Yellow Chaste Weed and Its Components, Apigenin and Galangin, Affect Proliferation and Oxidative Stress in Blue Light-Irradiated HaCaT Cells

While harmful effects of blue light on skin cells have been recently reported, there are few studies regarding natural products that alleviate its negative effects. Therefore, we investigated ameliorating effects of yellow chaste weed (YCW) (Helichrysum arenarium) extract and its components, apigenin and galangin, on blue light-irradiated HaCaT cells. In this study, we found that YCW extract improved the reduced proliferation of HaCaT cells induced by blue light-irradiation and reduced blue light-induced production of reactive oxygen species (ROS) levels. We also found that apigenin and galangin, the main components of YCW extract, showed the same activities as YCW extract. In experiments examining molecular mechanisms of YCW extract and its components such as apigenin and galangin, they all reduced expression of transient receptor potential vanilloid member 1 (TRPV1), its phosphorylation, and calcium ion (Ca2+) influx induced by blue light irradiation. In addition, apigenin and galangin regulated phosphorylation of mitogen-activated protein kinases (MAPKs). They also reduced phosphorylation of mammalian sterile 20-like kinase-1/2 (MST-1/2), inducing phosphorylation of Akt (protein kinase B), one downstream molecule of MST-1/2. Moreover, apigenin and galangin promoted translocation of Forkhead box O3 (FoxO3a) from the nucleus to the cytosol by phosphorylating FoxO3a. Besides, apigenin and galangin interrupted blue light influences on expression of nuclear and secretory clusterin. Namely, they attenuated both upregulation of nuclear clusterin and downregulation of secretory clusterin induced by blue light irradiation. We also found that they downregulated apoptotic protein Bcl-2 associated X protein (Bax) and conversely upregulated anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). Collectively, these findings indicate that YCW extract and its components, apigenin and galangin, antagonize the blue light-induced damage to the keratinocytes by regulating TRPV1/clusterin/FoxO3a and MAPK signaling.


Introduction
The skin is located at the outermost part of the human body, protecting us from a variety of external factors, including sun light, pollutants, and stresses. These various external Therefore, in this study, we investigated antagonizing activities of yellow chaste weed and its components against blue light and its mechanisms of action in human keratinocytes, HaCaT cells.
All types of antibodies were used in Western blot analysis. Antibodies for TRPV1   (10, 20, 30 µM) or galangin (1, 10, 20 µM) or vehicle (DMSO) for 24 h in phenol red-free culture medium (PRFCM) before blue light irradiation. On the second day, the culture media were removed, and the cells were incubated with fresh PRFCM. Then, the cells were irradiated with blue light with a photoreactor (CCP-4V, Luzchem, Ottawa, ON, Canada) that had an emission peak between 470-480 nm. The power density of LED blue light equipped in photoreactor was 76 W/m 2 , and it was exposed for 30 min. The dose (76 W × 30 min/m 2 = 13.68 J/cm 2 ) of blue light irradiation used is equivalent to those received by the skin during summer after proximately 1500 s of sun exposure. After the first blue light irradiation (13.68 J/cm 2 ), cells were incubated in the presence of the indicated concentration of YCW extract, apigenin, and galangin for 24 h at 37 • C. On the third day, the same process as on the second day was repeated. In order to consider temperature as a control variable, the control group was also left in the dark at room temperature, while the experimental groups were irradiated.

Cell Counting Kit-8 Assay for Cytotoxic Analysis
The cytotoxicity effect of apigenin and galangin on HaCaT cells were measured by cell counting kit-8 assay (CCK-8; CK04-11, Dojindo, Japan). Cells were cultured in 6-well plates. After changing culture media, the cells were incubated for 24 h with apigenin and galangin. These processes were repeated twice more to make a total treatment time of 72 h. After incubation, cells were washed with PBS and the media was changed to PRFCM. Next, the CCK-8 reagent (8 µL/well) was added to cells and incubated for 2 h at 37 • C. Then, the equal amount of supernatant (150 µL/well) was taken to measure the absorbance at 450 nm with microplate reader (Synergy HTX Multi-Mode Reader, BioTek, Winooski, VT, USA).

CellTiter-Glo ® 2.0 Assay for Cell Proliferation Analysis
Cells were cultured on 96-well opaque wall. After 4 days of rough processing (24 h pretreatment and blue light irradiation × 2 days), plates were placed at room temperature for 30 min to equilibrate the plate and its contents to optimal temperature. The CellTiter-Glo ® 2.0 reagent (G9242, Promega, Madison, WI, USA) equal to the volume of cell culture medium was presented in each well (100 µL). All the contents were mixed for 2 min on orbital shaker to induce cell lysis. Cells were incubated at room temperature for 10 min with light-blocked to stabilize the luminescent signal. Luminescence was recorded using a microplate reader (integration time of 0.5 s per well).

BrdU ELISA for Cell Proliferation Analysis
The cells were plated on 96 well black-wall/clear-bottom plates and proceeded 4 days repeated processing experiment. After the experiment, the cell proliferation was determined using the Cell Proliferation ELISA BrdU (colorimetric) kit (11647229001, Roche, Basel, Switzerland) in accordance with the manufacturer's instructions.

EdU Incorporation Imaging for Cell Proliferation Analysis
Click-iT ® EdU Imaging Kits (C10337, Invitrogen, Waltham, MA, USA) were used to examine the cell proliferation. Cells were cultured in confocal plates. Apigenin 30 µM, galangin 20 µM, and vehicle (DMSO) were pretreated for 24 h on the first day. The next day, PRFCM were changed, cells were irradiated blue light (13.68 J/cm 2 ) and treated with indicated concentrations of substances. On the third day, same processes as on the second day were proceeded. Twelve hours after blue light (13.68 J/cm 2 ) exposure, cells replaced half of the media with an equal volume of EdU labeling solution (final concentration of 10 µM) and incubated for 12 h at 37 • C. From this time on, cells were washed three times with 1 mL of PBS with 0.1% Tween-20 (PBST) before every steps. After incubation, cells were fixed with 1 mL of 4% formaldehyde in PBS for 15 min at room temperature. Fixative was removed, 1 mL of 0.1% Triton X ® -100 in PBS to permeabilize cells was added, and then incubated for 20 min at room temperature. After that, cells were blocked with 500 µL of 3% BSA in PBS for 1 h at room temperature. Next, cells were added with 200 µL of Click-iT ® reaction cocktail and incubated for 30 min at room temperature in the dark. After EdU detection, DNA staining was carried out by adding 200 µL of Hoechst 33342 (Invitrogen, Waltham, MA, USA) in PBS, and incubated for 10 min at room temperature in the dark. Thereafter, cells were washed three times with PBS and wet mounted with PBS for slide preparation. Finally, the cells were observed using an LSM 700-laser scanning confocal microscope (Zeiss, Jena, Germany). To measure the intensity of immunofluorescence, images were taken on the same laser power and the mean value of signals was evaluated. Signals from the images were measured by using ZEN 2012 Blue (Zeiss, Jena, Germany) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Flow Cytometry Analysis for Apoptosis
Cells were cultured in 60-mm plates and after 4 days of rough processing (24 h pretreatment and blue light irradiation × 2 days), suspended media were first collected, and cells were washed with 1 mL of PBS and collected on 15-mL tubes. Tubes were then centrifuged at 21,209× g for 3 min. Cells were harvested with 500 µL of trypsin and centrifuged at 1500 rpm for 3 min and removed supernatant. Cell pellets were resuspended with 100 µL of 1X annexin binding buffer with diethyl pyrocarbonate-treated double distilled water (DEPC-DW; C-9030, Bioneer, Daejeon, Korea). Cells were then stained with 100 µg/mL of propidium iodide (PI) and 5 µL of FITC-Annexin V using Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (V13242, Invitrogen, Waltham, MA, USA) and incubated for 15 min at room temperature in the dark. After the incubation, mix gently with 400 µL of 1X annexin binding buffer and keep the samples on ice. The stained cells were counted at least 10,000 cells and analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA).

Fluo-4 NW Calcium Assay for Intracellular Calcium Influx Analysis
Cells were cultured in 96-well black wall/clear bottom plates. Cells were pretreated with yellow chaste weed extract 0.1% or apigenin (10, 20, 30 µM) or galangin (1, 10, 20 µM) or vehicle (DMSO) for 1 h in PRFCM. Next, 100 µL of 1X Fluo-4 NW dye loading solution (F36205, Invitrogen, Waltham, MA, USA)/well were added to the cells and plates were incubated in the dark at 37 • C for 30 min, then at room temperature for an additional 30 min. After removing dye loading solution, 100 µL of assay buffer (1X HBSS, 20 mM HEPES) were added to cells and blue light (13.68 J/cm 2 ) were irradiated in plates except for control group. Then, cells were treated with indicated concentrations of yellow chaste weed extract or apigenin or galangin or vehicle (DMSO) dissolved in assay buffer. Fluorescence was measured using a microplate reader (excitation, 494 nm; emission, 516 nm).

Western Blotting
Cells were cultured in 60-mm plates and treated with indicated concentrations and incubation time of yellow chaste weed, apigenin, and galangin. After that, cells were har-vested and total proteins were extracted from the cells using RIPA buffer (9806S, Cell Signaling Technology, MA, USA), containing protease inhibitor cocktail (5872S, Cell Signaling Technology, Danvers, MA, USA). Nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific, Waltham, MA, USA), following manufacturer's instructions. Pierce TM BCA Protein Assay Kit (23227, Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify ac-curate protein sample concentrations. Protein samples to be loaded were mixed with 4X sample buffer (161-1747, Bio-Rad, Hercules, CA, USA) and heated at 95 • C for 5 min. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (162-0177, Bio-Rad, Hercules, CA, USA). After transfer, the proteins were dyed with Ponceau S solution (P7170, Sigma-Aldrich, St. Louis, MO, USA), and sufficiently washed with Tris-buffered saline and 0.1% Tween-20 (TBST). Membranes were blocked for 2 h using 2% BSA and were incubated overnight with the primary antibodies at 4 • C. Then, the membranes were incubated for more than 1 h with the secondary antibodies at room temperature. Protein bands were visualized by Pierce TM ECL Western blotting substrate (23227, Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis
The data are expressed as the mean ± standard error of the mean (SEM). Analyses of differences between two groups were performed using Student's t-test. The comparison between multiple groups was performed using one-way analysis of variance (ANOVA), followed by the Tukey's multiple-comparison test, for which the GraphPad Prism (5.0) Nutrients 2022, 14, 1217 6 of 19 (GraphPad, La Jolla, CA, USA) software was used. Statistical significance was considered when the p-value was less than 0.05.

Effect of YCW Extract on the Blue Light Irradiated HaCaT Cells
To examine the effects of YCW extract on the proliferation of HaCaT cells, cells were treated with various concentrations (0.01, 0.05, 0.1%) of YCW extract. When blue lightexposed HaCaT cells were treated with YCW extract, the proliferation was significantly increased ( Figure 1A,B). The effect of blue light on the activation of TRPV1 in HaCaT cells was demonstrated by our previous study [10]. Next, we investigated the influence of YCW extract on expression and phosphorylation of TRPV1. After blue light irradiation, the expression of TRPV1 and phospho-TRPV1 increased apparently ( Figure 1C,D). However, YCW extract-pretreated group has shown downregulation of TRPV1 and reduction of its phosphorylation ( Figure 1C,D). TRPV1 is a non-selective cation channel and the activation of TRPV1 elevates calcium influx [51]. To evaluate the inhibition effect of YCW extract on Ca 2+ influx, cells were pretreated with YCW extract 0.1% for 1 h and then irradiated with blue light. As shown in Figure 1E, while blue light increased Ca 2+ influx through the TRPV1 channel, YCW extract reduced Ca 2+ influx induced by blue light irradiation. This result indicates that YCW extract enhances cell proliferation decreased in blue light exposed-HaCaT cells and suggests its effect is mediated through downregulation of TRPV1 and inhibition of its phosphorylation.

Effects of Apigenin and Galangin on the Cytotoxicity of HaCaT Cells and the Proliferation of Blue Light-Irradiated HaCaT Cells
HaCaT cells were treated with different concentrations (10-50 µM) of apigenin ( Figure 2A) and (1-30 µM) of galangin ( Figure 2B) for 3 days and the percentage of cell viability was determined ( Figure 2C,D). As shown in Figure 2C, apigenin was cytotoxic at 40 µM and the cell viability of galangin was decreased at 30 µM. Therefore, the maximum final treatment concentrations for apigenin and galangin were 30 and 20 µM, respectively. To evaluate the effect of apigenin and galangin on proliferation of blue light-exposed keratinocytes, the cells were pretreated before blue light irradiation with 10, 20, and 30 µM of apigenin or 1, 10, and 20 µM of galangin for 24 h. As shown in Figure 2D, both apigenin and galangin enhanced the cell proliferation reduced by blue light. In the BrdU ELISA assay to confirm the proliferation effect of apigenin and galangin, both apigenin and galangin significantly attenuated the inhibitory effect of blue light on proliferation of HaCaT cells concentration-dependently ( Figure 2E). Similarly, EdU incorporation assay was conducted to prove cellular proliferation. As shown in Figure 2F, compared to the control group, blue light exposure remarkably reduced the population of EdU-positive cells. However, this effect was suppressed by pretreatment of apigenin (30 µM) and galangin (20 µM). In addition, this effect of apigenin and galangin was demonstrated using flow cytometry analysis after staining with FITC-annexin V and PI. Annexin V-stained group represents apoptotic cells, while PI staining is used to detect dead cells in a population. As shown in Figure 2G, the proportion of living cells in blue light-irradiated group reduced to 81.19% when compared to a non-irradiated group (96.20%). However, treatment with apigenin or galangin enhanced the ratio of living cells. Together, these indicate that blue light-irradiated reduction of cell proliferation is attenuated by either apigenin or galangin.

Apigenin and Galangin Suppress TRPV1-Mediated Signaling Induced by Blue Light-Irradiation
Previous studies in our laboratory have already shown that blue light upregulates TRPV1 and induce its phosphorylation [10]. Therefore, we examined effects of apigenin and galangin on blue light-induced upregulation of TRPV1. As shown in Figure 3A, various concentrations (10, 20, and 30 μM) of apigenin decreased the blue light-induced effect. Likewise, galangin also reduced the expression of TRPV1 induced by blue light irradiation ( Figure 3A). In addition, TRPV1 phosphorylation contributes the activation of TRPV1 [52]. Therefore, we investigated effects of apigenin and galangin on TRPV1 phosphorylation. As shown in Figure 3B, blue light-induced phosphorylation of TRPV1 was reduced by both apigenin (30 μM) and galangin (20 μM). These data indicate that apigenin and galangin can antagonize against blue light effect by suppressing phosphorylation and expression of TRPV1.
Studies have shown that activation of the TRPV1 induces Ca 2+ entry into the cytosol [51]. Therefore, we examined the involvement of apigenin and galangin in the blue lightinduced calcium influx. As shown in Figure 3C,D, while blue light irradiation increased the inflow of Ca 2+ compared to control group, apigenin and galangin reduced the calcium influx induced by blue light. These data indicate that apigenin and galangin antagonize

Apigenin and Galangin Suppress TRPV1-Mediated Signaling Induced by Blue Light-Irradiation
Previous studies in our laboratory have already shown that blue light upregulates TRPV1 and induce its phosphorylation [10]. Therefore, we examined effects of apigenin and galangin on blue light-induced upregulation of TRPV1. As shown in Figure 3A, various concentrations (10, 20, and 30 µM) of apigenin decreased the blue light-induced effect. Likewise, galangin also reduced the expression of TRPV1 induced by blue light irradiation ( Figure 3A). In addition, TRPV1 phosphorylation contributes the activation of TRPV1 [52]. Therefore, we investigated effects of apigenin and galangin on TRPV1 phosphorylation. As shown in Figure 3B, blue light-induced phosphorylation of TRPV1 was reduced by both apigenin (30 µM) and galangin (20 µM). These data indicate that apigenin and galangin can antagonize against blue light effect by suppressing phosphorylation and expression of TRPV1.
Studies have shown that activation of the TRPV1 induces Ca 2+ entry into the cytosol [51]. Therefore, we examined the involvement of apigenin and galangin in the blue light-induced calcium influx. As shown in Figure 3C,D, while blue light irradiation increased the inflow of Ca 2+ compared to control group, apigenin and galangin reduced the calcium influx induced by blue light. These data indicate that apigenin and galangin antagonize TRPV1-mediated signaling induced by blue light irradiation.

Apigenin and Galangin Reduce ROS Generation in Blue Light Irradiated HaCaT Cells
Next, we examined whether pretreatment of apigenin or galangin ameliorates the accumulation of ROS in blue light irradiated HaCaT cells using a DCFDA dye. Treatment with apigenin or galangin significantly decreased endogenous ROS production induced by blue light irradiation in dose dependent manner, which was amplified by the blue light irradiation ( Figure 4A,B). These data indicate that apigenin and galangin possess antioxidative activity.

Apigenin and Galangin Reduce ROS Generation in Blue Light Irradiated HaCaT Cells
Next, we examined whether pretreatment of apigenin or galangin ameliorates the accumulation of ROS in blue light irradiated HaCaT cells using a DCFDA dye. Treatment with apigenin or galangin significantly decreased endogenous ROS production induced by blue light irradiation in dose dependent manner, which was amplified by the blue light irradiation (Figure 4A,B). These data indicate that apigenin and galangin possess antioxidative activity. . After 24 h incubation, the cells were subjected to the same process twice and were finally subjected to DCF-DA assay. * p < 0.05 vs. control, *** p < 0.005, # p < 0.05 vs. B.L, ## p < 0.01%, ### p < 0.005%.

Apigenin and Galangin Antagonize against Blue Light by Regulating MAPKs and MST-1/2-Akt-FoxO3a Signaling
Previous studies have reported that Ca 2+ regulates MAPK functions [53]. Since apigenin and galangin were verified to lower Ca 2+ flux by blue light irradiation, Western blot was performed to examine the effect of apigenin and galangin on MAPK activation. As shown in Figure 5A,B, apigenin and galangin attenuated the blue light effects on MAPKs. Specifically, apigenin and galangin reduced phosphorylation levels of JNK induced by blue light. On the contrary, while blue light reduced phosphorylation levels of ERK and p38 MAPK, this blue light effect was suppressed by apigenin and galangin treatment (Figure 5A,B). These results suggest that apigenin and galangin antagonize blue light effect by regulating MAPKs.
To investigate the role of FoxO3a, which is a downstream signal of MAPKs and regulator of cell proliferation, we examined effects of apigenin and galangin on phosphorylation levels and nuclear translocation of FoxO3a under blue light-irradiated condition. As shown in Figure 5C, while blue light irradiation reduced the levels of phosphorylated FoxO3a (Ser253), apigenin and galangin increased its phosphorylation levels. Additionally, FoxO3a was detected at higher level in the nuclear fractions of blue light irradiated group ( Figure 5D). In contrast, apigenin and galangin treatment reduced the nuclear translocation of FoxO3a ( Figure 5D). These results indicate that apigenin and galangin reduce nuclear translocation of FoxO3a by phosphorylating FoxO3a.
Mst-1/2 is known to phosphorylate FoxO3a and dissociate FoxO3a from 14-3-3 protein, which lead to nuclear translocation of FoxO3a [54]. To examine effect of apigenin and galangin on MST-1/2 activation, Western blot analysis for phosphorylated MST-1/2 was carried out. As shown in Figure 5E,F, while blue light irradiation caused significant increase in phosphorylated MST-1/2, apigenin and galangin reduced its phosphorylation levels. This data indicates that apigenin and galangin regulate MST-1/2-FoxO3a signaling. Furthermore, to investigate relationship between Akt and both FoxO3a and MST-1/2 signaling, we performed Western blot analysis for phosphorylation forms of them. As shown in Figure 5G,H, while blue light decreased the phosphorylation levels of Akt, apigenin and galangin induced the phosphorylation of Akt. These results indicate that apigenin and galangin antagonize against blue light by regulating MST-1/2-Akt-FoxO3a signaling. . After 24 h incubation, the cells were subjected to the same process twice and were finally subjected to DCF-DA assay. * p < 0.05 vs. control, *** p < 0.005, # p < 0.05 vs. B.L, ## p < 0.01%, ### p < 0.005%.

Apigenin and Galangin Antagonize against Blue Light by Regulating MAPKs and MST-1/2-Akt-FoxO3a Signaling
Previous studies have reported that Ca 2+ regulates MAPK functions [53]. Since apigenin and galangin were verified to lower Ca 2+ flux by blue light irradiation, Western blot was performed to examine the effect of apigenin and galangin on MAPK activation. As shown in Figure 5A,B, apigenin and galangin attenuated the blue light effects on MAPKs. Specifically, apigenin and galangin reduced phosphorylation levels of JNK induced by blue light. On the contrary, while blue light reduced phosphorylation levels of ERK and p38 MAPK, this blue light effect was suppressed by apigenin and galangin treatment ( Figure 5A,B). These results suggest that apigenin and galangin antagonize blue light effect by regulating MAPKs.
To investigate the role of FoxO3a, which is a downstream signal of MAPKs and regulator of cell proliferation, we examined effects of apigenin and galangin on phosphorylation levels and nuclear translocation of FoxO3a under blue light-irradiated condition. As shown in Figure 5C, while blue light irradiation reduced the levels of phosphorylated FoxO3a (Ser253), apigenin and galangin increased its phosphorylation levels. Additionally, FoxO3a was detected at higher level in the nuclear fractions of blue light irradiated group ( Figure 5D). In contrast, apigenin and galangin treatment reduced the nuclear translocation of FoxO3a ( Figure 5D). These results indicate that apigenin and galangin reduce nuclear translocation of FoxO3a by phosphorylating FoxO3a.
Mst-1/2 is known to phosphorylate FoxO3a and dissociate FoxO3a from 14-3-3 protein, which lead to nuclear translocation of FoxO3a [54]. To examine effect of apigenin and galangin on MST-1/2 activation, Western blot analysis for phosphorylated MST-1/2 was carried out. As shown in Figure 5E,F, while blue light irradiation caused significant increase in phosphorylated MST-1/2, apigenin and galangin reduced its phosphorylation levels. This data indicates that apigenin and galangin regulate MST-1/2-FoxO3a signaling. Furthermore, to investigate relationship between Akt and both FoxO3a and MST-1/2 signaling, we performed Western blot analysis for phosphorylation forms of them. As shown in Figure 5G

Apigenin and Galangin Regulate Expression of Clusterin, Bax, and Bcl-2
Clusterin, Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) have been well known to be involved in cell survival and apoptosis. We examined whether apigenin and galangin affect blue light-induced expression of two clusterin isoforms (nuclear clusterin and secretory clusterin), Bax and Bcl-2. As shown in Figure 6A,B, while blue light irradiation increased the expression of nuclear clusterin (pro-apoptotic protein), protein levels of secretory clusterin (pro-survival protein) were decreased. However, apigenin and galangin decreased expression of nCLU and increased expression of sCLU. In addition, as shown in Figure 6C,D, blue light irradiation increased protein levels of Bax (apoptotic protein) and reduced protein levels of Bcl-2 (anti-apoptotic protein). These blue light effects on Bax and Bcl-2 was attenuated by apigenin and galangin. These data indicate that apigenin and galangin exert antagonizing activity by in-creasing expression of cell survival-related genes.

Apigenin and Galangin Regulate Expression of Clusterin, Bax, and Bcl-2
Clusterin, Bcl-2 associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) have been well known to be involved in cell survival and apoptosis. We examined whether apigenin and galangin affect blue light-induced expression of two clusterin isoforms (nuclear clusterin and secretory clusterin), Bax and Bcl-2. As shown in Figure 6A,B, while blue light irradiation increased the expression of nuclear clusterin (pro-apoptotic protein), protein levels of secretory clusterin (pro-survival protein) were decreased. However, apigenin and galangin decreased expression of nCLU and increased expression of sCLU. In addition, as shown in Figure 6C,D, blue light irradiation increased protein levels of Bax (apoptotic protein) and reduced protein levels of Bcl-2 (anti-apoptotic protein). These blue light effects on Bax and Bcl-2 was attenuated by apigenin and galangin. These data indicate that apigenin and galangin exert antagonizing activity by in-creasing expression of cell survival-related genes.  . After 24 h incubation, the cells were subjected to the same process twice and were finally subjected to Western blot analysis. The total proteins were extracted from the cells immediately after experimental conditions, and β-actin was used as a loading control. (C,D) Protein levels of Bax, apoptotic protein, and Bcl-2, anti-apoptotic protein, were determined by Western blotting. Cells were incubated with apigenin (10, 20, 30 µM) (C) or galangin (1, 10, 20 µM) (D) for 24 h and were then irradiated with blue light (30 min, 76 W/m 2 ). After 24 h incubation, the cells were subjected to the same process twice and were finally subjected to Western blot analysis. The total proteins were extracted from the cells immediately after experimental conditions, and β-actin was used as a loading control.

Discussion
In everyday life, a large amount of blue light is irradiated to our skin through not only sunlight, but also electronic devices. The blue light is well known to reduce cell proliferation and induce oxidative damage, which in turn could cause photoaging [13,[55][56][57][58]. Nevertheless, little is known about the mechanism for cell damage caused by blue light. In addition, only few substances have been reported to prevent the adverse effects of blue light. In this study, yellow chaste weed (YCW), and its main components such as apigenin and galangin were found to exert anti-blue light effect through regulation of TRPV1-mediated signaling.
First, our results showed that the YCW (Helichrysum arenarium), an herbaceous perennial plant, enhanced cell proliferation of human keratinocytes decreased by blue light irradiation. Next, according to our previous study, cellular damage caused by blue light irradiation in human keratinocyte is due to TRPV1 regulation [10]. TRPV1 cation channel can be activated by various stimuli and ligands such as heat (>43 • C), acid, capsaicin, and other vanilloid compounds (e.g., endocannabinoid, anandamide) [59]. Moreover, many studies have elucidated that TRPV1 expression is related to photoaging of the skin by UV [60][61][62]. Similar to UV results in these studies, our report has also proved that the expression of TRPV1 was increased by blue light irradiation [10]. Likewise, in this paper, we found that the TRPV1 expression elevated by the blue light was downregulated by high concentration of YCW extract ( Figure 1C). In addition, phosphorylation of TRPV1 mediate channel's activation [63]. Our data showed that blue light enhanced responsiveness of TRPV1 and yellow chaste weed extract dephosphorylated TRPV1 ( Figure 1D). Further, TRPV1 channel opening stimulates Ca 2+ entry into cytosol [14]. Blue light exposure increased Ca 2+ influx, which was suppressed by YCW extract ( Figure 1E). These data suggest that YCW extract could antagonize negative effects of blue light irradiation.
Apigenin and galangin, main flavonoids of YCW, showed the same effects as YCW extract. Specifically, while blue light inhibited keratinocyte proliferation, this blue light effect was attenuated by apigenin and galangin (Figure 2). Apigenin and galangin reduced expression and phosphorylation of TRPV1 as well as intracellular Ca 2+ influx induced by blue light irradiation ( Figure 3). These data suggest that apigenin and galangin has anti-blue light activity by regulating TRPV1 signaling.
ROS are produced during normal intracellular activation and are associated with various biological processes, including cell differentiation, gene expression, and response to cytokines [64,65]. Therefore, maintaining homeostasis of these ROS is critical for cell growth and survival. Oxidative stress is increased due to the imbalance between the production of ROS and the antioxidant reaction to remove it [64,66]. Thus, ROS has so far been recognized as a substance that simply oxidizes proteins, DNA, and lipids, causing cell necrosis, but also plays an important role in certain cytokines or growth factors signaling as an essential second messenger within cells [67,68]. In our study, we demonstrated that YCW extract, apigenin, and galangin can reduce intracellular ROS levels increased by blue light irradiation, suggesting that they possess anti-oxidative activity (Figure 4).
In addition, under various external stimuli, such as oxidative stress, mitogen-activated protein kinases (MAPKs) are involved in cell growth, apoptosis, and differentiation. MAPK subfamily consists of c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase, and regulates the expression of various genes by phosphorylating transcriptional regulatory factors [69][70][71]. Activated JNK stimulates an apoptotic signaling by regulating expression of p53-dependent genes, such as Bcl-2 associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA) [72,73]. JNK can also induce apoptosis by suppressing BH3-only family of Bcl-2 proteins [74]. In this study, apigenin and galangin reduced levels of phosphorylated-JNK (p-JNK), which can lead to anti-apoptotic signal ( Figure 5A,B). In addition, both ERK and p38 kinase are known to promote cell survival, proliferation, and development [75][76][77]. In this study, apigenin and galangin also increased phosphorylation of ERK and p38 kinase which were reduced by blue light irradiation ( Figure 5A,B).
MAPKs are known to regulate FoxO3a phosphorylation [78][79][80]. For example, under stress condition, activated JNK suppresses Akt activities which dephosphorylate FoxO3a and promotes FoxO3a nuclear translocation [78]. Specifically, phosphorylated Ser473 Akt can phosphorylate FoxO3a at Ser 253, leading to FOXO3a export from nucleus to cytosol and decreases its transactivation activity [32][33][34]. In addition, MST-1/2 is a positive regulator of FoxO3a activity [54]. MST-1/2 activation translocate FoxO3a from cytosol to nucleus and increase its transcription. Akt and MST-1/2 signaling negatively crosslink each other, and suppression of either Akt or MST-1/2 inhibits cell proliferation [81][82][83]. Activated p-Akt moves into nucleus, which phosphorylates FOXO3a and release from DNA to lower transcription activities [84,85]. The p-FoxO3a is exported out of the nucleus by binding to the 14-3-3 protein and remains inactive in the cytosol [86]. The FoxO3a transcription factors in the nucleus regulate target genes related to cell death, cell cycle arrest and oxidative stress [27]. In this study, we found that apigenin and galangin reduced phosphorylation of MST-1/2, and increased phosphorylation of Akt, leading to phosphorylation of FoxO3a and its export from nucleus ( Figure 5C-H).
Nuclear and secretory clusterin respectively carry out apoptotic or anti-apoptotic functions, by regulating Ku70, Bax, Bcl-2, and caspase activities [23,26,[87][88][89]. In this study, blue light irradiation increased nuclear clusterin and reduced secretory clusterin, leading to reduced cell growth. Apigenin and galangin antagonized the blue light effect on expression of two isoforms of clusterin, consequently promoting cell proliferation ( Figure 6). Collectively, these results indicate that apigenin and galangin exert anti-blue light effect by enhancing cell proliferation and decreasing ROS production through regulation of TRPV1 signaling and its related genes shown in Figure 7. However, since the connection between the molecules involved in the blue light-induced signaling was not clear, further study is needed to demonstrate it. apigenin and galangin also increased phosphorylation of ERK and p38 kinase which were reduced by blue light irradiation ( Figure 5A,B).
MAPKs are known to regulate FoxO3a phosphorylation [78][79][80]. For example, under stress condition, activated JNK suppresses Akt activities which dephosphorylate FoxO3a and promotes FoxO3a nuclear translocation [78]. Specifically, phosphorylated Ser473 Akt can phosphorylate FoxO3a at Ser 253, leading to FOXO3a export from nucleus to cytosol and decreases its transactivation activity [32][33][34]. In addition, MST-1/2 is a positive regulator of FoxO3a activity [54]. MST-1/2 activation translocate FoxO3a from cytosol to nucleus and increase its transcription. Akt and MST-1/2 signaling negatively crosslink each other, and suppression of either Akt or MST-1/2 inhibits cell proliferation [81][82][83]. Activated p-Akt moves into nucleus, which phosphorylates FOXO3a and release from DNA to lower transcription activities [84,85]. The p-FoxO3a is exported out of the nucleus by binding to the 14-3-3 protein and remains inactive in the cytosol [86]. The FoxO3a transcription factors in the nucleus regulate target genes related to cell death, cell cycle arrest and oxidative stress [27]. In this study, we found that apigenin and galangin reduced phosphorylation of MST-1/2, and increased phosphorylation of Akt, leading to phosphorylation of FoxO3a and its export from nucleus ( Figure 5C-H).
Nuclear and secretory clusterin respectively carry out apoptotic or anti-apoptotic functions, by regulating Ku70, Bax, Bcl-2, and caspase activities [23,26,[87][88][89]. In this study, blue light irradiation increased nuclear clusterin and reduced secretory clusterin, leading to reduced cell growth. Apigenin and galangin antagonized the blue light effect on expression of two isoforms of clusterin, consequently promoting cell proliferation (Figure 6). Collectively, these results indicate that apigenin and galangin exert anti-blue light effect by enhancing cell proliferation and decreasing ROS production through regulation of TRPV1 signaling and its related genes shown in Figure 7. However, since the connection between the molecules involved in the blue light-induced signaling was not clear, further study is needed to demonstrate it.

Conclusions
In conclusion, these findings indicate that apigenin and galangin, active components of YCW, can ameliorate negative effects of blue light through TRPV1/clusterin/FoxO3a and MAPK signaling pathways, suggesting the possibility of apigenin and galangin as an anti-blue light agent for human skin.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.