Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 9616574, 13 pages https://doi.org/10.1155/2018/9616574

Research Article Aster glehni Extract Containing Caffeoylquinic Compounds Protects Human Keratinocytes through the TRPV4-PPAR�-AMPK Pathway

Yong-Jik Lee ,1 Yoo-Na Jang,1 Yoon-Mi Han,1 Hyun-Min Kim,1,2 Changbae Jin ,3 Hyoung Ja Kim ,3 and Hong Seog Seo 1,2

1 CardiovascularCenter,KoreaUniversityGuroHospital,148,Gurodong-ro,Guro-gu,Seoul08308,RepublicofKorea 2Department of Medical Science, Korea University College of Medicine (BK21 Plus KUMS Graduate Program), Main building 6F Room 655. 73, Inchon-ro (Anam-dong 5-ga), Seongbuk-gu, Seoul 136-705, Republic of Korea 3Molecular Recognition Research Center, Materials and Life Science Research Division, Korea Institute of Science and Technology, Hwarangno 14 gil 5, Seoul 136-791, Republic of Korea

Correspondence should be addressed to Hyoung Ja Kim; [email protected] and Hong Seog Seo; [email protected]

Received 29 August 2018; Revised 21 October 2018; Accepted 12 November 2018; Published 9 December 2018

Academic Editor: Simona Martinotti

Copyright © 2018 Yong-Jik Lee et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Aster glehni (AG) has been used in cooking and as a medicine to treat various diseases for over hundreds of years in Korea. To speculate the protective efects of AG on skin barrier, we estimated the levels of biomarkers related to skin barrier protection in human keratinocytes, HaCaT cells treated with sodium dodecyl sulfate (SDS), or 2,4-dinitrochlorobenzene (DNCB). Te protein levels for keratin, involucrin, defensin, tumor necrosis factor alpha (TNF�), peroxisome proliferator-activated receptor � delta (PPAR�), 5 adenosine monophosphate-activated protein kinase (AMPK), palmitoyltransferase long chain base subunit 2 (SPTLC2), and transient receptor potential cation channel subfamily V member 4 (TRPV4) were evaluated using western blotting or immunocytochemistry in HaCaT cells. AG extract increased the protein levels of PPAR�, phosphorylated AMPK, SPTLC2, keratin, involucrin, and defensin compared to the SDS or DNCB control group. However, TNF� expression increased by SDS or DNCB was decreased with AG extract. Te order of action of each regulatory biomarker in AG pathway was identifed TRPV4�→ PPAR��→AMPK from antagonist and siRNA treatment studies. AG can ameliorate the injury of keratinocytes caused by SDS or DNCB through the sequential regulation of TRPV4�→ PPAR��→AMPK pathway.

1. Introduction corneodesmosome, and intercorneocyte lipid. Te cornifed envelope comprises , involucrin, loricrin, Te skin, also called integument, consists of three major and flaggrin, and it serves as a complete permeability barrier layers, namely epidermis, dermis, and hypodermis (subcutis). through the formation of the multilayered structure of inter- In particular, the epidermis is the outer region of the skin and corneocyte lipid. Interconeocyte lipids comprise ceramide, has four layers—stratum basale, stratum spinosum, stratum cholesterol, free fatty acids, and cholesterol sulfate, and, granulosum, and stratum corneum (the outermost thin layer among them, ceramide content is the highest. Te main of the skin). Keratinocytes are the main cells that make functions of the skin are to prevent the loss of body fuids, up the epidermis, and they contribute to the construction attack of toxins, and invasion of pathogens [1–3]. Terefore, of a defense wall for the body through keratinization. In the impairment of skin permeability barrier function is not stratum spinosum, keratinocytes produce keratin1 and 10, a simple mechanical destruction of the outer layer of the and keratin10 is a main constituent of desmosome. In general, skin. Te damaged skin can cause severe immune reactions the permeability barrier of skin is provided by stratum through the invasion and colonization of various pathogens. corneum and is composed of corneocytes, cornifed envelope, Sodium dodecyl sulfate (SDS) is generally known as a skin 2 Evidence-Based Complementary and Alternative Medicine irritant as well as an anionic surfactant. As a detergent, SDS Waters, Worcester, MA, USA). Separation was performed induces skin surface polarity and skin barrier disruption and using a Luna C18 column (5 �m, 250 × 4.6 mm, Phenomenex, can facilitate epidermal lipid extraction [4]. In addition, SDS Torrance, CA, USA) with a sample injection volume of 10 ∘ elevates transepidermal water loss from the stratum corneum �Lat30C. Te mobile phase was a gradient of acetonitrile and induces infammation [5, 6]. and 1% phosphoric acid. Te gradient system was as follows: Although atopic dermatitis (AD) develops in early in- 20% acetonitrile (0 min), 20% acetonitrile (0∼10 min), 30% fancy, it is also seen in adults. In general, AD is a chronic and acetonitrile (10∼20 min), 40% acetonitrile (20∼30 min), 80% pruritic infammatory skin disease characterized by increased acetonitrile (30∼40 min), and 100% acetonitrile (40∼50 min). immunoglobulin E (IgE) production, relapsing eczematous Te fow rate of the mobile phase was 1.0 ml/min. Organic skin lesions, infltration of infammatory immune cells, and solvents used in extraction procedure and HPLC analysis defective skin barrier, and its underlying cause is almost were purchased from Sigma-Aldrich (St. Louis, MO, USA). unknown [7, 8]. With the advancement of industrialization, AD has become more prevalent. Terefore, fnding efective 2.2. Cell Culture. HaCaT cells and human keratinocytes were remedies for AD is very urgent and socioeconomically cultured in Dulbecco’s modifed Eagle’s medium (DMEM) important. 2,4-Dinitrochlorobenzene (DNCB) is reported to containing 10% fetal bovine serum (FBS) and 1% antibiotic- ∘ induce contact dermatitis and AD [9, 10]. antimycotic solution (AA) in 37 Cand5%CO2 incubator. Aster glehni (AG) has been used as an edible food, and Te cell culture medium was replaced with new DMEM every medicine for over hundreds of years in Korea. 48-72 h. HaCaT cells within 5∼17 passages were plated at a 4 6 In Korean traditional medicine, Aster glehni (AG) has density of 1×10 cellsperwellin8-wellchamberslide,or1×10 been used to cure fever, pain, phlegm, and cough. Other cells per well in a six-well culture plate in DMEM containing functions of AG have been reported previously as follows. 10%FBSand1%AA.Tecellswereculturedfor24to48hin ∘ An ethyl acetate extract of AG inhibited the protein expres- 5% CO2 incubator at 37 C. Ten, the medium was changed to sion of tyrosinase and tyrosinase-related protein 1, which DMEM containing 1% FBS. Tereafer, the cells were treated are involved in melanin biosynthesis in melanocytes [11]. by DNCB (5 �mol; Sigma-Aldrich, Louis, MO, USA) or SDS In another study, the ethyl acetate extract of AG showed (30 �mol; Sigma-Aldrich) along with AG extract (50 �g) antioxidant efect and inhibited the protein expression of and PPAR� antagonist, GSK0660 (50 �mol; Sigma-Aldrich), induced nitric oxide synthase (iNOS), which is involved in AMPK antagonist, Compound C (1 �mol; Sigma-Aldrich), or infammation [12]. TRPV4 antagonist, GSK2193874 (50 nmole; Sigma-Aldrich) To clarify the efects and mechanism of AG on skin for 24 hours. HaCaT cells were donated from Dr. Sang- permeability barrier protection, we estimated the expression Wook Son. All reagents for cell culture were purchased from levels of biomarkers related to permeability barrier mainte- WELGENE Inc. (Daegu, Republic of Korea). Te treating nance in HaCaT cells treated with SDS or DNCB which is concentrations of all reagents were fnal concentrations. a skin irritant or an inducer of dermatitis containing atopic dermatitis. Particularly, in previous studies, peroxisome 2.3. Immunocytochemistry (ICC). Te cells in chamber slide proliferator-activated receptor � (PPAR�), AMP-activated were fxed by ice-cold methanol for 15 min. Te fxed protein kinase (AMPK), and transient receptor potential cells reacted with 0.3% hydrogen peroxide (H2O2)solution cation channel subfamily V member 4 (TRPV4) participated containing 0.3% normal horse serum for 5 min to remove in the maintaining of structural integrity of keratinocytes peroxidase activity in cells. Afer washing with phosphate- [13–17] , and in general the impairment of skin permeability bufered saline (PBS) for 5 min, the cells reacted with diluted barrier mainly composed by keratinocytes induces dermatitis normal blocking serum for 20 min, followed by treatment containing atopic dermatitis [18, 19]. Terefore this study was with diluted primary antibodies for 1 h. Te primary antibod- conducted with a focus on the roles of PPAR�,AMPK,and ies for involucrin and TNF� were obtained from Novus (Lit- TRPV4 as the regulators in the functions of AG. tleton, CO 80120, USA). Te primary antibody for defensin�1 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, 2. Materials and Methods Texas, USA) and that for pan-keratin was purchased from Abcam (Cambridge, UK). Afer washing with PBS, the cells 2.1. Plant Materials, Extraction Procedure, and High-Per- were treated with secondary antibody for 30 min. Afer PBS formance Liquid Chromatography (HPLC) Analysis. Plant washing, the cells were treated with premixed VECTAS- materialsusedinthisexperimentswereparboiledanddried TATIN ABC reagent solution for 30 min. Te cells were Aster glehni F. Schmidt (Compositae), and the extraction washed with PBS and allowed to react with DAB substrate procedure and high-performance liquid chromatography solution until proper color change was observed. Afer wash- (HPLC) analysis referred to our already published paper [20]. ing with tap water for 3 min, the cells were counterstained To acquire a methanol-soluble extract, 12 kg chopped AG with hematoxylin. Te cells were washed with tap water, air- were extracted three times with 70 L methanol at room tem- dried, and fnally mounted. Te number of each group was perature. Te dried extract residue of 2.6 kg was suspended three every experiments. All experiments were repeated over in water and then partitioned successively with ethyl acetate. three times. Te validity of ICC results was proved by the Te ethyl acetate extract of AG was analyzed by reverse-phase comparison with negative control. Te immunocytochem- high-performance liquid chromatography (HPLC) (Waters istry kit (containing secondary antibody) was purchased 1500 Series System), with a 2996 PDA Detector (254 nm, from Vector laboratories (Burlingame, CA, USA). Evidence-Based Complementary and Alternative Medicine 3

2.4. Western Blot Analysis. Protein concentration in sample size- 212 base pairs); forward 5’- ACAAGTTGTGGCTCA- was estimated by the Bradford method. Te extracted pro- CCCAA-3’ and reward 5’-TGGCACATGGTCATCATCAA- teins (10 �g) were loaded onto 10% SDS-polyacrylamide gels, 3’ for human AMPK�1 (product size- 147 base pairs); forward and protein blotting on nitrocellulose membranes was per- 5’- GCTTGGTCACTTCGTGGCTA-3’ and reward 5’-CAA- formed for 90 min. Te membranes were blocked overnight ACCGCTTCCAACTCAAA-3’ for human �-actin (product with 5% skim milk and washed three times for 10 min with size- 281 base pairs). Te reaction mixture containing cDNA ∘ tris-bufered saline containing 0.05% tween 20 (TBS-T). Te was preheated for 5 minutes at 95 C as an initial denaturation primary antibodies were allowed to bind to the membranes at step. Polymerase chain reaction consisted of denaturation ∘ room temperature for 2 h. Te primary antibody for PPAR� step for 20 seconds at 95 C, annealing step for 10 seconds ∘ ∘ was supplied by Abcam. Te primary antibodies for total at 55 C, extension step for 30 seconds at 72 C, and fnal ∘ and phosphorylated forms of AMPK (p-AMPK), and cleaved extension step for 5 minutes at 72 C. Te number of each poly-ADP-ribose polymerase (cPARP) were purchased from group was three every experiment. All experiments were Cell Signaling Technology, Inc. (Danvers, MA, USA). Te repeated over three times. Te normalization for RT-PCR primary antibody for serine palmitoyltransferase 2 (SPTLC results were done with �-actin and GAPDH. TRIzol5 reagent 2)waspurchasedfromNovusandthatfor�-actin was was purchased from Invitrogen (Grand Island, NY, USA). procured from Santa Cruz Biotechnology, Inc. Te dilution Power cDNA Synthesis kit, PCR premix, and DNA ladder conditions for primary antibodies were as follows: PPAR�, (100 ) were bought from Intron Biotechnology 1:500; AMPK, p-AMPK (at Tr172), cPARP, and SPTLC 2, (Seongnam-si, Gyeonggi-do, Korea) 1:1000; and �-actin, 1:800. Afer washing three times with TBS-T for 10 min, the membranes were incubated with sec- 2.6. Small Interfering RNA (siRNA) Transfection. siRNA solu- ondary antibodies (Santa Cruz Biotechnology, Inc.) at room tions for TRPV4, PPAR�,andAMPK�1/2 were prepared temperature for 1 h. Te dilution conditions for secondary as follows: solution A is composed of 6 ul siRNA plus 100 antibodies were as follows: anti-rabbit IgG antibodies for ul transfection medium (DMEM having no FBS and AA, PPAR�, AMPK, p-AMPK, cPARP, and SPTLC 2, 1:5000 and WELGENE Inc.) per well, and solution B is composed of 6 anti-mouse IgG antibody for �-actin, 1:5000. Afer three ul transfection reagent plus 100 ul transfection medium per washes with TBS-T for 10 min and a single TBS wash for well. Te mixture of solutions A and B was stirred thoroughly another 10 min, chemiluminescent substrate and enhancer and then incubated for 30 min at room temperature. Te solution (Bio-Rad, Hercules, CA, USA) were applied to the incubated mixture was added to cells, the total volume was 1 membranes to determine the protein expression rate. Images ml together with medium, and the cells treated with siRNA ∘ were processed manually with Kodak GBX developer and were incubated for 6 hr in CO2 incubator at 37 C. Afer fxer reagents (CARESTREAM HEALTH, INC., Rochester, incubation in CO2 incubator, 1 ml DMEM containing 20% NY, USA) and analyzed using ImageJ program. Te num- FBSwasaddedtothewell.siRNAsandtransfectionreagent ber of each group was three every experiments. All experi- were purchased from Santa Cruz Biotechnology. ments were repeated over three times. �-Actinwasusedasan Te sequences for siRNAs are as follows. Te human internal control to normalize the loaded . TRPV4 siRNA is a pool of 3 diferent siRNA duplexes: the sequences of frst duplex are sense 5’-CCUUCCGUGACA- 2.5. Semiquantitative Reverse Transcription Polymerase Chain UCUACUATT-3’ and antisense 5’-UAGUAGAUGUCA- Reaction (RT-PCR). Total RNA was extracted via TRIzol5 CGGAAGGTT-3’; the sequences of second duplex are sense reagent according to the manufacturer’s instructions. Com- 5’- GAGAACACCAAGUUUGUUATT-3’ and antisense 5’- plementary DNA was synthesized from total RNA using UAACAAACUUGGUGUUCUCTT-3’; the sequences of the Power cDNA Synthesis kit, and polymerase chain third duplex are sense 5’-CUGCUCUACUUCAUCUAC- reactions for keratin, involucrin, defensin�1, TNF�,TRPV4, UTT-3’ and antisense 5’- AGUAGAUGAAGUAGAGCA- PPAR�,AMPK�1, and �-actin were performed using PCR GTT -3’. Te human PPAR� siRNA is a pool of 3 diferent Premix kits. Te primer sequences used were as follows: siRNA duplexes: the sequences of frst duplex are sense forward 5’-CCCTTCTTTCGAGGTCTTGG-3’ and reward 5’-GGUUACCCUUCUCAAGUAUTT-3’ and antisense 5’- 5’-TGCTGGAAGGTAGGCACAAG-3’ for human keratin AUACUUGAGAAGGGUAACCTT-3’; the sequences of (product size- 239 base pairs); forward 5’- CACCTAGAG- second duplex are sense 5’- CCUUCAGUGAUAUCAUUG- GAGCAGGAGGG-3’ and reward 5’-CAGGTGCTTTGG- ATT-3’ and antisense 5’-UCAAUGAUAUCACUGAAG- CTGTCCTA-3’ for human involucrin (product size- 216 GTT-3’; the sequences of third duplex are sense 5’-CUC- base pairs); forward 5’- TGAGATGGCCTCAGGTGGTA- CUGUCUUCAGAGCAAATT-3’ and antisense 5’- UUU- 3’ and reward 5’-AGCTCACTTGCAGCACTTGG-3’ for GCUCUGAAGACAGGAGTT-3’. Te human AMPK�1/2 human defensin�1 (product size- 163 base pairs); forward siRNA is a pool of 3 diferent siRNA duplexes: the sequences 5’- CCCTCAACCTCTTCTGGCTC-3’ and reward 5’-AGC- of frst duplex are sense 5’-GAUGUCAGAUGGUGAAUU- TGTAGGCCCCAGTGAGT-3’ for human TNF� (product UTT-3’ and antisense 5’-AAAUUCACCAUCUGACAU- size- 187 base pairs); forward 5’- GTTCATGAAGAAATG- CTT-3’; the sequences of second duplex are sense 5’- CCA- CCCTG-3’ and reward 5’-GTGAGGATGATGTAGGTCAC- CUGCAAUACUAAUUGATT-3’ and antisense 5’-UCA- 3’ for human TRPV4 (product size- 522 base pairs); forward AUUAGUAUUGCAGUGGTT-3’; the sequences of third 5’- AAGGCCTTCTCCAAGCACAT-3’ and reward 5’-AAG- duplex are sense 5’-CUACUGGAUUUCCGUAGUATT-3’ ACGTGCACGCTGATCTC-3’ for human PPAR� (product and antisense 5’- UACUACGGAAAUCCAGUAGTT-3’. Te 4 Evidence-Based Complementary and Alternative Medicine

(1) 5-CQA: 5-caffeoylquinic acid 0.60 (1) (2) 3,4-DCQA: 3,4-dicaffeoylquinic acid O２ 2 OR (3) 3,5-epi-DCQA: 3,5-epi-dicaffeoylquinic acid 6 5 4 3 0.50 (4) 3,5-DCQA: 3,5-dicaffeoylquinic acid O２ 4 OH ２ OOC 4 3 HO (5) 4,5-DCQA: 4,5-dicaffeoylquinic acid 1 6 5 (6) 3,5-DCQA-Me: methyl 3,4-dicaffeoylquinate 1 2 3 0.40 (7) 4,5-DCQA-Me: methyl 4,5-dicaffeoylquinate COOH OR OH O２2 (4) . R = Caffeoyl 0.30 . ２1 =２2 =２3 =（, ２4 =＃；ff？ＩＳＦ AU (3) (5) . ２1 =（, ２2 =２3 =＃；ff？ＩＳＦ, ２4 =（ OH 0.20 . ２1 =（, ２2 =＃；ff？ＩＳＦ, ２3 =（, ２4 =＃；ff？ＩＳＦ ＃；ff？ＩＳＦ = . ２1 =２2 =（, ２3 =２4 =＃；ff？ＩＳＦ OH 0.10 (2) . ２ =－？, ２ =＃；ff？ＩＳＦ, ２ =（, ２ =＃；ff？ＩＳＦ O (6) 1 2 3 4 (7) 0.00 . ２1 =－？, ２2 =（, ２3 =２4 =＃；ff？ＩＳＦ 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Minutes (a) (b)

Figure 1: HPLC analysis chromatogram (a) and the structures (b) of constituent chemicals for Aster glehni ethyl acetate extract. Te main phytochemicals identifed from Aster glehni are cafeoylquinic acids of seven kinds: (1) 5-CQA: 5-cafeoylquinic acid, (2) 3,4-DCQA: 3,4-dicafeoylquinic acid, (3) 3,5-epi-DCQA: 3,5-epi-dicafeoylquinic acid, (4) 3,5-DCQA: 3,5-dicafeoylquinic acid, (5) 4,5-DCQA: 4,5- dicafeoylquinic acid, (6) 3,5-DCQA-Me: methyl 3,4-dicafeoylquinic acid, and (7) 4,5-DCQA-Me: methyl 4,5-dicafeoylquinic acid. number of each group was three every experiment. All exper- of serine palmitoyltransferase. Terefore, we estimated the iments were repeated over three times. Te experimental protein levels of PPAR�,AMPK,andSPTLC2inHaCaT validity was supported by control siRNA treatment group. cells treated with SDS or DNCB along with AG. AG extract increased the protein levels for PPAR�,p-AMPK,SPTLC2, 2.7. Statistics. Data are presented as mean ± SE (Stan- and TRPV4 compared to DNCB- and SDS-treated groups. dard Error of Measures). Statistically signifcant diferences However, the elevated protein levels were reversed by the � between two groups were calculated by the unpaired t-test, PPAR antagonist GSK0660 (Figure 2). and one-way ANOVA test was used to compare means of three or more groups with GraphPad Prism sofware. A value 3.3. Aster glehni Extract Increased the Protein and mRNA � of p<0.05 was considered signifcant. Expressions for Keratin, Involucrin, and Defensin 1 of HaCaT Cells in Pathological Conditions; however, TNF� Protein and 3. Results mRNA Expressions Were Decreased by the Extract. Te mRNA Levels for TRPV4, PPAR�,andAMPK�1 Were also Increased 3.1. Te Ethyl Acetate Fraction Extracted from Aster glehni by AG Treatment. Defensins are antimicrobial, antifungal, Consists of Cafeoylquinic Compounds of Seven Kinds Mainly. and antiviral small cationic peptides produced by diferent In our previously published paper, the cafeoylquinic com- cell types, and they include three subfamilies of �-, �-, and pounds of six kinds in AG were discovered by HPLC �-defensins. In keratinocytes, �-defensins are the mainly analysis [20]. But, through further analysis, it was confrmed secreted subtypes [26]. Terefore, in this study, we evaluated that the ethyl acetate fraction extracted from AG mainly the expression of defensin�1, a representative defensin�, consists of cafeoylquinic compounds of seven kinds which together with skin barrier constituents such as keratin and consisted of 5-cafeoylquinic acid, 3,4-dicafeoylquinic acid, involucrin and infammatory cytokine TNF�. 3,5-epi-dicafeoylquinic acid, 3,5-dicafeoylquinic acid (3,5- AG extract increased generally the mRNA levels of ker- DCQA), 4,5-dicafeoylquinic acid, methyl 3,4-dicafeoyl- atin, involucrin, defensin�1, TRPV4, PPAR�,andAMPK�1 quinate, and methyl 4,5-dicafeoylquinate (Figure 1). Te 3,5- related to the protection of keratinocytes in SDS or DNCB epi-dicafeoylquinic acid was newly discovered in further treated condition. But the mRNA level of TNF� was HPLC analysis. 3,5-DCQA was the most abundant in the decreased by AG treatment (Figure 3). AG extract increased seven cafeoylquinic compounds of the AG ethyl acetate the protein expressions of keratin, involucrin, and defensin�1, fraction. which were decreased by DNCB and SDS; however, the elevated protein levels were ofset by the PPAR� antagonist 3.2. Aster glehni Extract Increased the Protein Levels for GSK0660. TNF� expressions increased by DNCB and SDS PPAR�, AMPK, SPTLC2, and TRPV4 of HaCaT Cells in and were decreased by AG extract, but the ameliorating efect the Condition of DNCB or SDS Treatment. Previous studies of AG extract was reversed by GSK0660 (Figure 4). reported that PPAR� and AMPK are involved in cell survival and anti-infammatory response [21–24]. Ceramide is an 3.4. Te mRNA and Protein Expressions of Keratin, Involucrin, important and main lipid constituent in skin barrier, and and Defensin�1 in HaCaT Cells Were Decreased by Antag- serine palmitoyltransferase is an enzyme catalyzing a rate onists for TRPV4 and AMPK. However, TNF� Protein and limiting step in ceramide biosynthesis. In addition, TRPV4 mRNA Expressions Were Increased. To investigate the efects is known to involve in the formation of intercellular junction of TRPV4 and AMPK on the expressions of biomarkers in keratinocytes [25]. SPTLC2 is a long chain base subunit related to skin permeability barrier constituents, defense, Evidence-Based Complementary and Alternative Medicine 5

(p=0.0051) (p=0.0021) ∗∗ ∗∗ (p=0.0086) (p=0.0135) ∗∗ ∗ Ctrl DNCB D+AG D+A+GSK (p=0.0377)(p=0.0223) . (p=0.0206)(p=0.0451) . ∗∗ ∗ ∗ ) PPARδ ) .

ctin . ctin

. -A -A  /  ensity ensity . D D . SPTLC2 AR  . P T LC 2/ ( PP (S

. . TRPV4 Ctrl DNCB D+AG D+A+GSK Ctrl DNCB D+AG D+A+GSK Treatments Treatments (p<0.0001) ∗∗∗ (p=0.0015) (p=0.0099) ∗∗ p-AMPK ∗∗ (p=0.0046) (p=0.0028)(p=0.0246) ∗∗ ∗∗ ∗ . . (p=0.0101) ∗

AMPK ) . K)

ctin . . -A  K/AM P ensity  ensity . D -Actin . D . (TR P V4/ P (p-AM . . Ctrl DNCB D+AG D+A+GSK Ctrl DNCB D+AG D+A+GSK Treatments Treatments

(a) (p=0.0080) (p=0.0152) ∗∗ ∗ (p=0.0148) (p=0.0345) ∗ ∗ (p=0.0178)(p=0.0297)(p=0.0486) Ctrl SDS S+AG S+A+GSK . (p=0.0496) ∗ . ∗∗∗ ) ) PPARδ . ctin ctin . -A -A  /  ensity ensity D D SPTLC2 AR  . . P T LC 2/ ( PP (S . . TRPV4 Ctrl SDS S+AG S+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments (p=0.0006) ∗∗∗ (p=0.0082) ∗∗ p-AMPK (p=0.0024) ∗∗ (p=0.0087) ∗∗ (p=0.0094)(p=0.0269) . ∗∗ ∗ (p=0.0031)(p=0.0310) . ∗∗ ∗ ) AMPK K) . . ctin -A  . K/AM P ensity ensity

 D

-Actin D . . P (p-AM (TR P V4/

. . Ctrl SDS S+AG S+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments

(b)

Figure 2: Western blot analyses for PPAR�, AMPK,andSPTLC2 in HaCaT cells treated with Aster glehni extract, SDS, DNCB,and GSK0660. AG increased the protein levels of biomarkers related to the protection of keratinocytes in DNCB (a) or SDS (b) treated condition. At large, the efects of AG were ofset by PPAR� antagonist treatment. Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test and one-way ANOVA. All experiments were repeated over three times. Te dash-dotted line means the one-way ANOVA result between ctrl and all other groups. Te dotted line means the one-way ANOVA result among all groups except for control. Meaning of indications: Ctrl is an untreated control group, DNCB is a DNCB treated group, D+AG is a DNCB+AG treated group, D+A+GSK is a DNCB+AG+GSK0660 treated group, SDS is a SDS-treated group, S+AG is a SDS+AG treated group, and S+A+GSK is a SDS+AG+GSK0660 treated group. and infammation, the expressions for keratin, involucrin, and defensin�1 markedly decreased compared to the control defensin�1, and TNF� were evaluated via RT-PCR and (Figure 5). immunocytochemistry in HaCaT cells treated with antago- nists for TRPV4 and AMPK. 3.5. Te mRNA and Protein Expressions of TRPV4, PPAR�, Te mRNA and protein levels for TNF� were elevated and AMPK�1 in HaCaT Cells Were Diferently Changed by by TRPV4 antagonist or AMPK antagonist compared to the Antagonists and siRNAs for TRPV4, PPAR�,andAMPK.Te control in HaCaT cells. However, under the same condition, mRNA Expressions of TRPV4, PPAR�,andAMPKIncreased the mRNA and protein expressions of keratin, involucrin, by AG in HaCaT Cells in Pathological Conditions Were 6 Evidence-Based Complementary and Alternative Medicine

Ctrl SDS S+AG DNCB D+AG Keratin

Involucrin C vs.(p=0.0014) C vs.(p=0.0123) ∗∗ ∗ (p=0.0106) (p=0.0419) Defensin ∗ ∗ (p=0.0036)(p=0.0059) TNF-  ∗∗ ∗∗

TRPV4 )  ctin

 -A

PPAR /  ensity

D  AMPK1 eratin (K -Actin  Ctrl SDS S+AG DNCB D+AG Treatments C vs.(p=0.0012) C vs.(p=0.0010) C vs.(p=0.0011) C vs.(p=0.0268) C vs.(p=0.0019) C vs.(p=0.0125) ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ (p 0.0279)(p 0.0133) (p=0.0027) (p=0.0027) (p=0.0204)(p=0.0498) = = ∗∗ ∗∗ ∗∗ ∗∗ . (p=0.0204) (p=0.0498) . (p=0.0039) ∗ ∗ ∗∗ . ) ) ) ctin . . ctin . -A ctin -A  -A / . ensity /  ensity ensity D D

. . D olucrin /  efensin v

n . (D  (TNF- (I

. . . Ctrl SDS S+AG DNCB D+AG Ctrl SDS S+AG DNCB D+AG Ctrl SDS S+AG DNCB D+AG Treatments Treatments Treatments C vs.(p=0.0116) C vs.(p=0.0073) C vs.(p<0.0001) C vs.(p=0.0010) ∗ ∗∗ ∗∗∗ ∗∗ (p 0.0412)(p 0.0141) (p<0.0001)(p 0.0102) (p=0.0119) = = = ∗ ∗∗ ∗∗∗ ∗ . . . (p=0.0212) ∗ ) ) ) . . ctin . ctin ctin -A -A -A /  /  ensity ensity ensity K/  D D P D . . AR  . (TR P V 4 ( PP (AM

. . . Ctrl SDS S+AG DNCB D+AG Ctrl SDS S+AG DNCB D+AG Ctrl SDS S+AG DNCB D+AG Treatments Treatments Treatments Figure 3: RT-PCR for keratin, involucrin, defensinn, TNF�, TRPV4, PPAR�,andAMPK in HaCaT cells treated with Aster glehni extract, SDS,andDNCB. AG generally increased the mRNA levels of biomarkers related to the protection of keratinocytes in SDS or DNCB treated condition. But the mRNA level of TNF� was decreased by AG treatment. Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test and one-way ANOVA. All experiments were repeated over three times. Te dash-dotted line means the one-way ANOVA result between ctrl and other groups. Meaning of indications: Ctrl is an untreated control group, DNCB is a DNCB treated group, D+AG is a DNCB+AG treated group, SDS is a SDS-treated group, and S+AG is a SDS+AG treated group.

Decreased in TRPV4 Antagonist Treated Group. To deter- levels for PPAR and AMPK�1. But AMPK�1/2 siRNA lowered mine the priority of major regulators which are TRPV4, only AMPK mRNA expression (Figure 6(d)). In pathological PPAR�, and AMPK, we estimated the mRNA and protein conditions of HaCaT cells induced by DNCB and SDS levels of TRPV4, PPAR�, and AMPK in HaCaT cells treated treatments, the mRNA levels for TRPV4, PPAR�,andAMPK with siRNAs and antagonists for TRPV4, PPAR�,orAMPK. were decreased compared to control group. But the mRNA In HaCaT cells treated with TRPV4 antagonist, all the expressions of TRPV4, PPAR�,andAMPKloweredinthe protein levels for TRPV4, PPAR�,andp-AMPKweresignif- pathological conditions were increased by AG treatment. But icantly decreased (Figure 6(a)). PPAR� antagonist treatment the increasing efects of AG on the mRNA expressions for lowered the protein levels for PPAR� and p-AMPK; however, TRPV4, PPAR�, and AMPK in pathological conditions were it did not change the TRPV4 protein expression (Figure 6(b)). ofset by TRPV4 antagonist treatment (Figure 6(e)). Te AMPK antagonist, Compound C, decreased the protein expressionforonlyp-AMPK,butitdidnotafecttheprotein 3.6. 3, 5-DCQA Increased the Protein Levels for TRPV4, expressions for TRPV4 and PPAR� (Figure 6(c)). Te results PPAR�, AMPK, SPTLC2, Keratin, Involucrin, and Defensin�1 ofthesiRNAexperimentswereidenticaltothoseoftheantag- of HaCaT Cells. However, TNF� Expression Was Decreased. onists. TRPV4 siRNA treatment decreased all the mRNA 3, 5-DCQA as the most abundant cafeoylquinic acid con- levels for TRPV4, PPAR, and AMPK�1 compared to control stituent of AG extract increased the protein levels for TRPV4, siRNA treated group. And PPAR� siRNA decreased mRNA PPAR�, AMPK, SPTLC2, keratin, involucrin, and defensin�1 Evidence-Based Complementary and Alternative Medicine 7

Keratin Negative Ctrl Ctrl Negative Ctrl Ctrl

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ (p<0.0001) (p<0.0001) (p<0.0001) 100 (p<0.0001) (p<0.0001) (p<0.0001) 100 ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 80 80 ) )

unit 60 unit 60 eratin eratin

K 40 K 40 mage density mage density I I ( arbitrarry ( arbitrarry 20 20

0 0 Ctrl DNCB D+AG D+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments

DNCB D+AG D+A+GSK SDS S+AG S+A+GSK

Involucrin Negative Ctrl Ctrl Negative Ctrl Ctrl

(p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ 100 (p<0.0001) (p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ ∗∗∗ 100 (p<0.0001) (p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ ∗∗∗ 80 80 ) )

unit 60 unit 60 olucrin olucrin v v

n 40 40 n I I mage density mage density I I ( arbitrarry ( arbitrarry 20 20

0 0 Ctrl DNCB D+AG D+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments DNCB D+AG D+A+GSK SDS S+AG S+A+GSK

Defensin Negative Ctrl Ctrl Negative Ctrl Ctrl

(p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p=0.0001) (p<0.0001) (p<0.0001) 100 ∗∗∗ ∗∗∗ ∗∗∗ (p<0.0001) (p<0.0001) (p<0.0001) 100 ∗∗∗ ∗∗∗ ∗∗∗ 80 ) ) 80 unit 60 unit 60 efensin efensin

D 40 D 40 mage density mage density I I ( arbitrarry ( arbitrarry 20 20

0 0 Ctrl DNCB D+AG D+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments DNCB D+AG D+A+GSK SDS S+AG S+A+GSK

TNF Negative Ctrl Ctrl Negative Ctrl Ctrl

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ (p<0.0001) (p<0.0001) (p<0.0001) (p<0.0001) (p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 100 100

80 80 ) )

60 unit 60 unit TNF  TNF  40 40 mage density mage density I I ( arbitrary ( arbitrary 20 20

0 0 Ctrl DNCB D+AG D+A+GSK Ctrl SDS S+AG S+A+GSK Treatments Treatments

DNCB D+AG D+A+GSK SDS S+AG S+A+GSK

Figure 4: Immunocytochemistry for keratin, involucrin, defensin, and TNF� in HaCaT cells treated with Aster glehni extract, SDS, DNCB,andGSK0660. Te abnormal expressions for keratin, involucrin, defensin, and TNF� by SDS or DNCB treatment were normalized with AG treatment; however, the efects of AG were ofset by PPAR�antagonist. Images were taken at 200 × magnifcation. Densities for images were analyzed with Image J program. Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test and one-way ANOVA. All experiments were repeated over three times. Te dash-dotted line means the one-way ANOVA result between ctrl and all other groups. Te dotted line means the one-way ANOVA result between all groups except for control. Meaning of indications: Ctrl is an untreated control group, DNCB is a DNCB treated group, D+AG is a DNCB+AG treated group, D+A+GSK is a DNCB+AG+GSK0660 treated group, SDS is a SDS-treated group, S+AG is a SDS+AG treated group, and S+A+GSK is a SDS+AG+GSK0660 treated group. 8 Evidence-Based Complementary and Alternative Medicine

(p=0.0007) (p<0.0001) (p=0.0257) ∗∗∗ (p=0.0191) TRPV4 AMPK ∗∗∗ ∗ ∗ Ctrl antagonist antagonist (p=0.0027) (p=0.0002) ∗∗ (p=0.0483) ∗∗∗ (p=0.0259) Keratin . ∗ . ∗ (p=0.0021) (p=0.0001) . ∗∗ . ∗∗∗ (p=0.0472)

) ∗ ) Involucrin . ) ) . ctin 8 . . -A ctin ctin ctin /  -A . -A Defensin -A . /  / 

ensity . /  ensity ensity D ensity . D D

D . olucrin TNF v . eratin efensin n .  (TNF-

. (I (K (D

-Actin . . . . Ctrl TRPV4 AMPK Ctrl TRPV4 AMPK Ctrl TRPV4 AMPK Ctrl TRPV4 AMPK Treatment (antagonists) Treatment (antagonists) Treatment (antagonists) Treatment (antagonists)

(a)

Keratin Involucrin Negative Ctrl Ctrl Negative Ctrl Ctrl

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ (p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ 80 80

60 60 ) unit ) unit 40 40 olucrin eratin v K n I mage density mage density

I 20

I 20 ( arbitrarry ( arbitrarry

0 0 Ctrl TRPV4 AMPK Ctrl TRPV4 AMPK Treatments (antagonists) Treatments (antagonists) TRPV4 antagonist AMPK antagonist TRPV4 antagonist AMPK antagonist

Defensin TNF Negative Ctrl Ctrl Negative Ctrl Ctrl (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p<0.0001) ∗∗∗ (p=0.0073) 100 ∗∗ (p<0.0001) 80 ∗∗∗ 80 60 ) unit 60 ) unit 40 TNF  efensin 40 D mage density mage density

I 20 I ( arbitrary ( arbitrarry 20 0 0 Ctrl Trpv4 AMPK Ctrl TRPV4 AMPK Treatments (antagonists) Treatments (antagonists) TRPV4 antagonist AMPK antagonist TRPV4 antagonist AMPK antagonist

100

(b)

Figure 5: RT-PCR and ICC results for keratin, involucrin, defensin, and TNF� in HaCaT cells treated with antagonists for TRPV4and AMPK. Te antagonists for TRPV4 and AMPK decreased mRNA (a) and protein (b) expressions of keratin, involucrin, and defensin, but they increased TNF� expression (a, b). Images were taken at 200 × magnifcation. Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test and one-way ANOVA. All experiments were over repeated three times. Te dash-dotted line means the one-way ANOVA result between ctrl and other groups.

likewise the AG ethyl acetate extract in HaCaT cells (Figures Did Not Afect the Apoptotic Marker, cPARP. AG Did Not 7(a) and 7(b)). But TNF� expression related to skin infam- Signifcantly Afect AMPK�1mRNALevel.Te treatments mation was decreased by the substance (Figure 7(b)). of AG extract and 3, 5-DCQA increased the proliferation of HaCaT cells (Additional Figure 1A); also they elevated 3.7. Aster glehni Extract and 3, 5-DCQA Increased the Number the total AMPK protein level (Additional Figure 1B). AG and AMPK Protein Level of HaCaT Cells. However, Tey and 3, 5-DCQA did not induce the apoptosis in HaCaT Evidence-Based Complementary and Alternative Medicine 9

Ctrl TRPV4 Ctrl PPAR Ctrl AMPK

TRPV4 TRPV4 TRPV4 PPAR TRPV4 AMPK antagonist antagonist antagonist -Actin -Actin -Actin

(p=0.0336) (p=0.0022) . ∗ ∗∗ . . )

) . )

ctin .

. ctin -A

ctin .  -A  -A ensity  ensity . . D . ensity D D

. . (TR P V4/ (TR P V4/

(TR P V4/ . . . Ctrl AMPK Ctrl PPAR Ctrl TRPV4 Treatments (antagonist) Treatment (antagonist) Treatments (antagonist) Ctrl AMPK Ctrl TRPV4 Ctrl PPAR  PPARδ PPAR PPAR

 -Actin -Actin -Actin

. (p=0.0035) (p=0.0261) . ∗∗ ∗ . ) . ) . ) . ctin ctin -A -A . ctin /  .

. /  -A ensity ensity /  D D ensity AR  . AR 

. D . AR  ( PP . ( PP ( PP . . . Ctrl TRPV4 Ctrl AMPK  Treatment (antagonist) Ctrl PPAR Treatments (antagonist) Treatment (antagonist) Ctrl TRPV4 Ctrl AMPK Ctrl PPAR p-AMPK p-AMPK p-AMPK AMPK AMPK AMPK (p 0.0022) = (p=0.0309) ∗∗ . ∗ (p=0.0176) . . ∗ ) K) . K) . . ctin -A  K/AM P

. K/AM P ensity P ensity P ensity D

. D D . -AM

. -AM ( P ( P (TR P V4/ . . . Ctrl TRPV4 Ctrl PPAR Ctrl AMPK Treatment (antagonist) Treatments (antagonist) Treatments (antagonist)

(a) (b) (c) R

R Ctrl SDS S+AG S+A+Ta

R Ctrl DNCB D+AG D+A+Ta - si R

K- si TRPV4 TRPV4 - si

siRNAs AR  AM P Ctrl Ctrl si TR P V4- PP PPAR PPAR TRPV4 AMPK1 AMPK1 PPAR -Actin -Actin

 (p=0.0023) AMPK 1 (p=0.0444) ∗∗ ∗ (p=0.0238) (p=0.0029) ∗ -Actin ∗∗ . (p<0.0001) (p=0.0495) . (p=0.0338) (p=0.0121) ∗∗∗ ∗ ∗ ∗ ) (p=0.0149) ) ∗ . ctin

. ctin -A (p 0.0331) -A =  ∗  . ensity

. ensity D

. D

) . . (TR P V4/ (TR P V4/ ctin . . . -A  Ctrl DNCB D+AG D+A+Ta Ctrl SDS S+AG S+A+Ta ensity .

D Treatments Treatments . (p=0.0030) (p<0.0001)

(TR P V4/ ∗∗ ∗∗∗ . (p=0.0118) (p=0.0080) ∗ ∗∗ (p<0.0001) ∗∗∗ R R R R . . - si - si Ctrl (p=0.0025) (p<0.0001) ) K- si

) ∗∗ ∗∗∗

Ctrl . AR  ctin ctin . AM P PP si TR P V4- -A . -A / 

Treatments /  ensity ensity D

D . (p 0.0003) (p 0.0062) AR  = = AR  ∗∗∗ ∗∗ . ( PP ( PP (p=0.0005) (p=0.0004) ∗∗∗ ∗∗∗ . .

(p=0.0071) (p 0.0008) Ctrl DNCB D+AG D+A+Ta Ctrl SDS S+AG S+A+Ta ∗∗ = ∗∗∗ Treatments Treatments (p=0.0232) (p=0.0132) ∗ ∗ (p 0.0163) (p=0.0002) . (p 0.0460) . (p=0.0049) = = ∗ ∗∗ ∗ ∗∗∗ ) ) (p 0.0009) . (p=0.0041) = ∗∗ ∗∗∗ ctin ctin . . (p 0.0356) (p 0.0046) (p=0.0339) (p=0.0090) (p=0.0014)

-A = = . ∗ ∗∗ . ∗ ∗∗ ∗∗ /  ensity ) ensity K/  -A . )

D . D . . AR  ctin . ctin . (AM P . ( PP . . ensity ensity K/  -A . K/  -A . R R R R R R R R D D - si - si Ctrl - si - si Ctrl . . K- si K- si (AM P (AM P Ctrl Ctrl AR d AR d . . AM P AM P PP PP si TR P V4- si TR P V4- Ctrl DNCB D+AG D+A+Ta Ctrl SDS S+AG S+A+Ta Treatments Treatments Treatments Treatments (d) (e)

Figure 6: Western blot analyses and RT-PCR for TRPV4, AMPK,andPPAR� in HaCaT cells treated with antagonists or siRNAsfor TRPV4, PPAR�,andAMPK. RT-PCR for TRPV4, AMPK,andPPAR� in HaCaT cells treated with TRPV4antagonistinpathological conditions. TRPV4 antagonist and siRNA decreased the protein levels of TRPV4, PPAR�, and AMPK (a, d). PPAR� antagonist and siRNA decreased the protein levels PPAR� and AMPK (b, d). AMPK antagonist and siRNA decreased only AMPK (c, d). In pathological conditions, the TRPV4 antagonist reversed the AG efects on TRPV4, PPAR�, and AMPK (e). Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test and one-way ANOVA. All experiments were repeated over three times. Te dash- dotted line means the one-way ANOVA result between ctrl and all other groups. Te dotted line means the one-way ANOVA result among groups except for control. Meaning of indications: Ctrl is an untreated control group, DNCB is a DNCB treated group, D+AG is a DNCB+AG treated group, D+A+Ta is a DNCB+AG+TRPV4 antagonist treated group, SDS is a SDS-treated group, S+AG is a SDS+AG treated group, and S+A+Ta is a SDS+AG+TRPV4 antagonist treated group. 10 Evidence-Based Complementary and Alternative Medicine

Ctrl CQA Ctrl CQA

TRPV4 PPARδ

-Actin -actin

(p=0.0083) . (p=0.0477) . ∗∗ ∗ ) . )

ctin . ctin -A -A

 . /  ensity ensity D D

AR  . . ( PP (TR P V4/

. . Ctrl CQA Ctrl CQA Treatment Treatment

Ctrl CQA Ctrl CQA

p-AMPK SPTLC2

AMPK -Actin

(p=0.0007) (p=0.0087) . ∗∗∗ . ∗∗ ) K . . AM P K/ ensity ensity D . D . -AM P P T LC 2/  - actin ) ( P (S

. . Ctrl CQA Ctrl CQA Treatment Treatment

(a)

Keratin Involucrin

Negative Ctrl Negative Ctrl

(p<0.0001) (p<0.0001) ∗∗∗ ∗∗∗ 100 100

) 80 80 ) unit unit 60 60 olucrin eratin 40 v 40 K n I rbitrary mage density mage density I I

( arbitrarry 20 (A 20

0 0 Ctrl CQA Ctrl CQA Treatment Treatment

Ctrl CQA Ctrl CQA

Denfensin TNF

Negative Ctrl Negative Ctrl (p=0.0007) ∗∗∗ (p<0.0001) 100 150 ∗∗∗

80 100 ) unit 60 ) unit efensin 40 TNF  D 50 mage density mage density I I ( arbitrarry 20 ( arbitrarry

0 0 Ctrl CQA Ctrl CQA Treatment Treatment Ctrl CQA Ctrl CQA

(b)

Figure 7: Western blot analyses for TRPV4, AMPK,PPAR�,andSPTLC2, and ICC for keratin, involucrin, defensin�1, and TNF� in HaCaT cells treated with cafeoylquinic acid. CQA increased protein levels for TRPV4, PPAR�, AMPK, SPTLC2, keratin, involucrin, and defensin�1 (a, b); however, it decreased TNF� expression (b). Images were taken at 200 × magnifcation. Te results are expressed as means ± SEM. N=3 for each group. Values were statistically analyzed by unpaired t-test. All experiments were repeated over three times. Evidence-Based Complementary and Alternative Medicine 11 cells (Additional Figure 1B). Additionally, AG treatment did protection of skin permeability barrier, and it functions as not signifcantly afect the mRNA expression of AMPK�1in a regulator in the action of AG extract. Furthermore, the HaCaT cells (Additional Figure 2). results acquired from HaCaT cells treated by antagonists and siRNAs for PPAR�,AMPK,andTRPV4suggestthat 4. Discussion AG extract can protect keratinocytes through the sequential regulation of TRPV4�→ PPAR��→AMPK pathway. PPAR� as a nuclear receptor alleviates metabolic diseases Te 3,5-dicafeoylquinic acid, the most plentiful sub- such as obesity and atherosclerosis [27, 28]. PPAR� partic- stance in seven cafeoylquinic compounds of AG extract, ipates in skin barrier protection via the following mecha- showed equivalent efects with the AG ethyl acetate extract nisms. PPAR� ligand treatment stimulates stratum corneum in the expressions of biomarkers related to the integrity formation and permeability barrier development in a fetal and infammation of permeability barrier in HaCaT cells. rat explant culture model [13]. In addition, the epidermal Terefore, we can suggest that the function of AG extract is integrity is disrupted and infammation is increased in attributed to cafeoylquinic acids. PPAR� knockout mice [14]. Generally, AMPK is involved Moreover, when AG or CQA treated HaCaT cells, the cell in decreasing infammation as well as ameliorating the number was increased. Terefore, it is supposed that one of symptoms of metabolic diseases, which is also regulated by protecting mechanisms of AG on keratinocytes may be the PPAR� [20, 29]. Activated AMPK suppresses the increase of stimulation of cell proliferation (additional Figure 1A). matrix metalloprotenase1 induced by ultraviolet radiation in It is generally known that atopic dermatitis is incurred HaCaT cells [15]. Moreover, in human keratinocytes, AMPK through various causes such as the impairment of skin is involved in autophagy regulation through the inhibition of permeability barrier by irritant, allergen, and other external mTOR signal pathway by apigenin [30]. materials, genetic defects, immunological imbalance, and In this study, SDS- and DNCB-induced decreases in environmental pollution and it is easily recurred [36, 37]. protein expression of keratin, involucrin, and defensin�1 Remedies for atopic dermatitis include steroids, antihis- were recovered by AG extract. In addition, SDS- and DNCB- tamines, and immunomodulators. But, the need for more induced increases in expression of TNF-� were normalized efective new drugs having less side efects compared to by AG treatment. Te levels for PPAR� and AMPK were existingmedicinesisveryhighnow. elevated by AG extract treatment compared to SDS or DNCB Terefore, this study may be a small stepping stone in treatment alone. Furthermore, the positive efects of AG the development of drugs for contact dermatitis and atopic extract were ofset by PPAR� antagonist, GSK0660; therefore, dermatitis. we hypothesized that the efects of AG on keratinocytes are Tis study had done the efects and the mechanism of dependent on PPAR�. In the condition of AG and DNCB total AG extract and 3,5-dicafeoylquinic acid in only in or AG and SDS treatment, the pattern of AMPK protein vitro experiments, so, in our further study, animal study expression was diferent from the mRNA expression of and experiments for other cafeoylquinic acids should be AMPK in normal condition. From the data on AMPK additionally done to prove more precise mechanisms of AG. expression, we can suppose that the AMPK protein expres- Consequently, our study suggests that cafeoylquinic sion by AG at pathological conditions may be attributed compound-rich AG extract can protect the permeability to the translational modifcation or stability of protein. barrier through the sequential regulation of TRPV4-PPAR�- Besides PPAR� and AMPK, TRPV4 also plays important AMPK pathway in human keratinocytes HaCaT cells treated role in the maintenance or protection of skin permeability with SDS or DNCB (Figure 8). ++ barrier. TRPV4 is generally known as a calcium ion (Ca )- permeable cation transporter, and it responds to mechanical Data Availability stress such as swelling [31]. In dermatology, it protects the skin permeability barrier via strengthening of tight junctions We can provide the data when there is a request from other [16, 17] and increased keratin synthesis in HaCaT cells treated researcher (data available on request). Te e-mail addresses ++ with baicalein [32]. Te role of TRPV4 as a Ca transporter for the data request are as follows: [email protected] and suggests that it participates in calcium signal transduction [email protected]. ++ in keratinocytes. Ca generally functions as a signaling molecule in cells and stimulates the expression of biomarkers Conflicts of Interest such as keratin1/10, involucrin, loricrin, and transglutaminase 1 involved in keratinocyte diferentiation [33]. Moreover, Te authors declare that there are no conficts of interest ++ Ca facilitates the transition of proflaggrin to flaggrin regarding the publication of this paper. All authors have read [34]. When the skin permeability barrier is disrupted, Ca++ and agreed to the publication of the manuscript and confrm gradient is lost. Subsequently, the expression of loricrin, that the manuscript has not been submitted elsewhere. flaggrin, and involucrin is decreased [35]. In our study, TRPV4 protein level was elevated by AG extract compared Authors’ Contributions to DNCB or SDS-treated group; furthermore, TRPV4 antagonist negatively afected the biomarkers involved in Yong-Jik Lee designed the research, performed all experi- the maintenance of permeability barrier. Terefore, from ments, and wrote the article, Yoo-Na Jang, Yoon-Mi Han, these results, we can suppose that TRPV4 is involved in the and Hyun-Min Kim were involved in molecular biological 12 Evidence-Based Complementary and Alternative Medicine

SDS, DNCB

Keratin Aster glehni (Cafeoylquinic compounds) Involuclin rotection  Integrity of p TRPV4 PPAR AMPK Defensin skin barrier

SPTLC2

TNF Skin infammation ermeability barrier p kin S Keratinocyte

Figure 8: Schematic diagram for the functional mechanism of Aster glehni extract in keratinocytes. Te efects of Aster glehni on skin barrier protection are mainly mediated via the sequential regulation of TRPV4�→ PPAR��→p-AMPK�→ skin barrier constituting elements; also it is partly done by anti-infammation through the inhibition of TNF�. Meaning of symbols: arrow means activation, up and horizontal line means inhibition, and double vertical line means blocking.

experiments, Changbae Jin contributed to extraction and [5]C.M.DeJongh,M.M.Verberk,C.E.T.Withagen,J.J.L. preparation of experimental sample, and Hong Seog Seo and Jacobs,T.Rustemeyer,andS.Kezic,“Stratumcorneumcyto- Hyoung Ja Kim engaged in the research design and set the kines and skin irritation response to sodium lauryl sulfate,” study direction. Contact Dermatitis,vol.54,no.6,pp.325–333,2006. [6]C.M.deJongh,M.M.Verberk,S.W.Spiekstra,S.Gibbs,andS. Acknowledgments Kezic, “Cytokines at diferent stratum corneum levels in normal and sodium lauryl sulphate-irritated skin,” Skin Research and Tis research was supported by an intramural grant Technology,vol.13,no.4,pp.390–398,2007. (2E28030) from the Korea Institute of Science and Tech- [7] B. S. Kim, “Atopic Dermatitis,” http://emedicine.medscape nology, a grant from the National Research Foundation of .com/article/1049085-overview#a4, (accessed 15.02.2017). Korea(NRF-2016R1A2B3013825),agrantfromtheMinistry [8] T. Bieber, “Atopic dermatitis,” Te New England Journal of Med- of Future Creation and Science of Korea (2018K000255), a icine,vol.358,no.14,pp.1483–1494,2008. Korea University grant, a Korea University Guro Hospital [9]S.I.White,P.S.Friedmann,C.Moss,andJ.M.Simpson,“Te grant (O1801781), and a grant from BK21 Plus Korea Uni- efect of altering area of application and dose per unit area on versity Medical Science graduate program. sensitization by DNCB,” British Journal of Dermatology,vol.115, no. 6, pp. 663–668, 1986. Supplementary Materials [10] Y. Fujii, H. Takeuchi, S. Sakuma, T. Sengoku, and S. Takakura, “Characterization of a 2,4-dinitrochlorobenzene-induced Supplementary Figure 1: cell number profle and protein chronic dermatitis model in rats,” Skin Pharmacology and Phy- levels for AMPK and cPARP in HaCaT cells treated with siology,vol.22,no.5,pp.240–247,2009. AG or CQA. Supplementary Figure 2: RT-PCR for AMPK in [11] H. H. Kim, G. H. Park, K. S. Park, J. Y. Lee, T. H. Kim, and B. J. HaCaT cells treated with AG. (Supplementary Materials) An, “Te Efect of Aster glehni Fr. Schm. Extracts on Whitening and Anti-Wrinkle,” Life Science Journal,vol.20,no.7,pp.1034– References 1040, 2010. [12] H. H. Kim, G. H. Park, K. S. Park, J. Y. Lee, and B. J. An, “Anti- [1] “Shimizu’s Textbook of Dermatology,” Department of Derma- oxidant and Anti-infammation Activity of Fractions from tology, Faculty of Medicine and Graduate School of Medicine Aster glehni Fr. Schm,” Korean Journal of Microbiology and Hokkaido University, http://www.derm-hokudai.jp/shimizu- Biotechnology,vol.38,pp.434–441,2010. dermatology/ch01, (accessed 13.02.2017). [13] Y. J. Jiang, G. Barish, B. Lu et al., “PPAR� Activation Promotes [2] H. H. Jang and S. N. Lee, “Epidermal Skin Barrier,” Asian Journal Stratum Corneum Formation and Epidermal Permeability Bar- of Beauty and Cosmetology,vol.14,no.3,pp.339–347,2016. rier Development during Late Gestation,” Journal of Investiga- [3] J. S. Oh and H. H. Jang, “Epidermal Diferentiation and Skin tive Dermatology,vol.130,no.2,pp.511–519,2010. Barrier,” Korean Journal of Aesthetic and Cosmetology,vol.13, [14] M. Man, G. D. Barish, M. Schmuth et al., “Defciency of no.6,pp.713–720,2015. PPAR�/� in the Epidermis Results in Defective Cutaneous Per- [4] E. Lemery, S. Brianc¸aon, Y. Chevalier et al., “Surfactants have meability Barrier Homeostasis and Increased Infammation,” multi-fold efects on skin barrier function,” European Journal of Journal of Investigative Dermatology,vol.128,no.2,pp.370–377, Dermatology,vol.25,no.5,pp.424–435,2015. 2008. Evidence-Based Complementary and Alternative Medicine 13

[15] M. Kim, Y. G. Park, H.-J. Lee, S. J. Lim, and C. W. Nho, Hypertensive Conditions,” PPAR Research,vol.2014,ArticleID “Youngiasides A and C isolated from Youngia denticulatum 8048720, 14 pages, 2017. inhibit UVB-induced MMP expression and promote Type I [30]X.Tong,K.A.Smith,andJ.C.Pelling,“Apigenin,achemo- procollagen production via repression of MAPK/AP-1/NF-�B preventive biofavonoid, induces AMP-activated protein kinase and activation of AMPK/Nrf2 in HaCaT cells and human activation in human keratinocytes,” Molecular Carcinogenesis, dermal fbroblasts,” Journal of Agricultural and Food Chemistry, vol.51,no.3,pp.268–279,2012. vol.63,no.22,pp.5428–5438,2015. [31] D. Becker, C. Blase, J. Bereiter-Hahn, and M. Jendrach, “TRPV4 [16] N. Kida, T. Sokabe, M. Kashio et al., “Importance of transient exhibits a functional role in cell-volume regulation,” Journal of receptor potential vanilloid 4 (TRPV4) in epidermal barrier Cell Science, vol. 118, no. 11, pp. 2435–2440, 2005. functioninhumanskinkeratinocytes,”Pfugers¨ Archiv - Euro- [32]K.-F.Huang,K.-H.Ma,P.-S.Liu,B.-W.Chen,andS.-H.Chueh, pean Journal of Physiology,vol.463,no.5,pp.715–725,2012. “Baicalein increases keratin 1 and 10 expression in HaCaT [17] Y. Akazawa, T. Yuki, H. Yoshida, Y. Sugiyama, and S. Inoue, keratinocytes via TRPV4 receptor activation,” Experimental “Activation of TRPV4 strengthens the tight-junction barrier Dermatology,vol.25,no.8,pp.623–629,2016. in human epidermal keratinocytes,” Skin Pharmacology and [33]R.L.Eckert,J.F.Crish,andN.A.Robinson,“Teepidermal Physiology, vol. 26, no. 1, pp. 15–21, 2012. keratinocyte as a model for the study of regulation and [18]I.Jakasa,J.P.Tyssen,andS.Kezic,“Teroleofskinbarrier cell diferentiation,” Physiological Reviews,vol.77,no.2,pp.397– in occupational contact dermatitis,” Experimental Dermatology, 424, 1997. vol.27,no.8,pp.909–914,2018. [34] K. A. Resing, N. Al-Alawi, C. Blomquist, P.Fleckman, and B. A. [19] M. C. G. Winge, “Atopic dermatitis severity and skin barrier Dale, “Independent regulation of two cytoplasmic processing impairment,” British Journal of Dermatology,vol.170,no.3,pp. stages of the intermediate flament-associated protein flaggrin 490-491, 2014. and role of Ca2+ in the second stage,” Te Journal of Biological [20] Y.-J. Lee, Y.-N. Jang, Y.-M. Han et al., “Cafeoylquinic Acid-Rich Chemistry,vol.268,no.33,pp.25139–25145,1993. Extract of Aster glehni F. Schmidt Ameliorates Nonalcoholic [35]P.M.Elias,S.K.Ahn,M.Dendaetal.,“Modulationsinepi- Fatty Liver through the Regulation of PPAR� and Adiponectin dermal calcium regulate the expression of diferentiation-speci- in ApoE KO Mice,” PPAR Research,vol.2017,ArticleID3912567, fc markers,” Journal of Investigative Dermatology,vol.119,no.5, 19 pages, 2017. pp. 1128–1136, 2002. [21] E. Bell, F. Ponthan, C. Whitworth et al., “Cell Survival Signalling [36] A. Wollenberg, S. Kraf, T. Oppel, and T. Bieber, “Atopic der- through PPAR� and Arachidonic Acid Metabolites in Neurob- matitis: Pathogenetic mechanisms,” Clinical and Experimental lastoma,” PLoS ONE, vol. 8, no. 7, Article ID e68859, 2013. Dermatology,vol.25,no.7,pp.530–534,2000. [22] L. Salvad´o, E. Barroso, A. M. G´omez-Foix et al., “PPAR�/� pre- [37] W. David Boothe, J. A. Tarbox, and M. B. Tarbox, “Atopic Der- vents endoplasmic reticulum stress-associated infammation matitis: Pathophysiology,” Advances in Experimental Medicine and insulin resistance in skeletal muscle cells through an ampk- and Biology,vol.1027,pp.21–37,2017. dependent mechanism,” Diabetologia,vol.57,no.10,pp.2126– 2135, 2014. [23] Y.-Y. Xu, F.-L. Chen, F. Ji, H.-D. Fei, Y. Xie, and S.-G. Wang, “Activation of AMP-activated protein kinase by compound 991 protects osteoblasts from dexamethasone,” Biochemical and Bio- physical Research Communications,vol.495,no.1,pp.1014–1021, 2018. [24] Y. Lim, J. Park, O. Kwon et al., “Anti-infammatory efects of a methanolic extract of Castanea seguinii Dode in LPS-induced RAW264.7 macrophage cells,” International Journal of Mole- cular Medicine,2017. [25] T. Sokabe, T. Fukumi-Tominaga, S. Yonemura, A. Mizuno, and M. Tominaga, “Te TRPV4 channel contributes to intercellular junction formation in keratinocytes,” Te Journal of Biological Chemistry,vol.285,no.24,pp.18749–18758,2010. [26]K.YamasakiandR.L.Gallo,“Antimicrobialpeptidesinhuman skin disease,” European Journal of Dermatology,vol.18,pp.11– 21, 2008. [27] H. He, D. Yang, L. Ma et al., “Telmisartan prevents weight gain and obesity through activation of peroxisome proliferator-acti- vated receptor-�-dependent pathways,” Hypertension, vol. 55, no.4,pp.869–879,2010. [28] Y. Takata, J. Liu, F. Yin et al., “PPARdelta-mediated antiinfam- matory mechanisms inhibit angiotensin II-accelerated athero- sclerosis,” Proceedings of the National Acadamy of Sciences of the United States of America,vol.105,no.11,pp.4277–4282,2008. [29]Y.J.Lee,Y.N.Jang,Y.M.Han,H.M.Kim,J.M.Jeong,and H. S. Seo, “Fimasartan Ameliorates Nonalcoholic Fatty Liver Disease through PPAR� Regulation in Hyperlipidemic and