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

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.


Introduction
The older population suffers from a variety of skin disorders such as eczema, actinic keratosis, and infectious diseases [1]. Most people over 65 years have a minimum of two skin disorders that require clinical attention [2]. Skin disorders in older individuals progress with aging of skin. There are two types of skin aging: intrinsic and extrinsic. Intrinsic aging affects all skin areas and is mainly visible in photo-protected areas [3], while intrinsic aging is characterized by thin, dry, fine wrinkles, and irregular hair growth. On the other hand, extrinsic aging affects exposed skin areas due to environmental factors such as solar radiation, smoking, and pollution. Extrinsic aging, especially photoaging, is characterized by deep and thick wrinkles, loss of elasticity, dryness, laxity, and actinic lentigines [4].
In skin aging, several factors, including ultraviolet (UV) irradiation, pollution, reactive oxygen species (ROS), telomere attrition, matrix metalloproteinase (MMP) up-regulation, and decreased collagen and elastin, alter the cellular condition of the skin, resulting in the formation of wrinkles [5]. Skin aging of the face is mainly caused by UV irradiation [6]. CCD-986sk fibroblasts or HaCaT cells were plated in 96-well plates and after adhesion to plates treated with extracts or AMF for 24 h. UVB-irradiation was conducted using a UVB lamp (Sankyo-Danki, Hiratsuka, Japan) which emits wavelengths between 280 nm to 360 nm and peaked at 305-310 nm. Cell viability was detected using the D-Plus™ CCK kit (Donginls, Daejeon, Republic of Korea).

Measurement of MMP-1 and Procollagen Secretion Levels
MMP-1 and procollagen secretion levels were measured as previously reported [24]. CCD-986sk or HaCaT cells were irradiated with UVB and treated with SRE or AMF for 24 h. MMP-1 levels in CCD-986sk or HaCaT cells were measured using the MMP-1 ELISA kit (Abcam, Cambridge, MA, USA). Concentration of PIP in CCD-986sk cells was detected by procollagen type I C-peptide (PIP) EIA kit (Takara Bio Inc., Shiga, Japan).

Quantitative Real-Time RT-PCR
SRE or AMF treated cells were prepared for total RNA using TRI solution (Ambion, Carlsbad, CA, USA). Total RNA was used to synthesize cDNA using a High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For detection of mRNA expression level, a quantitative Real-Time PCR system (Life Technologies, Grand Island, NY, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems, Woolston, Warrington, UK) were used to conduct Real-Time RT-PCR. The used primers are shown in Table S1.

Statistical Analysis
Values of results were given as the means ± standard deviations (SD) and the significant differences between the control and treatment groups were analyzed using Student's t-tests. A p-value < 0.05 was considered significant.

Selaginellaceae Inhibited MMP-1 Expression in CCD-986sk Fibroblasts
The potential cytotoxicity of Selaginellaceae plants, including ST, SI and SR, were evaluated in CCD-986sk fibroblasts. After treatment with 50 µg/mL of each extract, none were toxic to CCD-986sk fibroblasts under normal conditions without UVB irradiation ( Figure 1A). Under UVB irradiation, the viability of the fibroblasts decreased significantly to 82.9%. However, treatment with SR95E or SRM, but not the STM or SIM, markedly restored UVB-induced cell cytotoxicity ( Figure 1A). SR extracts exhibited more potent activity on MMP-1 inhibition than the other Selaginella species ( Figure 1B). These results correlate with the AMF contents of the Selaginellaceae extracts [14]. Hence, this study focused on the anti-wrinkle effects of SR against UVB-exposed fibroblasts and keratinocytes.

Statistical Analysis
Values of results were given as the means ± standard deviations (SD) and the significant differences between the control and treatment groups were analyzed using Student's t-tests. A p-value < 0.05 was considered significant.

Selaginellaceae Inhibited MMP-1 Expression in CCD-986sk Fibroblasts
The potential cytotoxicity of Selaginellaceae plants, including ST, SI and SR, were evaluated in CCD-986sk fibroblasts. After treatment with 50 μg/mL of each extract, none were toxic to CCD-986sk fibroblasts under normal conditions without UVB irradiation ( Figure 1A). Under UVB irradiation, the viability of the fibroblasts decreased significantly to 82.9%. However, treatment with SR95E or SRM, but not the STM or SIM, markedly restored UVB-induced cell cytotoxicity ( Figure 1A). SR extracts exhibited more potent activity on MMP-1 inhibition than the other Selaginella species ( Figure 1B). These results correlate with the AMF contents of the Selaginellaceae extracts [14]. Hence, this study focused on the anti-wrinkle effects of SR against UVB-exposed fibroblasts and keratinocytes.

Phytochemical Components of SR Extracts
The phytochmical and HPLC profiles analyses showed that SR95E and SR70E illustrated the presence of alkaloids, flavonoids, glycosides, phenolics and pheophorbides (products of chlorophyll breakdown). Among the 11 species of Selaginella, biflavonoid content was high in S. sinensis, S. davidii and S. mollendorfii (ranging from 1.0 to 1.1%) [25]. Compared with other Selaginellaceae species such as ST and SI, SR95E and SR70E contained markedly higher AMF [14]. The AMF contents of SR95E and SR70E were 66.6 mg AMF/g extract and 46.8 mg AMF/g extract, respectively, as quantified by HPLC analysis ( Figure S1). The analytical HPLC profile showed that SR95E contained a large number of its natural derivatives and different flavonoid glycosides in addition to AMF ( Figure S2) [25].

Phytochemical Components of SR Extracts
The phytochmical and HPLC profiles analyses showed that SR95E and SR70E illustrated the presence of alkaloids, flavonoids, glycosides, phenolics and pheophorbides (products of chlorophyll breakdown). Among the 11 species of Selaginella, biflavonoid content was high in S. sinensis, S. davidii and S. mollendorfii (ranging from 1.0 to 1.1%) [25]. Compared with other Selaginellaceae species such as ST and SI, SR95E and SR70E contained markedly higher AMF [14]. The AMF contents of SR95E and SR70E were 66.6 mg AMF/g extract and 46.8 mg AMF/g extract, respectively, as quantified by HPLC analysis ( Figure S1). The analytical HPLC profile showed that SR95E contained a large number of its natural derivatives and different flavonoid glycosides in addition to AMF ( Figure S2) [25].

SR Inhibited MMP-1 Secretion and MMP Expression in CCD-986sk Fibroblasts
Next, MMP-1 concentration and MMP expression levels were measured to determine the anti-wrinkle effects of SR95E and SR70E on UVB-irradiated CCD-986sk fibroblasts. Treatment with 50 µM SR70E or SR95E significantly reduced UVB-induced MMP-1 secretion by 57.1% and 84.4%, respectively ( Figure 2A). In addition, the UVB-induced mRNA expression of MMP-1, MMP-2, MMP-3 and MMP-9 was significantly reduced by treatment with SR70E and SR95E ( Figure 2B-E). SR95E exhibited much higher AMF content than SR70E, as quantified by HPLC analysis. These results confirmed that the inhibitory effects of MMPs expression depended on the AMF contents of the SR extracts. Thus, SRE can effectively inhibit skin wrinkle formation by inhibiting UVB-induced MMP expression in CCD-986sk cells.
Next, MMP-1 concentration and MMP expression levels were measured to determine the anti-wrinkle effects of SR95E and SR70E on UVB-irradiated CCD-986sk fibroblasts. Treatment with 50 μM SR70E or SR95E significantly reduced UVB-induced MMP-1 secretion by 57.1% and 84.4%, respectively ( Figure 2A). In addition, the UVB-induced mRNA expression of MMP-1, MMP-2, MMP-3 and MMP-9 was significantly reduced by treatment with SR70E and SR95E ( Figure 2B-E). SR95E exhibited much higher AMF content than SR70E, as quantified by HPLC analysis. These results confirmed that the inhibitory effects of MMPs expression depended on the AMF contents of the SR extracts. Thus, SRE can effectively inhibit skin wrinkle formation by inhibiting UVB-induced MMP expression in CCD-986sk cells.

SR Enhanced Procollagen Expression in CCD-986sk Fibroblasts
Skin fibroblast-synthesized procollagen is a major component in the formation of collagen fibrils in the intracellular matrix. The PIP production in UVB-irradiated CCD-986sk cells was detected to determine whether SR extract treatment could regulate procollagen synthesis. UVB irradiation significantly decreased PIP levels. However, treatment with 50 μM SR70E or SR95E significantly increased PIP concentrations compared to UVB-irradiated CCD-986sk cells ( Figure 3A). Further, the mRNA expression levels of collagen type I alpha (COL1A)1 and COL1A2 genes related to procollagen synthesis were significantly increased by SR70E or SR95E treatment ( Figure 3B,C). In addition, SR95E was more effective in promoting procollagen synthesis than SR70E. These

SR Enhanced Procollagen Expression in CCD-986sk Fibroblasts
Skin fibroblast-synthesized procollagen is a major component in the formation of collagen fibrils in the intracellular matrix. The PIP production in UVB-irradiated CCD-986sk cells was detected to determine whether SR extract treatment could regulate procollagen synthesis. UVB irradiation significantly decreased PIP levels. However, treatment with 50 µM SR70E or SR95E significantly increased PIP concentrations compared to UVBirradiated CCD-986sk cells ( Figure 3A). Further, the mRNA expression levels of collagen type I alpha (COL1A)1 and COL1A2 genes related to procollagen synthesis were significantly increased by SR70E or SR95E treatment ( Figure 3B,C). In addition, SR95E was more effective in promoting procollagen synthesis than SR70E. These results suggest that SR extract may exert anti-wrinkle effects by enhancing procollagen synthesis in skin fibroblasts.

SR Inhibited MMP-1 Secretion and MMP Expression in HaCaT Keratinocytes
Under UV irradiation, epidermal keratinocytes are a major source of MMP production in human skin. Therefore, the MMP-inhibitory activity of SR95E was determined in HaCaT keratinocytes. SR95E reversed UVB-induced cell damage at concentrations ranging from 10 to 50 µg/mL ( Figure 4A). Treatment with SR95E significantly reduced UVB-induced MMP-1 secretion in a dose-dependent manner ( Figure 4B). In addition, the mRNA expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 were significantly reduced by SR95E results suggest that SR extract may exert anti-wrinkle effects by enhancing procollagen synthesis in skin fibroblasts.

SR Inhibited MMP-1 Secretion and MMP Expression in HaCaT Keratinocytes
Under UV irradiation, epidermal keratinocytes are a major source of MMP production in human skin. Therefore, the MMP-inhibitory activity of SR95E was determined in HaCaT keratinocytes. SR95E reversed UVB-induced cell damage at concentrations ranging from 10 to 50 μg/mL ( Figure 4A). Treatment with SR95E significantly reduced UVB-induced MMP-1 secretion in a dose-dependent manner ( Figure  4B). In addition, the mRNA expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 were significantly reduced by SR95E treatment in UVB-treated HaCaT cells ( Figure 4C-F). These results indicate that SR95E inhibits UVB-induced MMP expression in HaCaT keratinocytes.

SR Inhibited MMP-1 Secretion and MMP Expression in HaCaT Keratinocytes
Under UV irradiation, epidermal keratinocytes are a major source of MMP production in human skin. Therefore, the MMP-inhibitory activity of SR95E was determined in HaCaT keratinocytes. SR95E reversed UVB-induced cell damage at concentrations ranging from 10 to 50 μg/mL ( Figure 4A). Treatment with SR95E significantly reduced UVB-induced MMP-1 secretion in a dose-dependent manner ( Figure  4B). In addition, the mRNA expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 were significantly reduced by SR95E treatment in UVB-treated HaCaT cells ( Figure 4C

SR Regulated MAPK and NF-κB Signaling in HaCaT Keratinocytes
Under UVB irradiation, increased cellular ROS levels enhanced MMP expression through the activation of the MAPK and NF-κB signaling pathways. Therefore, we determined the effects of SR95E on ROS levels and the MAPK/NF-κB signaling pathways. UVB-induced ROS levels decreased significantly in a dose-dependent manner by SR95E treatment ( Figure 5A). Furthermore, UVB-induced phosphorylation levels of JNK and p38 were markedly decreased by SR95E treatment, but not by ERK ( Figure 5B). In addition, the phosphorylation of NF-κB p65 markedly increased after UVB irradiation. However,

SR Regulated MAPK and NF-κB Signaling in HaCaT Keratinocytes
Under UVB irradiation, increased cellular ROS levels enhanced MMP expression through the activation of the MAPK and NF-κB signaling pathways. Therefore, we de- termined the effects of SR95E on ROS levels and the MAPK/NF-κB signaling pathways. UVB-induced ROS levels decreased significantly in a dose-dependent manner by SR95E treatment ( Figure 5A). Furthermore, UVB-induced phosphorylation levels of JNK and p38 were markedly decreased by SR95E treatment, but not by ERK ( Figure 5B). In addition, the phosphorylation of NF-κB p65 markedly increased after UVB irradiation. However, SR95E treatment decreased the phosphorylation of NF-κB p65 ( Figure 5B). These results suggest that SR95E can inhibit UVB-induced MMP expression by inhibiting ROS accumulation and the JNK MAPK, p38 MAPK, and NF-κB p65 signaling pathways.
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.

SR Regulated MAPK and NF-κB Signaling in HaCaT Keratinocytes
Under UVB irradiation, increased cellular ROS levels enhanced MMP expression through the activation of the MAPK and NF-κB signaling pathways. Therefore, we determined the effects of SR95E on ROS levels and the MAPK/NF-κB signaling pathways. UVB-induced ROS levels decreased significantly in a dose-dependent manner by SR95E treatment ( Figure 5A). Furthermore, UVB-induced phosphorylation levels of JNK and p38 were markedly decreased by SR95E treatment, but not by ERK ( Figure 5B). In addition, the phosphorylation of NF-κB p65 markedly increased after UVB irradiation. However, SR95E treatment decreased the phosphorylation of NF-κB p65 ( Figure 5B). These results suggest that SR95E can inhibit UVB-induced MMP expression by inhibiting ROS accumulation and the JNK MAPK, p38 MAPK, and NF-κB p65 signaling pathways.

AMF Inhibited UVB-Induced Skin Aging in HaCaT Keratinocytes
AMF is the main component of SRE, as shown in Figure S1. Therefore, the effect of AMF on MMP expression was determined to confirm whether AMF serves as an active compound in SRE. AMF reversed UVB-induced cell damage at concentrations ranging from 2 to 10 µM ( Figure 6A). AMF treatment significantly reduced UVB-induced MMP-1 secretion in a dose-dependent manner ( Figure 6B). In addition, the mRNA expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 were significantly reduced by AMF treatment in UVB-treated HaCaT cells ( Figure 6C-F). Furthermore, AMF treatment significantly decreased UVB-induced ROS accumulation in a dose-dependent manner ( Figure 7A). AMF treatment decreased the phosphorylation of JNK and p38 MAPK, but not that of ERK ( Figure 7B). UVB-induced phosphorylation of NF-κB p65 was markedly decreased by AMF treatment ( Figure 7B). These results indicate that AMF may serve as an active compound of SRE in inhibiting UVB-induced MMP expression in HaCaT keratinocytes.

SR and AMF Protected against AAPH-Induced Senescence in HaCaT Keratinocytes
Senescent cells accumulate in aged skin tissues and cause skin aging. Cellular senescencerelated markers were investigated in 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)treated HaCaT cells. Prior to the detection of cellular senescence markers, the viability of AAPH-treated HaCaT cells was measured. The viability of the AAPH-treated HaCaT cells was significantly decreased to 81.4%. Treatment with AMF, but not SR95E, slightly restored AAPH-induced cytotoxicity ( Figure 8A). In addition, treatment with SR95E or AMF significantly decreased AAPH-induced ROS accumulation in HaCaT keratinocytes ( Figure 8B). Furthermore, AAPH-induced mRNA expression of cellular senescence-related cell cycle regulators, including p16 and p21, was significantly reduced by treatment with SR95E or AMF ( Figure 8C,D). Nuclear lamin B1 (LMNB1) expression was decreased in AAPH-treated HaCaT cells. However, treatment with SR95E or AMF significantly upregulated LMNB1 expression ( Figure 8E). These results suggest that SR95E and its active compound AMF can improve oxidative stress-induced cellular senescence.
AMF on MMP expression was determined to confirm whether AMF serves as an active compound in SRE. AMF reversed UVB-induced cell damage at concentrations ranging from 2 to 10 μM ( Figure 6A). AMF treatment significantly reduced UVB-induced MMP-1 secretion in a dose-dependent manner ( Figure 6B). In addition, the mRNA expression levels of MMP-1, MMP-2, MMP-3 and MMP-9 were significantly reduced by AMF treatment in UVB-treated HaCaT cells ( Figure 6C-F). Furthermore, AMF treatment significantly decreased UVB-induced ROS accumulation in a dose-dependent manner ( Figure 7A). AMF treatment decreased the phosphorylation of JNK and p38 MAPK, but not that of ERK ( Figure 7B). UVB-induced phosphorylation of NF-κB p65 was markedly decreased by AMF treatment ( Figure 7B). These results indicate that AMF may serve as an active compound of SRE in inhibiting UVB-induced MMP expression in HaCaT keratinocytes.

SR and AMF Protected against AAPH-Induced Senescence in HaCaT Keratinocytes
Senescent cells accumulate in aged skin tissues and cause skin aging. Cellular senescence-related markers were investigated in 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-treated HaCaT cells. Prior to the detection of cellular senescence markers, the viability of AAPH-treated HaCaT cells was measured. The viability of the

Discussion
Although many Selaginellaceae plants including ST and SI have been used as traditional medicines in Asia, little is known about the bioactivities of SR. In a previous study, we reported that SRE and SRM were much more enriched in AMF than the methanol extracts of other Selaginellaceae plants, ST and SI [14]. In addition, in our previous study we also found that AMF-enriched SR extracts inhibited high-fat dietinduced obesity and hyperglycemia by suppressing intestinal lipid absorption [14]. During the progression of skin photoaging, AMF inhibits MMP-1 expression through the inhibition of ERK/activator protein (AP)-1 activation (but not via JNK and p38) in normal human fibroblasts [22]. In addition, AMF protects against nuclear aberrations in UVBinduced human fibroblasts [26]. Our results confirmed that the MMPs-inhibitory activities of SR70E and SR95E were dependent on their AMF content. Both SR70E and SR95E inhibited MMP expression and increased collagen synthesis in CCD-986sk fibroblasts.

Discussion
Although many Selaginellaceae plants including ST and SI have been used as traditional medicines in Asia, little is known about the bioactivities of SR. In a previous study, we reported that SRE and SRM were much more enriched in AMF than the methanol extracts of other Selaginellaceae plants, ST and SI [14]. In addition, in our previous study we also found that AMF-enriched SR extracts inhibited high-fat diet-induced obesity and hyperglycemia by suppressing intestinal lipid absorption [14]. During the progression of skin photoaging, AMF inhibits MMP-1 expression through the inhibition of ERK/activator protein (AP)-1 activation (but not via JNK and p38) in normal human fibroblasts [22]. In addition, AMF protects against nuclear aberrations in UVB-induced human fibroblasts [26]. Our results confirmed that the MMPs-inhibitory activities of SR70E and SR95E were dependent on their AMF content. Both SR70E and SR95E inhibited MMP expression and increased collagen synthesis in CCD-986sk fibroblasts.
Selaginella species are rich sources of bioflavonoids. Among the bioflavonoids identified from ST, AMF (3 ,8 -biapigenin), and sumaflavone (6 -O-hydroxy AMF) showed significant inhibitory activity against MMP-1 in UV-irradiated human fibroblasts, while 2 ,8 -biapigenin, robustaflavone (3 ,6 -biapigenin) and taiwaniaflavone (6-methy-7,4 -di-O-methyl AMF) had no significant effects. These differences in MMP-1 inhibitory activity are likely due to structural differences, particularly those related to the location of the C-C bond and number of hydroxyl groups in the flavonoid skeleton, which can cause potent nitric oxide blocking effects [27]. After oral administration, AMF was rapidly metabolized into 39 products via oxidation, internal hydrolysis, hydrogenation, methylation, sulfation, glucuronidation, glucosylation, O-aminomethylation, and degradation in rats. Moreover, O-aminomethylation and glucosylation are metabolic pathways unique to AMF [28]. AMF metabolites should be further investigated to provide more information on the safety and efficacy of SR extracts with high AMF content. The bioavailability of AMF with oral administration (0.04% ± 0.01% at 300 mg/kg) was much lower than that of intraperitoneal injection (77.4% ± 28.0% at 10 mg/kg) [29]. Pheophorbides, especially pheophorbode a and pyropheophorbide a, exert anti-wrinkle effects in UVB-induced fibroblasts by suppressing MMPs expression and MAPK/NF-κB signaling [24]. The pheophobide contents of SR aerial extracts were not high.
In aged skin, changes in the ECM composition, including fragmented collagen and elastin fibrils, can affect the phenotypes of skin cells [5]. Fibroblasts in aged skin are characterized by a collapsed cytoplasm and lack of connections to surrounding collagen fibrils, with decreased cell surface area. The fibroblasts in age-associated dermal microenvironments produce ROS, MMPs, and cysteine-rich proteins [30,31]. Moreover, there is a higher load of age-related biomolecules, including ROS, MMPs, and cysteine-rich proteins, in the epidermis compared to the dermis [9,32,33]. Among MMPs, only MMP-1 can degrade intact collagen fibrils, whereas other types of MMPs, including MMP-2, MMP-3 and MMP-9, can further decompose degraded collagen fragments [7]. SR extracts effectively suppressed UVB-induced MMP-1, MMP-2, MMP-3 and MMP-9 expression and MMP-1 secretion in CCD-986sk fibroblasts and HaCaT keratinocytes.
UV-induced ROS can upregulate MMP expression through activation of MAPK, AP-1, nuclear factor erythroid 2-related factor 2 (Nrf2), and NF-κB signaling in keratinocytes, melanocytes, and fibroblasts [34]. MAPK signaling includes the ERK, JNK and p38 signaling pathways. Mitogenic stress activates the ERK MAPK pathway, whereas cellular stress activates the JNK and p38 MAPK pathways [35]. UVB irradiation leads to activation of the JNK and p38 MAPK pathways, but there are several factors which may lead to the activation of ERK MAPK dependent on cell conditions [36][37][38]. Under UVB irradiation, normal human keratinocytes exhibit suppressed activation of ERK MAPK, whereas HaCaT cells exhibit enhanced activation of ERK MAPK [36]. In this study, UVB irradiation also induced all of the above MAPKs, including ERK, JNK and p38, in HaCaT cells. However, SR95E and AMF dose-dependently suppressed UVB-induced ROS accumulation by inhibiting JNK and p38 MAPK activation, but not ERK MAPK activation signaling in HaCaT keratinocytes. Keratinocytes are the most abundant cells in the epidermis, and UV-induced NF-κB signaling is more rapid in keratinocytes than in human dermal fibroblasts [39]. In UVB-irradiated keratinocytes, activation of NF-κB signaling is dependent on activation of p38 MAPK [40,41]. In this study, SR95E and AMF markedly inhibited the UVB-induced activation of p38 MAPK and NF-κB signaling in HaCaT keratinocytes.
In aged skin, senescent cells promote skin aging. Senescent cells display various changes, such as irregular shape, loss of lamin B1 with compromised nuclear integrity, and increased cytoplasmic chromatin fragments [42]. Cellular senescence is accompanied by decreased proliferation, resistance to apoptosis, and altered gene expression [43]. Altered gene expression in senescent cells includes alteration of genes related to cell cycle regulation, inflammation, the cytoskeleton, and metabolism [44,45]. Some well-known cell cycle regulatory genes include p53, p16 and p21 [44,46,47]. Under conditions such as UV irradiation and oxidative stress, p53 plays a critical role in the cell cycle arrest of senescent cells [48]. p53 acts as a transcription factor and p21 is the target gene of p53. Activated p21 inhibits cyclin-dependent kinases in cell cycle arrest at the G1/S or G2/S phase [49,50]. In addition, p16 induces the accumulation of the phosphorylated retinoblastoma protein (pRB) to stop cell proliferation [51]. Briefly, senescence growth arrest is mainly controlled by p53/p21 and p16/pRB signaling. SR95E and its compound AMF increased AAPHinduced LMNB1 expression and suppressed that of AAPH-induced cell cycle regulators including p21 and p16. This study is the first to indicate the anti-senescence effects of SR and AMF. These results indicate that SR can also prevent cellular senescence in oxidative stress-induced keratinocytes.
In summary, in this study, the anti-aging effects of Selaginellaceae plants, including ST, SI and SR were comparatively evaluated. Among them, SR was the most effective for inhibiting UVB-induced MMP-1 expression and exhibited the highest AMF content. Both SR70E and SR95E exerted anti-wrinkle effects by suppressing MMP-1 secretion and upregulating procollagen synthesis in UVB-induced CCD-986sk fibroblasts. The antiwrinkle activity of SR95E was also confirmed in UVB-irradiated HaCaT keratinocytes. SR95E decreased MMP-1 secretion and the expression of MMP-1, MMP-2, MMP-3 and MMP-9 by suppressing the JNK/p38 MAPKs and NF-κB signaling in a dose-dependent manner. The anti-wrinkle activity of SRE is mainly attributed to its main component, AMF.
In addition, SR95E and AMF effectively suppressed AAPH-induced cellular senescence by suppressing p21 and p16 expression and enhancing LMNB1 expression. Therefore, these findings indicate that SR has the potential to be used in functional cosmetics to prevent skin aging and wrinkles.