ISSN 2466-2046 (Print) ISSN 2466-2054 (Online) Asian J Beauty Cosmetol 2017; 15(4): 513-522 http://dx.doi.org/10.20402/ajbc.2017.0177 R E S E A R C H A R T I C L E Open Access Anti-oxidant and Anti-inflammatory Effects of Sinapic Acid in UVB Irradiation-Damaged HaCaT Keratinocytes Kye Hwa Lim 1, Jung-Eun Ku 2, Sung-Ja Rhie 3, Ji Young Ryu 3, Seunghee Bae 4, Young-Sam Kim 5* 1 Department of Beauty Care, Sangji Youngseo College, Wonju-si, Gangwon-do, Korea 2 Department of Cosmetology, Kyung-In Women s University, Incheon, Korea 3 Department of Beauty Care Design, Halla University, Wonju-si, Gangwon-do, Korea 4 Research Institute for Molecular-Targeted Drugs, Department of Cosmetics Engineering, Konkuk University, Seoul, Korea 5 Department of Image Industry, Graduate School of Engineering, Konkuk University, Seoul, Korea * Corresponding author: Young-Sam Kim, Department of Image Industry, Graduate School of Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel.: +82 2 450 3595 Email: gracehelen@konkuk.ac.kr Received August 22, 2017 Revised September 1, 2017 Accepted September 18, 2017 Published December 30, 2017 Abstract Purpose: The purpose of the present study was to explore the possibility of using sinapic acid as a skin anti-aging cosmetic by demonstrating its antioxidant and anti-inflammatory effects in keratinocytes damaged by ultraviolet (UV) irradiation. Methods: The effects of sinapic acid against oxidative stress were examined in a UVB-induced aging model of human keratinocyte HaCaT cells. The water-soluble tetrazolium salt (WST-1) assay was employed to examine protective effects against UVB. Changes in the concentration of reactive oxygen species (ROS) produced by UVB were measured using 2,7 -dichlorofluorescin diacetate (DCF-DA). Expression of genes associated with anti-oxidant and anti-inflammatory effects were confirmed using quantitative real-time polymerase chain reaction (qrt-pcr). Results: Sinapic acid protected cells from UVB-induced cytotoxicity and reduced ROS produced by UVB irradiation. Expression of genes associated with antioxidant effects including, superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase 1 (GPX1), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and heme oxygenase 1 (HO1), increased and lipid peroxidation decreased in a dose-dependent manner. The expression of cyclooxygenase 2 (COX2), interleukin 6 (IL6), and interleukin 8 (IL8) genes, which are associated with inflammation, also decreased in a dose-dependent manner. Conclusion: These results suggest that sinapic acid has potential as an anti-aging cosmetic ingredient with anti-oxidant, anti-inflammatory properties against UVB irradiation. Keywords: Sinapic acid, Anti-oxidant, Anti-inflammation, UVB, HaCaT Introduction 피부는외부로부터표피, 진피, 피하층의독특한 3개층으로구성되어있으며, 그중피부의최외측에존재하는표피층은장벽기능을수행하는일차적방어막이며대사상활동성인중층편평각화상피 (stratified squamous epithelium) 로서주로각질형성세포 (keratinocyte) 로구성되어있다 (Lee et al., 2001). 이러한피부는외부환경에항상노출되어있어물리적자극, 자외선, 열, 다양한오염등에영향받기쉽다. 특히일상의대사과정중발생되는활성산소종 (reactive oxygen species, ROS; Lee et al., 2012) 과 UV에의해발 생하는 ROS (Kulms et al., 2002) 에의한산화적스트레스 (oxidative stress) 는피부노화의주된원인으로작용된다 (Kujoth et al., 2005). ROS의종류로는 superoxide anion (O 2- ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH ), singlet oxygen ( 1 O 2 ) 등이대표적이다 (Thannickal & Fanburg, 2000). 이러한 ROS로인한산화적스트레스로인해야기되는산화물질의불균형및산화억제기능에대응하기위하여체내에는이를방어하는 SOD, CAT, GPX, glutathione reductase (GR), HO1 등의효소가존재한다 (Jakus, 2000). 산화적스트레스환경은다양한세포보호유전자들의발현을유 Copyright c Korea Institute of Dermatological Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
UVB Protective Effects of Sinapic Acid in HaCaT Cells 도하는생리적반응을유발하게한다. NRF2는핵전사인자로서발암성물질, 독성물질, 산화적손상의노출에반응하여활성화되고, 항산화반응요소 (antioxidant response element, ARE) 의결합을통한해독및 Phase Ⅱ 항산화관련효소의발현을촉진하여세포를보호하는역할을수행한다 (Kensler et al., 2007). NRF2는정상적인조건에서는 Kelch-like ECH-associated protein 1 (KEAP1) 에결합되어세포질내에존재하나 ROS와같은자극을받게되면 KEAP1 으로부터유리되어핵내로이동하여핵내에존재하는 Maf 단백질과결합하여 dimer 를이루고항산화효소의 promoter 부위에존재하는 ARE와결합함으로써 GR, HO1, NAD(P)H quinone dehydrogenase 1 (NQO1), gamma-glutamate cysteine ligase (γgcl), glutathione S-transferase (GST) 와같은항산화효소들의발현을증가시킨다 (Kang et al., 2005; Lee & Surh, 2005; Lee et al., 2017). ROS에의해생성되는산화적스트레스와미토콘드리아의산소대사산물에의한신호전달과조직의파괴는 nuclear factor kappa B (NFκB) 의염증매개유전자를발현시켜체내염증반응을유발하게한다. NFκB는세포내에서염증반응을조절하는대표적인전사조절인자이다. 세포질에서 NFκB는활성화를억제하는단백질 inhibitor of kappa B (IκB) 와함께존재한다. UV나 ROS와같은다양한자극에의해서 IκB kinase (IKK) 에의해인산화되어 NF κb로부터 IκB의결합이분해되면서떨어지게된다. IκB로부터자유로워진 NFκB는핵속으로이동하며핵속에서 κb binding site 에결합하여 inducible nitric oxide synthase (inos), COX2, tumor necrosis factor-alpha (TNFα), interferon gamma (IFNγ), IL6, IL8, interleukin 1 beta (IL1β), 등의발현을유도한다 (Baeuerle, 1998; Longley et al., 1988; Takeda et al., 2003). Sinapic acid 는주로향신료, 베리류의과일, 감귤류, 채소, 곡물, 오일씨드 (oil seed) 등의다양한식물에서발견되는페놀화합물이다 (Sawa et al., 1999; Shahidi & Naczk, 2003). Sinapic acid 는항암 (Balaji et al., 2014), 고혈당완화 (Cherng et al., 2013), 신경세포보호 (Zare et al., 2015), 항혈전 (Kim et al., 2016), 항불안 (Yoon et al., 2007), 간세포보호 (Shin et al., 2013) 등의효과가보고되어있으며, 인간모낭모유두세포 (human hair follicle dermal papilla cells, HFDPC) 의모발에서성장인자의발현을유도하여모발성장을유도함으로써탈모치료를위한잠재적화합물로서의가능성도제시되었다 (Woo et al., 2017). 본연구에서는 sinapic acid 처리시 UVB 에의해발생하는산화적스트레스에인간각질형성세포 (HaCaT) 에서항산화제활성을증가시킬수있는지를알아보기위하여세포내 ROS 제거를하는대표적인항산화효소인 SOD1, GPx1, CAT, NRF2 그리고 HO1 유전자의발현변화를확인하여항산화효능을확인하고염증관련유전자인 COX2, IL6 및 IL8 mrna 발현의변화를확인하여피부노화예방에도움을줄수있는기능성을가진화장품원료로서의가능성을제시하고자한다. Methods 1. 세포배양본연구에서사용한인간각질형성세포주인 HaCaT keratinocytes는 American Type Culture Collection (ATCC; USA) 에서구매하여사용하였고, HaCaT 세포주의배양은 Dulbecco s modified Eagles medium (DMEM; HyClone, GE Healthcare Life Sciences, UK) 에 10% fetal bovine serum (FBS; HyClone ) 과 1% penicillin (100 IU/mL)/streptomycin (100 μ g/ml) (Invitrogen, Thermo Fisher Scientific, USA) 을함유한배지를사용하였고, 세포배양기내의조건은 37, 5% CO 2 를유지시키며배양하였다. 2. 시료처리및자외선조사 Sinapic acid는순수정제 (>90%) 된분말형태로 Sigma-Aldrich (USA) 에서구매하였으며, 실험에사용할때에는 dimethyl sulfoxide (DMSO; Sigma-Aldrich) 에적정농도로용해하여서사용하였다. 세포배양접시에 HaCaT (1 10 6 cells/well) 을 24 h 배양한후적정농도의 sinapic acid를배지에첨가하여 6 h 동안전처리하였으며, 세포에 UV-B lamp (UVP, USA) 를사용하여 UVB를조사하였다. UVB 조사전우선세포배양접시의배지제거를위해 ph7.4의 phosphate-buffered saline (PBS; Thermo Fisher Scientific) 로 2회세척을하였으며세포의건조를막기위해 PBS를넣은후세포배양접시의뚜껑을열고 UVB를조사하였다. UVB 파장의측정은 Fiber Optic Spectrometer System USB2000 (Ocean Optics, USA) 을사용하였으며, UVB 조사후에 PBS를제거하였고새로운배양배지를첨가하여 24 h 배양기에서추가배양한후실험에사용하였다. 3. 세포생존율측정세포생존율의실험방법으로는 water-soluble tetrazolium salt (WST-1) assay 원리를이용하였다. 96-well plate에 HaCaT (3 10 3 cells/well) 을 100 μl씩접종하여 24 h을배양한후, HaCaT에 sinapic acid를 5, 10, 20, 40, 80 μm로처리하고 24h 추가배양한후세포생존율을측정하였으며, 같은조건으로추가배양한다른배양접시의 HaCaT에는 sinapic acid를각각 10, 20, 40 μm로처리하고, 40 mj/cm 2 UVB 조사후 24 h 동안추가배양하였다. 배양된세포에 EZ-Cytox cell viability assay kit reagent (itsbio, Korea) 을 10 μl 첨가하여 1 h 후에 microplate reader (Bio-Rad Laboratories, USA) 로 490 nm에서흡광도를측정하였다. 4. 세포내 ROS 정량분석 HaCaT (2 10 5 cells/well) 을 60 mm 배양접시에접종하여 514 http://dx.doi.org/10.20402/ajbc.2017.0177
Sinapic acid 의인간각질형성세포에서 UVB 의손상에대한항산화및항염증효과 Figure 1. Cell survival analysis of UVB-irradiated and sinapic acid treated HaCaT cells. HaCaT cells were inoculated into 96-well plate at a 3 10 3 cells/well and incubated for 24 h. (A) Sinapic acid was added at various concentrations and cells were cultured for 24 h. The survival rate did not decrease compared with the control group until sinapic acid treatment at 40 μm. (B) The cells were pretreated with sinapic acid at 10, 20, and 40 μm for 6 h and irradiated with 40 mj/cm² of UVB. After 24 h of incubation, cell viability was determined using the WST-1 assay. Cell survival was restored in a dose-dependent manner. The results show the mean and standard deviation for three independent experiments ( ** p<0.01; # p<0.05). UVB, ultraviolet B; WST-1, watersoluble tetrazolium salt. Table 1. List of primers used in this study Gene Forward primer (5 3 ) Reverse primer (5 3 ) β-actin GGATTCCTATGTGGGCGACGA CGCTCGGTGAGGATCTTCATG SOD1 GGGAGATGGCCCAACTACTG CCAGTTGACATCGAACCGTT GPX1 TTCCCGTGCAACCAGTTTG GGACGTACTTGAGGGAATTCAGA CAT ATGGTCCATGCTCTCAAACC CAGGTCATCCAATAGGAAGG NRF2 TACTCCCAGGTTGCCCACA CATCTACAAACGGGAATGTCTGC HO1 GCCTGCTAGCCTGGTTCAAG AGCGGTGTCTGGGATGAACTA COX2 CGCGGATCCGCGGTGAGAACCGTTTAC GCGAGGAAGCGGAAGAGTCTAGAGTCGACC IL6 TAACAGTTCCTGCATGGGCGGC AGGACAGGCACAAACACGCACC IL8 CTCTCTTGGCAGCCTTCC CTCAATCACTCTCAGTTCTTTG β-actin, beta-actin; SOD1, superoxide dismutase 1; GPX1, glutathione peroxidase 1; CAT, catalase; NRF2, nuclear factor (erythroid-derived 2)- like 2; HO1, heme oxygenase 1; COX2, cyclooxygenase 2; IL6, interleukin 6; IL8, interleukin 8. 24 h 배양후, 세포에 sinapic acid를처리한다음 24 h을추가로배양하였다. 세포내 ROS를측정하기위해 DCF-DA (Sigma- Aldrich) 를 10 μm를첨가하여 30 min 배양후에세포를수확하였다. PBS를첨가하여세포를풀어준다음 Flow Cytometer (BD Biosciences, USA) 를이용하여 ROS의변화량을측정하였다. Sinapic acid의 ROS 제거효능을검증하기위하여양성대조군으로 ROS scavenger 역할을하는 N-acetyl-L-cysteine (NAC; Calbiochem, USA) 을사용하였으며, sinapic acid와동일한방식으로처리후측정하였다. 5. Quantitative real-time polymerase chain reaction (qrt- PCR) 세포배양으로얻은세포를 TRIzol reagent (Invitrogen ) 를이용하여추출하였다. 추출된 RNA는 Nanodrop (Maestrogen, USA) 을이용하여 260/280 nm ratio 1.8 이상순도의 RNA 만을실험에사용하였다. cdna는 PCR tube에 1 μg RNA, 0.5 ng oligo dt18, diethyl pyrocarbonate (DEPC; Biopure, Canada) water를 total 10 μl로제조한후 70 에서 10 min 처리하여 RNA 변성을유도하여 M-MLV reverse transcriptase (Enzynomics, Korea) 를이용하여 37 에서 1 h 반응시켜 cdna 를합성하여사용하였다. qrt-pcr은 PCR tube에 5x HOT FIREPol EvaGreen qpcr Supermix (Solis Biodyne, Estonia) 을사용하여반응액을제조하였다. LineGene K (Hangzhou Bioer Technology, China) 를사용하여 PCR을진행하였으며 PCR의유효성은 melting curve로검증하였다. 각유전자의발현은 β-actin (beta-actin) 의발현을표준화하여비교분석하였다. 실험에사용된 primer 는 Table 1과같다. http://www.e-ajbc.org 515
UVB Protective Effects of Sinapic Acid in HaCaT Cells Figure 2. ROS scavenging in UVB-irradiated and sinapic acid treated HaCaT cells. Sinapic acid was added for 6 h, and cells were UVB-irradiated and cultured for 24 h. DCF-DA (10 μm) was added and the cells were harvested after incubating for 30 min. After the addition of PBS, the amount of ROS change was measured using flow cytometry. Compared with the control group that was not irradiated with UVB, ROS decreased in a dose-dependent manner when pretreated with sinapic acid. In addition, increased anti-oxidative effects were obtained when compared with 10 mm NAC control group. The results show the mean and standard deviation for three independent experiments ( *** p<0.001; ** p<0.01; ### p<0.001). ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; UVB, ultraviolet B; DCF-DA, 2,7 -dichlorofluorescin diacetate; PBS, phosphate buffered saline. 6. Lipid peroxidation 변화량분석 Lipid Peroxidation Assay Kit (Abcam, UK) 를사용하여제공 된실험방법에따라실험을수행하였다. Malondialdehyde (MDA) lysis buffer 로 cell을 ice에서균질화시키고, 원심분리 (1300 g, 10 min) 하여 200 μl 상층액만을분리하고, kit에있는 600 μl의 thiobarbituric acid (TBA) solution을각 test tube에넣고, 95 에서 1 h, ice에서 10 min 식힌뒤, 96-well plate에 200 μl씩분주하여 586 nm에서 microplate reader 로흡광도를측정하였다. 7. 통계분석 본연구에서의모든실험은동일한조건하에서독립적으로 3 회이상을실시하여실험결과를얻었으며, 실험의결과는평균 ± 표준편차 (mean±standard deviation, M±SD) 로나타내었다. 각실험에관하여 Student s t-test를이용하여모든실험결과의 p-value 값이 0.05 이하인경우에통계적으로유의하다고분석하였다. Results and Discussion 1. Sinapic acid 의세포생존율분석 Sinapic acid 가 HaCaT 세포에미치는세포독성을확인하기위해 WST-1 assay 를이용하여세포생존율을측정하였다. HaCaT 세포에서 sinapic acid 의독성을확인한결과 sinapic acid 를처리하지않은대조군의세포생존율 (100%) 을기준으로하였을때, sinapic acid 를 5, 10, 20, 40, 80 μm 처리시 105%, 121%, 130%, 136%, 97% 의생존율을보여 40 μm까지는생존율이감소하지않았으며, sinapic acid 80 μm 처리시세포생존율이다소감소하는것으로나타났다 (Figure 1A). 또한, UVB에의해손상된 HaCaT 세포에서 sinapic acid 의세포보호효능을알아보기위해세포생존율의변화를측정하였다. 그결과 UVB만조사된대조군의세포생존율 (59%) 에비해 sinapic acid 을 10 μm 전처리한경우 73%, 20 μm에서 92% 의세포생존율을보여 sinapic acid 에의해농도의존적으로세포생존율이회복되는것이확인되었다. 단 40 μm에서세포생존율이 79% 로감소하여향후실험은 10, 20 μm 농도를사용하기로결정하였다 (Figure 1B). 2. Sinapic acid의 ROS 제거효능분석본실험에서는 sinapic acid 가 HaCaT 세포내 ROS를제거하는능력을확인하기위해서 DCF-DA assay 를이용하였다. HaCaT에 sinapic acid 를처리하지않고 UVB를조사한대조군은 ROS량이 6.9 로증가하였으나 sinapic acid 를 10 μm 전처리시 3.6, 20 μm 전처리시 2.2로농도의존적으로감소하였다. 양성대조군으로사용한 ROS scavenger 역할을하는 NAC 의 10 mm의 1.5와비교하였을때보다뛰어난항산화효과가있는것을확인하였다 (Figure 2). 3. Sinapic acid의항산화유전자발현분석체내의여러대사반응의결과로생성되는 O 2-, H 2 O 2 등의과산화물은세포안의 DNA, 단백질, 지질등의고분자물질과반응하여다른 ROS를형성하는연쇄전파과정을일으킨다 (Niki et al., 1991). 이에대응하여인체내항산화물질들은산화적스트레스로인한손상으로부터신체를보호하며상호작용에의하여각각의항산화기능을상승시켜줌으로써산화적스트레스로부터인체를방어하는역할을하는것으로보고되고있다 (Frei et al., 1990, Schmidt, 1991). 본연구에서는 UVB로인해증가된산화적스트레스로인해야기되는산화물질의불균형및산화억제기능을위해서체내에서생성해내는여러항산화물질들의변화량을확인하기위하여 qrt-pcr을수행하였다. HaCaT에서 UVB로인해 SOD1, GPX1, CAT, NRF2 mrna 의발현량이감소하였으나 sinapic acid 를 10, 20 μm로전처리시발현량이 SOD1은 0.75으로감소하였으나 1.01, 1.37로농도의존적으로증가하였으며, GPX1은 0.31에서 0.90, 2.46로, CAT는 0.23에서 0.77, 0.97으로, NRF2는 0.46에서 0.75, 0.99로농도의존적으로증가되는것을확인할수있었다. HO1 mrna 의발현량은 UVB조사시 1.77으로증가하였으며 sinapic acid 를전처리시 2.51, 3.72로농도의존적으로발현량이더욱증가되어더많은항산화효 516 http://dx.doi.org/10.20402/ajbc.2017.0177
Sinapic acid 의인간각질형성세포에서 UVB 의손상에대한항산화및항염증효과 Figure 3. Anti-oxidant gene expression analysis of HaCaT cells treated with UVB irradiation and sinapic acid. The indicated concentrations of sinapic acid were added to the culture medium for 6 h then cells were irradiated with 40 mj/cm². After further incubation for 24 h, changes in anti-oxidant gene expression were analyzed using qrt-pcr. (A-D) The expression of SOD1, GPX1, CAT, and NRF2 mrna increased in a dose-dependent manner when cells were pretreated with sinapic acid before UVB irradiation. (E) The gene expression of HO1 mrna was also increased in a concentration-dependent manner when compared with the control group. The results show the mean and standard deviation for three independent experiments ( * p<0.05; ** p<0.01; *** p<0.001; ## p<0.01; ### p<0.001). UVB, ultraviolet B; qrt-pcr, quantitative real-time polymerase chain reaction; SOD1, superoxide dismutase 1; GPX1, glutathione peroxidase 1; CAT, catalase; NRF2, nuclear factor (erythroid-derived 2)-like 2; HO1, heme oxygenase 1. 능을나타내고있음을확인하였다 (Figure 3). 이것은 sinapic acid 가산화적스트레스로부터세포를보호할뿐아니라세포내의환원력을유지하는데효과가있을것으로생각된다. 4. Sinapic acid의 lipid peroxidation 변화량분석 ROS로인한산화적스트레스는단백질이나 DNA 를산화시켜서 구조적변화를일으키게하고, 세포막의지방산을산화시켜지질과산화물인 MDA 의함량을증가시키게된다. 이러한 MDA 의증가는세포의산화적손상을일으켜세포의기능을저하시킴으로써암과같은여러가지만성질환을유발하게한다 (Gloire et al., 2006). 그러므로본연구에서는 MDA assay 를이용하여지질산화의최종산물을측정하였다. Sinapic acid 와 UVB 모두처리하지않은대조군의 level http://www.e-ajbc.org 517
UVB Protective Effects of Sinapic Acid in HaCaT Cells Figure 4. Analysis of changes in lipid peroxidation of HaCaT cells treated with UVB and sinapic acid. HaCaT cells were inoculated into 6-well plates at 1 10⁶ cells/ well and were further pretreated with sinapic acid. The cells were UVB irradiated and cultured for 24 h. MDA assay was performed to determine the degree of lipid peroxidation in cells damaged caused by UVB irradiation. When cells were pretreated with sinapic acid lipid peroxidation decreased in a dose-dependent manner. The results show the mean and standard deviation for three independent experiments ( * p<0.05; *** p<0.001; ### p<0.001). UVB, ultraviolet B; MDA, malondialdehyde. 을 100% 로보았을때 UVB만처리시 388% 로증가하였으며, sinapic acid 전처리시농도의존적으로감소함을확인할수있었다 (Figure 4). 이러한결과는 sinapic acid 가 UVB로부터세포의지질산화물의생성을감소시켜세포내항산화효과가있을것으로사료된다. 5. Sinapic acid의항염증유전자발현분석체내의산화환원의불균형은 NFκB의활성화를일으키고활성화된 NFκB는 inos, COX2, TNFα, IL6, IL8, IL1β 등과같이여러노화와관련된유전자및사이토카인 (cytokine) 을발현시키고이로인해염증반응이유발되며노화로진행된다. 본연구에서는 sinapic acid 가이러한 COX2, IL6, IL8의발현에미치는영향을확인해보았다. UVB로유도된 HaCaT에 sinapic acid 를전처리한후, COX2, IL6, IL8 유전자발현을확인한결과 UVB만처리시각각 9.31, 6.50, 3.62으로증가되었으나 sinapic acid 를 10, 20 μm으로전처리시 COX2는 4.42, 2.27으로, IL6은 4.77, 2.00으로, IL8은 2.23, 1.65 로농도의존적으로발현량이감소됨을확인할수있었다 (Figure 5). 이를통하여 sinapic acid 가염증을억제하는효과가있을것으로사료된다. Conclusion ROS는산소원자를포함한짝지어지지않은불안정상태로화학적으로반응성이매우높다. 세포신호와항상성에중요한역할 Figure 5. Analysis of inflammatory gene expression in UVB irradiated HaCaT cells pretreated with sinapic acid. The indicated concentrations of sinapic acid were added to the culture medium for 6 h, and the cells were irradiated with 40 mj/cm² of UVB, After further incubation for 24 h changes in inflammatory gene expression were examined using qrt-pcr. (A C) The expressions of COX2, IL6, and IL8 mrna, which was increased by UVB irradiation, were found to decreased in a dosedependent manner when the cells were pretreated with sinapic acid. The results show the mean and standard deviation for three independent experiments ( * p<0.05; ** p<0.01; *** p<0.001; ### p<0.001). UVB, ultraviolet B; qrt-pcr, quantitative realtime polymerase chain reaction; COX2, cyclooxygenase 2; IL6, interleukin 6; IL8, interleukin 8. 518 http://dx.doi.org/10.20402/ajbc.2017.0177
Sinapic acid 의인간각질형성세포에서 UVB 의손상에대한항산화및항염증효과 을한다고알려져있지만산화적스트레스로인해세포의구조가손상될수도있다. 이러한산화적스트레스는세포내 DNA, 지질, 단백질등의변형및손상과세포괴사 (necrosis) 또는세포자멸사 (apoptosis) 등을초래하여노화뿐만이아닌질병을일으키는데도관여한다 (Sagara et al., 1998). 본연구에서이러한세포에손상을주는 ROS를제거하는 sinapic acid 의효능을확인하기위하여항산화표준물질로사용되는 NAC 과함께 DCF-DA 를이용하여비교실험을한결과 NAC 보다도더많은항산화효능이있음을확인할수있었다. 또한체내에서생성해내는여러항산화물질들의변화량을확인하기위하여 qrt-pcr을확인한결과 HaCaT에서 UVB로인해발현량이감소되었던 SOD1, GPX1, CAT, NRF2 mrna 의발현량을 sinapic acid 에의해농도의존적으로발현량이증가하였으며, HO1 mrna 의발현량또한대조군과비교하여농도의존적으로증가하였다. 산화적스트레스를측정하는지표로사용되는 lipid peroxidation 의변화량또한 UVB로인하여증가하였으나 sinapic acid 를전처리시농도의존적으로감소하였다. 항염증과관련하여 sinapic acid 의농도가증가할수록염증반응전사인자인 COX2와염증진단표지자인 IL6, IL8 mrna 발현량이감소하는것을확인하였다. 이는염증발생을억제시키는효능이있을것으로사료된다. 이렇듯 sinapic acid 는유전자레벨에서부터조절하여다각적인방법으로항산화효능및항염증효능을나타내고있다는것을확인할수있었다. 본연구는세포효능을위주로진행되었으며화장품소재로서활용하기위해서는화장품의기본적인성분이갖는화학적특성과 sinapic acid 의생화학적특성이잘배합되고조합되어피부에적용하였을때목적하는효과를나타낼가능성이있는지를평가하는향후연구가추가적으로진행되어야할것으로생각된다. This work is part of the Kye Hwa Lim s Ph.D. thesis at the Konkuk University, Seoul, Korea. Acknowledgements 본논문은보건복지부보건의료연구개발사업의지원 ( 과제번호 : HN13C0080) 에의한것이며, 이에감사드립니다. References Baeuerle PA. Pro-inflammatory signaling: last pieces in the NFκB puzzle? Current Biology, 8: R19-R22, 1998. Balaji C, Muthukumaran J, Nalini N. Chemopreventive effect of sinapic acid on 1,2-dimethylhydrazime-induced experimental rat colon carcinogenesis. Human & Experimental Toxicology, 33: 1253-1268, 2014. Cherng YG, Tsai CC, Chung HH, Lai YW, Kuo SC, Cheng JT. Antihyperglycemic action of sinapic acid in diabetic rats. Journal of Agricultural and Food Chemistry, 61: 12053-12059, 2013. Frei B, Stocker R, England L, Ames BN. Ascorbate: the most effective antioxidant in human blood plasma. Antioxidants in Therapy and Preventive Medicine, 264: 155-163, 1990. Gloire G, Legrand-Poels S, Piette J. NF-κB activation by reactive oxygen species: fifteen years later. Biochemical Pharmacology, 72: 1493-1505, 2006. Jakus V. The role of free radicals, oxidative stress and antioxidant systems in diabetic vascular disease. Bratislavské Lekárske Listy, 101: 541-551, 2000. Kang KW, Lee SJ, Kim SG. Molecular mechanism of Nrf2 activation by oxidative stress. Antioxidants & Redox Signaling, 7: 1664-1673, 2005. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annual Review of Pharmacology and Toxicology, 47: 89-116, 2007. Kim MS, Shin WC, Kang DK, Sohn HY. Anti-thrombosis activity of sinapic acid isolated from the lees of bokbunja wine. Journal of Microbiology and Biotechnology, 26: 61-65, 2016. Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science, 309: 481-484, 2005. Kulms D, Zeise E, Pöppelmann B, Schwarz T. DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way. Oncogene, 21: 5844-5851, 2002. Lee JS, Surh YJ. Nrf2 as a novel molecular target for chemoprevention. Cancer Letters, 224: 171-184, 2005. Lee MG, Jo GH, Kim MN. Structure and function of skin. In: Dermatology. Korean society of dermatology textbook compilation committee (ed.), Ryo Moon Gak, Seoul, pp1-29, 2001. Lee NK, Ku JE, Han HS. Cytoprotective and anti-inflammatory effects of 6-shogaol on human dermal fibroblasts. Asian Journal of Beauty and Cosmetology, 15: 367-376, 2017. http://www.e-ajbc.org 519
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Sinapic acid 의인간각질형성세포에서 UVB 의손상에대한항산화및항염증효과 국문초록 Sinapic acid 의인간각질형성세포에서 UVB 의손상에대한항산화및항염증효과 임계화 1, 구정은 2, 이승자 3, 류지영 3, 배승희 4, 김영삼 5* 1 상지영서대학교뷰티케어과, 강원도원주시, 한국 2 경인여자대학교피부미용과, 인천, 한국 3 한라대학교뷰티케어디자인학과, 강원도원주시, 한국 4 건국대학교화장품공학과질병분자표적신약연구소, 서울, 한국 5 건국대학교산업대학원이미지산업학과, 서울, 한국 목적 : 본연구는인간각질형성세포 (HaCaT) 에서자외선 (UV) 손상에대한항산화및항염증효과를확인하여피부노화예방화장품소재로서 sinapic acid 의가능성을확인하고자한다. 방법 : Sinapic acid 를농도별로 HaCaT에처리후 UVB로산화적스트레스를유도하였다. UVB에대한 sinapic acid 의세포보호효과를확인하기위하여 water-soluble tetrazolium salt (WST-1) 를이용하였으며, UVB에의하여생성된활성산소종 (reactive oxygen species, ROS) 의농도변화는 2,7 -dichlorofluorescin diacetate (DCF-DA) 를사용하여측정하였다. 항산화및염증과관련된유전자발현은 quantitative real-time polymerase chain reaction (qrt-pcr) 을통해확인하였다. 결과 : Sinapic acid 는 UVB에의한세포독성으로부터세포를보호하고, ROS의생성을감소시킨다. 항산화와관련한유전자인 superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase 1 (GPX1), nuclear factor (erythroid-derived 2)-like 2 (NRF2) 및 heme oxygenase 1 (HO1) 의발현을증가하게하였고, 지질과산화는농도의존적으로감소하였다. 또한, 염증과관련이있는 cyclooxygenase 2 (COX2), interleukin 6 (IL6) 와 interleukin 8 (IL8) 의유전자발현은농도의존적으로감소하였다. 결론 : 본연구를통하여 sinapic acid 가 UVB에대하여항산화및항염증의효과가있음을확인하였으며이는노화예방화장품성분으로서의가능성이있을것으로사료된다. 핵심어 : Sinapic acid, 항산화, 항염증, 자외선 B, 인간각질형성세포 본논문은보건복지부보건의료연구개발사업의지원 ( 과제번호 : HN13C0080) 에의한것이며, 이에감사드립니다. 참고문헌 이민걸, 조광현, 김명남. 피부의구조및기능. In: 피부과학. 대한피부과학회교과서편찬위원회 (ed.), 여문각, 서울, pp1-29, 2001. 이나경, 구정은, 한효선. 인간진피섬유아세포에서 6-shogaol 의세포보호및항염증효과. 아시안뷰티화장품학술지, 15: 367-376, 2017. http://www.e-ajbc.org 521
UVB Protective Effects of Sinapic Acid in HaCaT Cells 中文摘要 芥子酸 (Sinapic acid) 对 UVB 诱导损伤的人角质形成细胞的抗氧化以及抗炎功效 任桂華 1, 具貞恩 2, 李勝子 3, 柳枝榮 3, 裵承熙 4 5*, 金永三 1 尙志岭西大学美容学科, 江原道原州市, 韩国 2 敬仁女子大学皮肤美容科, 仁川, 韩国 3 汉拏大学美容设计学科, 江原道原州市, 韩国 4 建国大学化妆品工学科疾病分子靶标新药研究所, 首尔, 韩国 5 建国大学产业大学院影像产业学科, 首尔, 韩国 目的 : 探讨芥子酸对 UVB 诱导损伤的人角质形成细胞的抗氧化以及抗炎功效, 确认芥子酸作为抗衰老化妆品原料的可行性 方法 : 对 HaCaT 细胞用不同浓度的芥子酸处理后, 利用 UVB 进行氧化应激 为确认芥子酸的细胞保护作用, 利用 water-soluble tetrazolium salt (WST-1) 方法来评价 ; 为确认由 UVB 诱导产生的活性氧 (reactive oxygen species, ROS) 的浓度变化, 利用 2,7 -dichlorofluorescin diacetate (DCF-DA) 来进行测定 ; 为确认与抗氧化和抗炎相关的遗传因子的表达程度, 利用 quantitative real-time polymerase chain reaction (qrt-pcr) 方法来测定 结果 : 芥子酸由 UVB 产生的细胞毒性中保护细胞, 并减少 ROS 的生成 芥子酸增加与抗氧化相关的遗传因子 superoxide dismutase 1 (SOD1) catalase (CAT ) glutathione peroxidase 1 (GPX1) nuclear factor (erythroid-derived 2)-like 2 (NRF2) 以及 heme oxygenase 1 (HO1) 等的表达程度, 并以浓度依赖性方式减少脂质过氧化 此外, 与炎症相关的遗传因子 cyclooxygenase 2 (COX2) interleukin 6 (IL6) 以及 interleukin 8 (IL8) 的表达以浓度依赖性方式减少 结论 : 从以上的研究结果, 芥子酸对 UVB 具有抗氧化和抗炎作用, 因此作为预防衰老的化妆品原料充分具有可行性 关键词 : 芥子酸, 抗氧化, 抗炎,UVB, 人角质形成细胞 522 http://dx.doi.org/10.20402/ajbc.2017.0177