Fisetin Ameliorated Photodamage by Suppressing the Mitogen

Apr 16, 2015 - Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear ...
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Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways Hsiu-Mei Chiang,* Shih-Yun Chan, Yin Chu, and Kuo-Ching Wen* Department of Cosmeceutics, China Medical University, Taichung 404, Taiwan ABSTRACT: Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5−25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents. KEYWORDS: fisetin, photodamage, matrix metalloproteinase, MAP kinase, NF-κB, CREB



peroxynitrite and other reactive oxygen species (ROS),9 which upregulate COX-2 expression to stimulate the inflammation process.10 ROS drives MAP kinase (including ERK, JNK, and p38) activation, which transfers c-Fos and c-Jun to the nucleus, induces nuclear factor-κB (NF-κB) activation, and subsequently upregulates the gene related to pro-inflammatory reactions.11−13 NF-κB existed in the cytoplasm as an inactive complex with an inhibitor κB (IκB), and the degradation of IκB causes translocation of NF-κB to the nucleus. UV irradiation activates NF-κB, which increases MMP-1 in the dermis.14 UV irradiation is absorbed by skin molecules, which results in ROS generation and, subsequently, causes oxidative stress to cellular components involving cell walls, lipid membranes, mitochondria, and DNA.4,15 Fisetin (3,7,3′,4′-tetrahydroxyflavone) (Figure 1) is a bioactive flavonol molecule that exists in fruits and vegetables, such as strawberries, apples, grapes, and onions.16 It has been reported that fisetin exhibits neuroprotective, antitumor,17,18 antioxidative,19 and anti-inflammatory20,21 activities. In addition, fisetin can attenuate inflammation through the reduction of COX-2, iNOS, and NO levels in RAW 264.7 cells and mice.20,22 Fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells and may be developed as treatment for non-melanoma skin cancers.23 It has been reported that a wide variety of new cosmeceuticals and formulas can accelerate collagen synthesis, prevent aging and photoaging,

INTRODUCTION Ultraviolet (UV) irradiation is one of the most noxious environmental hazards and can induce inflammation and oxidative stress in human skin. UV irradiation that induces skin damage principally manifests as a degradation of extracellular matrix (ECM) proteins, including collagen, elastin, proteoglycans, and fibronectin, which are the main building blocks of the skin.1,2 Collagen, the most abundant ECM protein in the dermis, is derived from dermal fibroblasts and regulated by mitogenactivated protein (MAP) kinase. MAP kinase induces activator protein-1 (AP-1; a transcription factor) and promotes collagen breakdown by upregulating enzymes called matrix metalloproteinases (MMPs), especially MMP-1, the major collagen-degrading enzyme in the skin.3 MMPs are a family of structurally related matrix-degrading enzymes that play crucial roles in various destructive processes, including skin aging and photodamage. MMP-1, also known as interstitial collagenase, initiates the degradation of collagen types I, II, and III in the skin.4 In addition, MMP-1, MMP-3, and MMP-9 play vital roles in photodamage by degrading ECM in the dermis.3,5,6 In addition to regulating MMPs and collagen, excessive UV irradiation can cause acute skin inflammation and lead to the development of skin cancer. Prostaglandins (PGs) and nitric oxide (NO) play crucial roles in the inflammatory process.1,7 UVB induces cyclooxygenase-2 (COX-2) production, which is the rate-limiting enzyme in PG generation.8 UVB irradiation induces erythema in the skin (sunburn), which is related to nitric oxide synthase (NOS) and the up-expression of cyclooxygenase (COX). UV irradiation upregulates the expression of inducible nitric oxide synthase (iNOS) to generate NO, which reacts with superoxide. This leads to the production of © XXXX American Chemical Society

Received: November 25, 2014 Revised: April 15, 2015 Accepted: April 16, 2015

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(5, 10, and 25 μM) were added and then incubated for 2 h. The cells were incubated with 10 μM DCFH-DA for 30 min. The fluorescence intensity was determined at 488 nm excitation and 520 nm emission using a fluorescence microplate reader (Thermo Electron Corporation, Ratastie, Vantaa, Finland), and images were observed under a fluorescence microscope (Leica DMIL, Wetzla, Germany). Measurement of Total Collagen Synthesis. Total collagen synthesis in fibroblasts after UVB exposure was measured using the Sircol soluble collagen assay kit and modified on the basis of a previous study.6,25 Briefly, a cell culture medium was collected and mixed with Sircol dye reagent and incubated. After centrifugation, ice-cold acid− salt washing reagent was added to the precipitate and then the mixture was centrifuged. The precipitate was dissolved using alkali reagent, and the absorbance was determined at 555 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Grodig, Austria). Western Blot Analysis. The cells were harvested at the indicated time and washed twice with ice-cold PBS for western blot analysis. The cells were lysed in a cold lysis buffer; the lysates were centrifuged; and an aliquot of the lysate was used to determine the protein content by conducting a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins (30 μg) were separated using SDS− polyacrylamide gel electrophoresis (SDS−PAGE) and blotted onto polyvinyl difluoride membranes. The membranes were blocked with non-fat milk in a Tris-buffered saline and Tween 20 (TBST) buffer and then incubated overnight with specific antibodies. These were goat polyclonal antibodies, which are used against MMP-1 and type I procollagen, and mouse polyclonal antibodies, which are used against phosphor-CREB, CREB, MMP-3, MMP-9, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, phosphor-c-Jun, and NF-κB p65. The membranes were incubated with the corresponding horseradish-peroxidaseconjugated secondary antibody after washing with PBS buffer. Immunoreactive proteins were detected using the ECL western blotting detection system (Fujifilm, LAS-4000, Tokyo, Japan), and the signal intensity of each band was quantified using a densitometer system (Multi Gauge V2.2) and then normalized with the internal control (actin). Immunofluorescence Staining. For immunofluorescence staining, 1 × 105 cells were cultivated on glass coverslips overnight. The medium was removed, and the cells were washed twice with a PBS buffer. The cells were fixed with 4% paraformaldehyde in PBS for 30 min. The fixed cells were blocked with 5% non-fat milk with 0.3% Triton X-100/PBS buffer for 40 min and incubated overnight, separately, with the primary antibody. After washing, cells were incubated with the Alexa Fluor 488 anti-rabbit IgG secondary antibody for 2 h (Invitrogen, Carlsbad, CA). The unbound secondary antibody was removed by washing the cells 3 times with a PBS buffer. Thereafter, the samples were counterstained with ProLong Gold antifade reagent with DAPI and observed using a microscope.

Figure 1. Structure of fisetin (3,7,3′,4′-tetrahydroxyflavone).

and facilitate the reparation of wrinkles in the skin.24 Development of MMP inhibitors is a potential strategy for photoaging therapy.5,25,26 In the literature, it had been reported that fisetin inhibited UV-induced ROS and activation of NF-κB and MAP kinase in lens epithelial cells.27 However, few studies exist on the effect of fisetin against UV-induced photodamage in human skin and the related mechanisms. Therefore, this study investigates the potential mechanisms by which fisetin counteracts UVB-induced overexpression of MMPs/MAP kinases, COX-2, AP-1, and MMPs, the degradation of IκB and translocation of NF-κB, and the reduction in type I procollagen levels in human skin fibroblasts.



MATERIALS AND METHODS

Materials. Fisetin, DL-dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), and Triton X-100 were purchased from Sigma-Aldrich Chemicals Corporation (St. Louis, MO). Collagenase was purchased from Calbiochem, Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin− streptomycin, and trypsin−ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco, Invitrogen (Carlsbad, CA). Coomassie Blue R-250, dibasic sodium phosphate, lgepal CA-630, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tris(hydroxymethyl)aminomethane (Tris), and sodium dodecyl sulfate (SDS) were purchased from USB Corporation (Cleveland, OH). c-Jun, phosphor-c-Jun, NF-κB p65, and IκB antibodies were purchased from Cell Signaling Technology (Beverly, MA). Donkey anti-goat p38, p-p38, IgG-HRP, cAMP response element-binding protein (CREB), phosphor-CREB, ERK1, p-ERK 1/2, JNK1, p-JNK, MMP-1, MMP-3, and MMP-9 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cell Culture. Human foreskin fibroblasts (Hs68) were purchased from the Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. The cells were cultured in DMEM, which contained 10% FBS, 100 units/mL penicillin, and 100 units/mL streptomycin and were maintained at 37 °C in a humidified, 5% CO2 incubator. UVB Irradiation Dose. Cells were washed with phosphatebuffered saline (PBS) and covered with PBS for UV irradiation using a UV lighter (302 nm, CL-1000M, UVP, Upland, CA). The UVB irradiation dose was 40 mJ/cm2 (exposure time was 15 s) according to the previous studies.6,28 This dose equates to about 23 s exposure at the noontime on July in the middle of Taiwan measured by a UV meter (UVP, Upland, CA). At this UV dose, the cell viability does not reduce considerably, whereas the protein expression is induced. After UVB irradiation, PBS was replaced with a serum-free medium and then incubated for 24 h for the MTT and various assays. Cell Viability Test. A MTT assay was performed to examine the viability of the cells as previous studies described.25,29 After being irradiated with UVB, the cells were incubated with various concentrations of fisetin for 24 h and the culture medium was replaced with MTT solution (0.5 mg/mL) and incubated. The MTT solution was then removed, and 10% SDS−HCl was added. The absorbance of the dissolved formazan crystals was then determined at 570 nm using a spectrophotometer (Tecan, Grodig, Austria). Measuring Intracellular ROS. Intracellular ROS generation was measured using a 2′,7′-dichlorodihydro fluorescein diacetate (DCFHDA) fluorescence dye assay as a previous study described, with slight modification.6,30 Fibroblasts were seeded into 24-well plates and irradiated with UVB. Subsequently, various concentrations of fisetin

Figure 2. Cell viability of fisetin with or without UVB exposure on human fibroblasts. Significant difference versus control: (###) p < 0.001. Significant difference versus UV-exposed control: (∗∗∗) p < 0.001. B

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Figure 3. Repressive effect of fisetin on UVB-induced intracellular oxidative stress in human fibroblasts. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests.



NO Measurement. The measurement of NO generation in human skin fibroblasts was by a previous study, with slight modification.25 Cells were grown in a 6 well plate for 24 h before treatment. After UVB irradiation, cells were treated with different concentrations of fisetin for 24 h. Cell culture medium was then collected and mixed with Griess reagent (Promega, Madison, WI), and the absorbance of azo compound was determined at 540 nm using a spectrophotometer (Tecan, Grodig, Austria). Quantitation of Prostaglandin E2 (PGE2). PGE2 levels in cultured medium were determined using the protocol provided by the manufacturer of the kit (Cayman, Ann Arbor, MI). The cell culture and treatments were as described for the NO measurement. Statistical Analysis. All measurements in the present study were obtained as averages of experiments that were at least three independent experiments performed in triplicate and are expressed as means ± standard deviation (SD). Differences between groups in experiments were analyzed for statistical significance using analysis of variation (ANOVA) with least significant difference (LSD) posthoc tests or the Student’s t test. p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Effect of Fisetin on the Cell Viability and UVB-Induced Phototoxicity. Safety is the primary criterion for skin-care products. Hs68 cells were treated with various concentrations of fisetin, and cell viability was measured using the MTT assay. The resulting survival curve indicated that fisetin (5−50 μM) did not exhibit cytotoxic effects on the proliferation of cells (Figure 2). After UVB irradiation (40 mJ/cm2), the cell viability was significantly decreased to 82.7 ± 1.9% (p < 0.001). At 15−25 μM, fisetin did not significantly influence the cell viability of fibroblasts after UVB irradiation but decreased the cell viability to 64.8 ± 0.9% at 50 μM (Figure 2). Fisetin does not recover UVB-induced fibroblast mortality; indeed, the highest dose of fisetin (50 μM) even increases UVB damage. Therefore, 5−25 μM fisetin was used in the study of its antiphotoaging activity and mechanism. Fisetin Reduced UVB-Induced Intracellular ROS. DCFH-DA staining and fluorescence microscopy were used C

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Journal of Agricultural and Food Chemistry to qualitatively characterize the degree of ROS generation. ROS are potential inducers of skin-photoaging-related proteins and cause intracellular oxidative damage in human skin fibroblasts. Fibroblasts were exposed to UVB and then treated with various concentrations of fisetin for 2 h in a 24 well plate. After the fisetin-containing medium was removed, the cells were washed with PBS and incubated with 10 μM dichlorofluorescin diacetate (DCFDA) for 30 min. In this study, treatment with UVB significantly increased ROS generation 1.4-fold compared to control cells, whereas treatment with 5, 10, and 25 μM fisetin reduced UVB-induced ROS generation to 0.9-, 0.8-, and 0.7-fold, respectively (Figure 3). The results of fluorescence and immunofluorence staining indicate that fisetin protected Hs68 cells from the damage of UVB-induced ROS. UV irradiation induced ROS generation in living organisms, causing oxidative stress, especially when ROS is not scavenged by an antioxidant defense system. Antioxidants, such as N-acetyl cysteine, scavenged ROS to protect skin from UVBinduced oxidative stress and cell death.31,32 In this study, fisetin suppressed UV-induced intracellular ROS formation, protecting skin cells from oxidative damage and related diseases. In a previous study, fisetin exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity.33 It has been reported that the presence of a catechol group in the C-2 position and the number of hydroxyl substituents and their location in the molecule (especially at C-3, C-5, and/or C-7) are determinant structural factors for their ability to scavenge free radicals.33,34 Fisetin, which has C-2 catechol and hydroxyl groups at C-3 and C-7, is an excellent free-radical scavenger. Therefore, its protective effect on skin against UV damage may contribute to its ability to scavenge UV-induced radical generation. Anti-inflammatory Effect of Fisetin. Fisetin on UVBInduced COX-2 Expression. The levels of COX-2 were 8.31fold higher in fibroblasts exposed to UVB (40 mJ/cm2) than in control cells (Figure 4a). In addition, fisetin (5, 10, and 25 μM) exhibited a dose-dependent reduction in UVB-induced COX-2 expression and a reduction of the UVB-induced expression of COX-2, which were 3.57-, 2.94-, and 1.06-fold compared to the control, respectively. The effect was significant when the dose was higher than 5 μM (Figure 4a). Epigallocatechin gallate (EGCG, 1 μM; positive control) significantly suppressed UVBinduced COX-2 expression (from 8.31- to 2.66-fold of the control). Fisetin on UVB-Induced iNOS Expression. The result of fisetin on iNOS expression in fibroblasts is shown in Figure 4b. iNOS expression was increased to 1.6-fold after exposure to UVB; however, fisetin treatment (5, 10, and 25 μM) did not considerably reduce the levels of iNOS. Effect of Fisetin on NO and PGE2 Generation. As shown in Figure 5a, UVB exposure induced NO to 3-fold of the control and fisetin treatment reduced UVB-induced NO generation in human skin fibroblasts. UVB elevated PGE2 in human skin fibroblasts, and fisetin at 10 and 25 μM would significant reduce PGE2 production induced by UVB (Figure 5b). UV irradiation leads to direct or indirect DNA damage and the formation of ROS, which induces an inflammatory response (induced COX-2 and iNOS) and damages the integrity of the extracellular matrix.35 Our studies have shown that fisetin possesses anti-inflammatory activities by inhibiting UVBinduced PGE2 and NO. The results of this study indicated that fisetin reduced UVB-induced COX-2 expression but not iNOS. The suppressive effect on COX-2 induction might be an independent process or an indirect phenomenon via reduced NO production. In addition, NO was controversially reported

Figure 4. (a) Effect of fisetin on the UV-induced expression of COX-2 in human fibroblasts. (b) Effect of fisetin on the UVB-induced expression of iNOS in human fibroblasts. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD posthoc tests.

to affect the level of COX activity and/or COX-2 expression.36 It had been reported that flavonoids might directly affect COX-2 activity and/or expression, for example, wogonin, which was a direct COX-2 inhibitor but not an iNOS inhibitor.37 Therefore, fisetin might directly inhibit COX-2 but not iNOS. The effect mechanism of fisetin on iNOS and COX-2 needs further study. Effects of Fisetin on UVB-Induced Photodamage and Photodamage-Related Protein Expression. Fisetin Attenuated UVB-Induced Reduction in Total Collagen Synthesis. Fibroblasts were pretreated with fisetin (5−25 μM) for 1 h, exposed to UVB, and then treated with fisetin for 24 h. As shown in Figure 6a, fisetin treatment resulted in a dose-dependent D

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Figure 5. (a) Effect of fisetin on NO generation in human skin fibroblasts. Human fibroblasts were treated with/without UVB of 40 mJ/cm2 and fisetin of 5, 10, and 25 μM. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests. (b) Effect of fisetin on PGE2 generation in human skin fibroblasts. Human fibroblasts were treated with/without UVB of 40 mJ/cm2 and fisetin of 5, 10, and 25 μM. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests.

restoration of collagen and fisetin at 25 μM would significantly increase total collagen synthesis. Effect of Fisetin on MMP Expression. To examine the antidamage effects of fisetin on UVB-irradiated human skin fibroblasts, we measured cellular MMP-1, MMP-3, and MMP-9 protein expressions. As shown in Figure 6b, UVB caused significant elevation of MMP-1, MMP-3, and MMP-9 protein expression (1.33-, 1.42-, and 1.53-fold compared to the control, respectively), whereas fisetin attenuated MMP expression. As shown in Figure 6b, the UVB-induced MMP-1 expression was reduced to 0.64-, 0.51-, and 0.42-fold when using 5, 10, and 25 μM fisetin, respectively, and 1 μM EGCG reduced MMP-1 0.54-fold compared to the control. Fisetin (5, 10, and 25 μM) suppressed UVB-induced MMP-3 expression 1.22-, 1.19-, and 0.92-fold compared to the control, respectively, and EGCG decreased 0.93-fold (Figure 6b). Furthermore, fisetin (5, 10, and 25 μM) inhibited UVB-induced MMP-9 expression 1.41-,

1.38-, and 1.43-fold, respectively, and EGCG decreased 0.8-fold compared to the control (Figure 6b). These results provide evidence that fisetin prevents the UVB-induced elevation of MMP-1, MMP-3, and MMP-9 levels, thus protecting UVBinduced skin damage and photoaging. Effect of Fisetin on MAP Kinase Phosphorylation. In Figure 6c, the phosphorylation of ERK was increased after UVB irradiation, pretreatment with a high dose (25 μM) of fisetin diminished the effect (from 2.2- to 1.7-fold compared to the control), but pretreatment with lower doses (5 and 10 μM) did not. The results of the phosphorylation of JNK resembled those of the phosphorylation of ERK, whereas fisetin (10 and 25 μM) significantly reduced the phosphorylation of p-38 expression (Figure 6c). Effect of Fisetin on c-Jun and p-c-Jun Expression. As Figure 7a shows, UV induced p-c-Jun expression (3.28-fold compared to the control), whereas fisetin treatment reduced E

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Figure 6. (a) Effect of fisetin on the UV-suppressed total collagen synthesis in human fibroblasts. (b) Effect of fisetin on the UVB-induced expression of MMP-1, MMP-3, and MMP-9 in human fibroblasts. (c) Effect of fisetin on the UVB-induced ERK, JNK, and p38 phosphorylation in human fibroblasts. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests.

cytoplasma, whereas fisetin treatment significantly increased the expression of p65 in a dose-dependent manner (Figure 7b). Immunohistochemistry Assay of NF-κB. The immunohistochemistry staining assay of NF-κB was conducted in fibroblast cells to determine the degree of NF-κB activation. As shown in Figure 7c, UVB induced the translocation of NF-κB to the nucleus, whereas fisetin suppressed the translocation of NF-κB induced by UVB. Effect of Fisetin on the PI3K/AKT/CREB Pathway. Treatment of skin fibroblasts with UVB irradiation resulted in a

the c-Jun phosphorylation level in a dose-dependent manner. c-Jun expression was increased after UV irradiation, whereas fisetin inhibited the effect at 25 μM but not at 5 and 10 μM. Effect of Fisetin on the IκB/NF-κB Pathway. Western blot analysis revealed that the protein levels of IκB were inhibited after the cells were treated with UVB irradiation, whereas fisetin significantly increased IκB expression in a dose-dependent manner (Figure 7b). In addition, the level of the NF-κB subunit, p65, was reduced 0.66-fold compared to the control in F

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Figure 7. (a) Effect of fisetin on the UVB-induced c-Jun and c-Jun phosphorylation in human fibroblasts. (b) Effect of fisetin on the UVB-induced IκB degradation and the amount of NF-κB/p65 in cytoplasm. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests. (c) Fisetin inhibits UVB-induced NF-κB activation by fluorescein isothiocyanate (FITC) immunofluorescence labeling.

in peroxynitrite and other ROS formations.11 These ROS then induce an MAP kinase transcription factor, AP-1, phosphorylation of IκB, and then degradation, subsequently activating NF-κB and inducing COX-2 expression.38,39 Furthermore, UV exposure activates MAP kinase and PI3K/AKT pathways, leading to upregulation of CREB Ser-133 and the subsequent transcriptional activation of the COX-2 gene.40 In previous studies, the activation of p-38 resulted in AP-1 transcription, NF-κB expression and activation in the cytoplasma, and

2.21-fold increase in p-CREB Ser-133 levels (Figure 8). This showed the intense effect of UVB irradiation on the content of p-CREB Ser-133 in skin fibroblasts. This increase in p-CREB Ser-133 expression was significantly suppressed by fisetin treatment at 25 μM (p < 0.001). CREB expression was unchanged after either UVB or fisetin treatment and, similarly, was obtained after co-treatment of these two factors. UV irradiation has been shown to upregulate the expression of iNOS, producing NO, and to react with superoxide, resulting G

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Figure 9. Schematic diagram showing inhibitory effects of fisetin in UVB-induced skin damage.

by modulating the expression of MMPs, MAP kinases, AP-1, COX-2, and p-CREB Ser-133 (Figure 9). In addition, fisetin effectively restored UV-induced IκB degradation, resulting in NF-κB inhibition. Therefore, fisetin is a potential agent used on UV-induced skin damage.

Figure 8. Effect of fisetin on the UVB-induced CREB phosphorylation at Ser-133. Groups sharing the same letter are not significantly different (p > 0.05) as revealed by LSD post-hoc tests.



AUTHOR INFORMATION

Corresponding Authors

*Telephone: 886-4-22053366, ext. 5302. Fax: 886-4-22078083. E-mail: [email protected]. *Telephone: 886-4-22053366, ext. 5302. Fax: 886-4-22078083. E-mail: [email protected].

p-CREB Ser-133 expression.41 Results from this study indicated that fisetin attenuated the UV-induced overexpression of COX-2, p-CREB Ser-133, and MAP kinases and restored UV-induced IκB degradation, resulting in NF-κB downregulation. In addition, fisetin inhibited UVB-induced NF-κB activation by inhibiting the translocation of NF-κB into the nucleus. Fisetin also inhibited UVB-induced NO and PGE2 production. The inhibition of the phosphorylation of p-38, NF-κB transcription, and reduction of p-CREB Ser-133 by fisetin may contribute to the suppression of COX-2 expression. Inhibition of the MAP kinase pathway, therefore, prevents the phosphorylation of ERK, thereby enhancing the expression of type I procollagen. The scavenging of ROS by fisetin may lead to a blockage of the MAP kinase pathway, which, in turn, may inhibit the activation of NF-κB and AP-1 and, therefore, inhibit the expression of MMP-1, MMP-3, MMP-9, and COX-2. MMPs play a vital role in UV-induced skin aging and degrade ECM in the dermis, resulting in the structural dysfunction of the skin. MMP-1 is a major enzyme that degrades collagen; MMP-3 activates proMMP-1; and MMP-9 degrades collagen fragments already degraded by MMP-1.3 It has been shown that MMP-1 is expressed when the skin is exposed to UV, which induces a decrease of collagen and wrinkle formation.5,6,42,43 Therefore, fisetin-inhibited MMP-1, MMP-3, and MMP-9 attenuated collagen degradation, thereby protecting skin from aging and photoaging. With regard to MMP expression, MAP kinase signaling cascades activate AP-1, a transcription factor and a complex containing c-Fos and c-Jun, and activate AP-1dependent gene expression. The results indicate that fisetin suppressed the overexpression of UVB-induced MAP kinase/ AP-1/MMPs and increased collagen content in human fibroblasts. In summary, our results demonstrate that fisetin attenuated UVB-induced oxidative stress, photodamage, and inflammation

Funding

This study was sponsored by the National Science Council (NSC99-2320-B-039-012-MY3 and NSC99-2622-B-039-001CC3), Taipei, Taiwan, and the China Medical University (CMU99-S-39), Taichung, Taiwan. Notes

The authors declare no competing financial interest.



ABBREVIATIONS USED AP-1, activator protein-1; COX-2, cyclooxygenase-2; CREB, cAMP response element-binding protein; ECM, extracellular matrix; IκB, inhibitor κB; iNOS, inducible nitrite oxide synthase; MMP, matrix metalloproteinase; MAP kinase, mitogen-activated protein kinase; NO, nitric oxide; PG, prostaglandin; UV, ultraviolet



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DOI: 10.1021/jf502500t J. Agric. Food Chem. XXXX, XXX, XXX−XXX