Article pubs.acs.org/JAFC
Youngiasides A and C Isolated from Youngia denticulatum Inhibit UVB-Induced MMP Expression and Promote Type I Procollagen Production via Repression of MAPK/AP-1/NF-κB and Activation of AMPK/Nrf2 in HaCaT Cells and Human Dermal Fibroblasts Myungsuk Kim, Young Gyun Park, Hee-Ju Lee, Sue Ji Lim, and Chu Won Nho* Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 210-340, Republic of Korea ABSTRACT: This study investigated the effects of youngiaside A (YA), youngiaside C (YC), and Youngia denticulatum extract (YDE) on extrinsic aging and assessed its molecular mechanisms in UVB-irradiated HaCaT keratinocytes and human dermal fibroblasts (HDFs). The results showed that YA, YC, and YDE decreased matrix metalloproteinase (MMP) expression and production in HaCaT cell and HDFs and increased collagen expression and production in HDFs. In addition, YA, YC, and YDE significantly increased antioxidant enzyme expression, thereby down-regulating UVB-induced reactive oxygen species (ROS) production and ROS-induced mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) signaling in HaCaT cells. Furthermore, YA, YC, and YDE reduced phosphorylation of IκBα and IKKα/β, blocked nuclear factor-κB (NF-κB) p65 nuclear translocation, and strongly suppressed pro-inflammatory mediators. Finally, YA, YC, and YDE augmented UVB-induced adenosine monophosphate activated protein kinase (AMPK) phosphorylation and YA and YC did not inhibit MMP-1 production in AMPK inhibitor or nuclear factor-erythroid 2-related factor-2 (Nrf2) siRNA-treated HaCaT cells. The results suggest that these compounds could be potential therapeutic agents for prevention and treatment of skin photoaging. KEYWORDS: youngiaside, Youngia denticulatum, UV, MMPs, type I procollagen
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INTRODUCTION
The adenosine monophosphate activated protein kinase (AMPK) cascade is a regulator and sensor of cellular energy status. Stresses that consume cellular ATP, such as oxidative stress, hypoxia, nutrient deprivation, and metabolic poisoning, can activate AMPK.9 Recently, it was reported that AMPK activation plays an important role in MMP-1 expression in human dermal fibroblasts (HDFs) by regulating the upstream and downstream signaling of AMPK.10 Thus, AMPK activators could be used to prevent UV-induced skin photoaging. Nuclear factor-erythroid 2-related factor-2 (Nrf2) and Kelch-like ECH associated protein 1 (Keap1) coordinate the transcriptional induction of a large number of antioxidantmetabolizing enzymes, thus affirming the important role of these two proteins.11 Nrf2 was recently shown to play a central role in protecting cells from UV-induced apoptosis in vitro and acute sunburn reactions in vivo.12,13 Furthermore, the Nrf2-Keap1 pathway plays an important role in preventing photoaging by maintaining high levels of antioxidants, such as glutathione, in the skin.14 Thus, Nrf2 activators may also be beneficial for preventing skin photoaging. Youngia denticulatum is a plant native to Korea, the early sprouts of which have traditionally been eaten as wild vegetables.15 Y. denticulatum contains many phenolic compounds, including chlorogenic acid, dicaffeoylquinic acid, and chicoric acid, all of which exhibit antioxidant activity.15−18 We recently reported that youngiasides A, B, and C isolated from
Skin photoaging is classified into extrinsic aging and intrinsic aging. Extrinsic aging can be triggered by ultraviolet (UV) radiation, stress, or smoking and is characterized by wrinkles, laxity, and dryness.1 UV radiation consists of three components, UVA, UVB, and UVC. Whereas UVA and UVB reach the Earth in sufficient amounts to damage the skin, UVC is almost completely absorbed by the ozone layer.2 UVB is particularly damaging, as it penetrates the epidermis and the upper part of the dermis, where it damages keratinocytes and leads to sunburn, photoaging, and skin cancer.3 UVB radiation activates activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) in the skin, thereby increasing the production of inflammatory cytokines and matrix metalloproteinases (MMPs) in keratinocytes.4 These cytokines stimulate fibroblasts to promote the production of MMPs, which degrade the extracellular matrix (ECM).5 UVB radiation also promotes the generation of intracellular reactive oxygen species (ROS). ROS induce many harmful effects including DNA damage, inflammatory reactions, and damage to ECM integrity.6 UVB-induced ROS production triggers growth factor receptors, cell surface cytokines, and mitogen-activated protein kinase (MAPK) cascades such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase, which in turn regulate AP-1.7 Increased AP-1 activity increases MMP-1 production and decreases type I procollagen production.8 The transcriptional activity of AP-1, a heterodimer composed of c-Jun and c-Fos, is dependent on the degree of c-Jun phosphorylation and c-Fos expression.8 © 2015 American Chemical Society
Received: Revised: Accepted: Published: 5428
January 26, 2015 May 20, 2015 May 21, 2015 May 21, 2015 DOI: 10.1021/acs.jafc.5b00467 J. Agric. Food Chem. 2015, 63, 5428−5438
Article
Journal of Agricultural and Food Chemistry
amounts of protein (30 μg) from each sample were loaded and separated by 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). Membranes were initially blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and then incubated with primary antibodies for 16 h at 4 °C. After three washes in Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with horseradish peroxidaselinked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Proteins were detected with enhanced chemoluminescence reagents (Amersham Biosciences, Little Chalfont, UK) and visualized using an ECL Advanced system (GE Healthcare, Hatfield, UK). Enzyme-Linked Immunosorbent Assay (ELISA). HaCaT and HDF cells were cultured in 24-well plates (1 × 105 cells/well) and pretreated with YDE, YA, and YC for 1 h. After washing with PBS, cells were irradiated with UVB (15 mJ/cm2) through a thin layer of PBS. After UVB irradiation, HaCaT and HDF cells were incubated with serum-free DMEM containing YDE, YA, and YC. Cell culture medium was collected after 24 h, and MMP-1 and type I procollagen productions were quantified using a human MMP-1 ELISA kit (QIA55; Merck & Co. Inc., Whitehouse Station, NJ, USA) and a procollagen type I C-peptide enzyme immunoassay kit (MK101; Takara, Shiga, Japan), respectively. Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from cell pellets. Total RNA was quantified by spectrophotometry at 260 nm. The cDNA was synthesized in a 20 μL reaction containing 2 μg of total RNA, oligo (dT), and Reverse Transcription Premix (ELPIS-Biotech, Daejeon, Korea). PCR amplification of the cDNA products (3 μL) was performed with PCR premix (ELPISBiotech, Daejeon, Korea) and the following primer pairs (Bioneer, Daejeon, Korea): MMP-1 forward, 5′-ATT CTA CTG ATA TCG GGG CTT TGA-3′, and MMP-1 reverse, 5′-ATG TCC TTG GGG TAT CCG TGT AG-3′ (409 bp); MMP-2 forward, 5′-ATT CTA CTG ATA TCG GGG CTT TGA-3′, and MMP-2 reverse, 5′-ATG TCC TTG GGG TAT CCG TGT AG-3′ (409 bp); MMP-3 forward, 5′-TGA GGA CAC CAG CAT GAA CC-3′, and MMP-3 reverse, 5′-ACT TCG GGA TGC CAG GAA AG-3′ (248 bp); MMP-9 forward, 5′- GGG CCG CTC CTA CTC TGC CT-3′, and MMP-9 reverse, 5′-TCG AGT CAG CTC GGG TCG GG-3′ (296 bp); collagen, type I, α1 (COL1A1) forward, 5′-CTC GAG GTG GAC ACC ACC CT-3′, and COL1A1 reverse, 5′-CAG CTG GAT GGC CAC ATC GG-3′ (366 bp); collagen, type III, α1 (COL3A1) forward, 5′-TGG TGC CCC TGG TCC TTG CT-3′, and COL3A1 reverse, 5′-TAC GGG GCA AAA CCG CCA GC-3′ (87 bp); tumor necrosis factor α (TNF-α) forward, 5′-AGC CGC ATC GCC GTC TCC TA3′, and TNF-α reverse, 5′-CAG CGC TGA GTC GGT CAC CC-3′ (163 bp); prostaglandin-endoperoxide synthase 2 (COX-2) forward, 5′-GCC AGC TTT CAC CAA CGG GC-3′, and COX-2 reverse, 5′-CTC CTG CCC CAC AGC AAA CCG-3′ (220 bp); interleukin 6 (IL-6) forward, 5′-TCG AGC CCA CCG GGA ACG AAA-3′, and IL6 reverse, 5′-AGG CAA CTG GAC CGA AGG CG-3′ (94 bp); p65 forward, 5′-CGC GCC GCT TAG GAG GGA GA-3′, and p65 reverse, 5′-GGG CCA TCT GCT GTT GGC AGT-3′ (197 bp); p50 forward, 5′-AGC AGC GTG GGG ACT ACG AC-3′, and p50 reverse, 5′-GGC TGG GGT CTG CGT AGG GA-3′ (345 bp); superoxide dismutase 1 (SOD1) forward, 5′-ATG GCG ACG AAG GCC GTG TG-3′, and SOD1 reverse, 5′-GAC CAC CAG TGT GCG GCC AA3′ (360 bp); heme oxygenase 1 (HO-1) forward, 5′-AGG GAA TTC TCT TGG CTG GC-3′, and HO-1 reverse, 5′-GAC AGC TGC CAC ATT AGG GT-3′ (237 bp); Nrf2 forward, 5′-CAG CGA CCT TCG CAA ACA AC-3′, and Nrf2 reverse, 5′-CAT GAT GAG CTG TGG ACC GT-3′ (464 bp); and β-actin forward, 5′-TCA TGT TTG AGA CCT TCA A-3′, and β-actin reverse, 5′-GTC TTT GCG GAT GTC CAC G-3′ (517 bp). Prior to PCR amplification, primers were denatured at 94 °C for 5 min. Amplification consisted of 28 cycles as follows: 30 s denaturation at 94 °C, 1 min annealing at 56 °C, and 1 min extension at 72 °C, followed by a final 5 min extension at 72 °C. PCR was performed on a GeneAmp PCR System 2700
Y. denticulatum induce the detoxification enzymes, quinone reductase and CYP1A1, through activation of Nrf2 and the aryl hydrocarbon receptor (AhR).19 We also reported that Y. denticulatum protects liver and retinal cells from oxidative stress-induced damage in vitro and in vivo.15,20,21 However, it remains unknown whether Y. denticulatum and its constituents prevent skin photoaging in HaCaT and HDF cells. Thus, this study was performed to investigate the effects of Y. denticulatum extract (YDE) and its active compounds, youngiasides A (YA) and C (YC), on the production of MMPs and type I procollagen in UVB-irradiated HaCaT cells and HDFs and to determine their underlying mechanisms.
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MATERIALS AND METHODS
Chemical Reagents. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma Chemicals (St. Louis, MO, USA). Antibodies against superoxide dismutase (SOD) 1, heme oxygenase (HO)-1, Nrf2, cyclooxygenase (COX)-2, phospho-Akt, phosphoSAPK/JNK (Thr183/Tyr185), SAPK/JNK (Thr183/Tyr185), phosphop44/42 MAPK (Erk1/2), p44/42 MAPK (Erk1/2), phospho-p38, p38, phospho-IκBα (Ser32/36), IκBα, phospho-NF-κB p65 (Ser536), NF-κB p65, phospho-IκB kinase (IKK) α/β (Ser176/180), IKKα, phospho-c-Jun (Ser243), c-Jun, phospho-c-Fos (Ser32), phosphoAMPKα (Thr172), AMPKα, phospho-acetyl-CoA carboxylase (Ser79), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from HyClone Laboratories (Logan, UT, USA). All other chemicals and reagents used were of analytical grade. Plant Material and Preparation of YA and YC. Whole plants (the aerial component and roots) of Y. denticulatum were collected in August 2011 at the Wild Vegetable Experiment Station, Gangwon ARES, Korea, and were identified by one of the authors (C.Y.K.). A voucher specimen (D-043) was deposited at the Natural Product Research Center, KIST Gangneung Institute, Korea. Isolation of chemical constituents (Figure 1A) from Y. denticulatum was performed as previously described.19 Cell Culture and UVB Irradiation. HaCaT and HDF cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM supplemented with penicillin (120 units/mL), streptomycin (75 μg/mL), and 10% FBS in an atmosphere of 5% CO2 at 37 °C. HaCaT and HDF cells were maintained until 80% confluence and then pretreated with various concentrations of YDE, YA, and YC. After a 1 h incubation, the culture medium was replaced with 1.5 mL of phosphate-buffered saline (PBS). Cells were then exposed to UVB light (15 mJ/cm2) with a 312 nm UVB light source (VL-6.LM; Vilber Lourmat, Marne-la-Vallée Cedex 1, France). UV strength was measured with a Waldman UV meter (model 585100; Waldman, Wheeling, IL, USA). After UVB irradiation, cells were treated with various concentrations of YDE (5 or 10 μg/mL), YA, and YC (5 or 10 μM) in serum-free medium. Cell Viability. Cell viability was determined using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) colorimetric assay. HaCaT and HDF cells were maintained until 80% confluence and then pretreated with various concentrations of YDE, YA, and YC. After a 1 h incubation, the cells were exposed to UVB (15 mJ/cm2) and treated with various concentrations of YDE, YA, and YC for 24 h in serum-free medium. Cell medium was replaced with 200 μL of MTT (0.5 mg/mL) and incubated for an additional 3 h. After washing the cells, the insoluble formazan crystals were dissolved in 200 μL of dimethyl sulfoxide. Absorbance at 540 nm was measured by spectrophotometry using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Western Blot Analysis. HaCaT cells were lysed in RIPA buffer containing protease inhibitors and then incubated on ice for 10 min. Protein concentrations of cell lysates were determined by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal 5429
DOI: 10.1021/acs.jafc.5b00467 J. Agric. Food Chem. 2015, 63, 5428−5438
Article
Journal of Agricultural and Food Chemistry
Figure 1. continued
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DOI: 10.1021/acs.jafc.5b00467 J. Agric. Food Chem. 2015, 63, 5428−5438
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effects of Youngia dendiculatum extract (YDE), youngiaside A (YA), and youngiaside C (YC) on MMP-1 production and MMPs mRNA expression in UVB-irradiated HaCaT cells and human dermal fibroblasts (HDF). (A) Chemical structure of constituents from YDE. HaCaT cells and HDF were treated with different concentrations of YDE and its active compounds, chicoric acid (CRA), 3,5-dicaffeoylquinic acid (DCQA), chlorogenic acid (CA), luteolin 7-O-glucuronide (Lute-glu), youngiaside A (YA), youngiaside B (YB), youngiaside C (YC), for 1 h and then exposed to UVB irradiation (15 mJ/cm2). At 24 h after UVB exposure, (B, C) release of MMP-1 into the culture media was determined by ELISA in HaCaT cell and HDF, respectively. (D, E) mRNA expression of MMP-1, -2 ,-3, and -9 was measured by RT-PCR with regard to YDE, YA, and YC in HaCaT cells. β-Actin was used as an internal control for RT-PCR. Epigallocatechin gallate (EGCG) was used as a positive control. Results are expressed as the mean ± SD (% control) of three independent experiments. (#) P < 0.05 compared with vehicle control; (∗) P < 0.05 compared with UVBtreated cells. (Applied Biosystems, Foster City, CA, USA). PCR products were separated by 1.5% agarose gel electrophoresis and visualized with ethidium bromide. β-Actin was used as an internal control. A densitometer was used for RT-PCR on DIG chemiluminescent film (volume of all markers/volume of β-actin). Measurement of ROS Production. HaCaT cells were pretreated with YDE, YA, and YC for 1 h, washed with PBS, and stained with 40 μM DCFH-DA for 30 min. Cells were then irradiated with UVB (15 mJ/cm2), and changes in fluorescence intensity were measured using a microplate reader (Bio-Tek Instruments) at excitation and emission wavelengths of 485 and 530 nm, respectively. siRNA Treatment. The siRNAs targeted against Nrf2 were designed by and purchased from Cell Signaling (Beverly, MA, USA). Transfection of siRNAs (100 pmol) into HaCaT cells was performed with Lipofectamine RNAiMAX (Invitrogen). Statistical Analysis. All experiments were repeated at least three times, and each experiment was performed in triplicate. Results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Group differences were assessed by one-way analysis of variance (ANOVA), followed by Scheffe’s test. A P value of
