVB-induced MMP-1 concentration and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA expression. Moreover, SR95E treatment markedly inhibited UVB-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling and nuclear factor kappa-B signaling in HaCaT cells. Furthermore, SR95E and AMF markedly regulated the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced expression of cellular senescence-related markers, including p16, p21 and LMNB1, in HaCaT cells. Overall, this study indicates that SR may have potential as a functional material on preventing UVB- and AAPH-induced skin aging and wrinkles.

Keywords: Selaginella rossii; amentoflavone; anti-wrinkle; matrix metalloproteinases; skin aging.

Figure 1

Cell viability and MMP-1 expression of Selaginellaceae extracts in CCD-986sk cells. (A) Cells were treated with each extract (50 μg/mL) for 24 h with or without UVB-irradiation. Cell viability was measured by CCK kit (Donginls, Daejeon, Republic of Korea). (B) CCD-986sk cells were treated with the extracts of Selaginellaceae (50 μg/mL) and UVB-irradiation. After 24 h, the mRNA levels were measured by real-time qRT-PCR and normalized using ACTIN as a reference gene. Values are presented as means ± SD. ^# p < 0.01 vs. cells treated with media only; * p < 0.05, ** p < 0.01 vs. cells treated with UV only. SR95E, Selagenella rossii 95% ethanol extract; SRM, Selagenella rossii methanol extract; STM, Selaginella tamariscina methanol extract; SIM, Selaginella involvens methanol extract; Ex, extract; SD, standard deviations.

Figure 2

Effect of the SR extracts on matrix metalloproteinase (MMP)-1 concentration and MMPs expression in UVB-irradiated CCD-986sk fibroblasts. Cells were treated with SRE extracts and irradiated to UVB for 24 h. (A) MMP-1 concentrations were measured from cell culture medium. (B–E) The mRNA expressions of MMP-1, MMP-2, MMP-3 and MMP-9 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. UVB − indicates treatment without UVB-irradiation and UVB + indicates treatment with UVB-irradiation. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only. SR70E, Selagenella rossii 70% ethanol extract.

Figure 3

Effect of SR extracts on procollagen type I C-peptide (PIP) concentration and mRNA expression in UVB-induced CCD-986sk cells. Cells were treated with SR70E or SR95E and irradiated to UVB for 24 h. (A) PIP concentrations were measured from cell culture medium. (B,C) The mRNA expression of COL1A1 and COL1A2 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only.

Figure 4

Effect of SR on MMPs expression in UVB-induced HaCaT keratinocytes. (A) Cells were treated with SR95E (10, 20 and 50 μg/mL) for 24 h with or without UVB-irradiation. For detection of MMPs expression, cells were treated with SR95E extracts and irradiated to UVB for 16 h. (B) MMP-1 concentrations were measured from cell culture medium. (C–F) The mRNA expression of MMP-1, MMP-2, MMP-3 and MMP-9 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only.

Figure 4

Effect of SR on MMPs expression in UVB-induced HaCaT keratinocytes. (A) Cells were treated with SR95E (10, 20 and 50 μg/mL) for 24 h with or without UVB-irradiation. For detection of MMPs expression, cells were treated with SR95E extracts and irradiated to UVB for 16 h. (B) MMP-1 concentrations were measured from cell culture medium. (C–F) The mRNA expression of MMP-1, MMP-2, MMP-3 and MMP-9 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only.

Figure 5

Effect of SRE on reactive oxygen species (ROS) accumulation and mitogen-activated protein kinase (MAPK)/NF-κB signaling in UVB-induced HaCaT keratinocytes. (A) Cells were treated with SRE and irradiated to UVB. After 24 h, ROS accumulation was determined using 2′,7′-dichloro-fluorescin diacetate. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only. (B) The total and phosphorylation protein levels of ERK, JNK, p38, and NF-κB p65 were detected by Western blot.

Figure 6

Effect of amentoflavone (AMF) on MMPs expression in UVB-induced HaCaT keratinocytes. (A) Cells were treated with AMF (2, 5 and 10 μM) for 24 h with or without UVB-irradiation. For detection of MMPs expression, cells were treated with AMF (1, 2 and 5 µM) and irradiated to UVB for 16 h. (B) MMP-1 concentrations were measured from cell culture medium. (C–F) The mRNA expression of MMP-1, MMP-2, MMP-3 and MMP-9 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only.

Figure 7

Effect of AMF on reactive oxygen species (ROS) accumulation and mitogen-activated protein kinase (MAPK)/NF-κB signaling in UVB-induced HaCaT keratinocytes. (A) Cells were treated with AMF and irradiated to UVB. After 24 h, ROS accumulation was determined using 2′,7′-dichloro-fluorescin diacetate. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not irradiated; * p < 0.05, ** p < 0.01 vs. cells with UVB-irradiation only. (B) The total and phosphorylation protein levels of ERK, JNK, p38 and NF-κB p65 were detected by Western blot.

Figure 8

Effect of SR and AMF in AAPH-induced senescent HaCaT keratinocytes. (A) Cells were treated with SR95E or AMF for 24 h with 2 mM AAPH. For detection of MMPs expression, cells were treated with SRE or AMF and irradiated to UVB for 24 h. (B) Cells were treated with SRE or AMF and induced with AAPH. After 24 h, ROS accumulation was determined using 2′,7′-dichloro-fluorescin diacetate. (C–E) The mRNA expression of p65, p21 and LMNB1 were measured by real-time RT-PCR and normalized against those of ACTIN as reference gene. Data was presented as means ± SD (n = 3). ^# p < 0.01 vs. cells not induced; * p < 0.05, ** p < 0.01 vs. cells with AAPH-induced only.