ISSN 2466-2046 (Print) ISSN 2466-2054 (Online) Asian J Beauty Cosmetol 2017; 15(3): 367-376 http://dx.doi.org/10.20402/ajbc.2016.0158 R E S E A R C H A R T I C L E Open Access on Human Dermal Fibroblasts Na-Kyeong Lee 1, Jung-Eun Ku 2, Hyo Sun Han 3,4* 1 Department of Cosmetics, JEI University, Incheon, Korea 2 Department of Cosmetology, Kyung-In Women s University, Incheon, Korea 3 Korea Institute of Dermatological Sciences, Seoul, Korea 4 Department of Biological Engineering, Konkuk University, Seoul, Korea * Corresponding authors: Hyo Sun Han, Department of Biological Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel.: +82 70 7707 4564 Email: dang0602@hanmail.net Received June 19, 2017 Revised July 6, 2017 Accepted July 11, 2017 Published September 30, 2017 Abstract Purpose: 6-Shogaol is one of the spicy flavor ingredients of ginger and hydrolysis product of gingerols. The purpose of this study is to investigate the potential of 6-shogaol as a natural cosmetic raw material by examining cell protection and inflammation inhibitory activity in human dermal fibroblasts (HDFs). Methods: To verify the cytoprotective effects of 6-shogaol, cell viability was measured by watersoluble tetrazolium salt (WST-1) assay. To verify the anti-inflammatory effects of 6-shogaol, we performed nuclear factor kappa-light-chain-enhancer of activated B cells (NFΚB) promoter luciferase assay to check the expression of NFΚB. Additionally, the expression of inflammation related gene such as cyclooxygenase 2 (COX2), receptor for advanced glycation end product (RAGE), tumor necrosis factor alpha (TNFΑ), interleukin 6 (IL6), and interleukin 8 (IL8) mrna was measured by quantitative real-time polymerase chain reaction (qrt-pcr). Results: 6-Shogaol showed no toxicity in HDFs at concentrations of 5, 10, 20, and 40 μm, respectively and the cell viability was increased in a dose dependent manner. qrt-pcr analysis showed 6-shogaol treatment downregulated the expression of NFΚB, COX2, RAGE, TNFΑ, IL6, and IL8 in a dose dependent manner, resulting that 6-shogaol leads to protective activities against inflammatory responses in HDFs. Conclusion: As mentioned above, 6-shogaol restored cell viability and decreased the expression of inflammatory factors in a dose dependent manner. Consequently, these results suggest the possibility of 6-shogaol as cosmetic material preventing skin aging, through identified functions on cytoprotection and anti-inflammation. Keywords: Human dermal fibroblast, Cytoprotection, Anti-inflammation, 6-Shogaol, Cosmetic Introduction 피부에다양한산화적스트레스가증가하면홍반, 부종, 발열과같은염증반응이유발된다. 인체에서가장흔하게발생하는염증반응은조직에화학물질, 세균감염, 물리적작용등의기질적변화를가져오는자극이가해졌을때그상처부위를복구하고재생하는일련의과정중에일어난다 (Greaves & Søndergaard, 1970; Hruza & Pentland, 1993). 면역관련세포인대식세포 (macrophage) 는 lipopolysaccharide (LPS) 또는 interferon gamma (IFNγ) 와같은자극에의해염증반응전사인자인 NFκB를활성화시키고, 결과적으로 inducible nitric oxide synthase (inos), prostaglandins (PGs), nitric oxide (NO), COX2를만들어내어염증반응을일으킨다 (Nathan, 1987). NFκB는염증반응에관여하는대표적인전사인자로 (Gomez-Nicola et al., 2010), 평상시에는 inhibitor of kappa B (IκB) 와복합체를이루어비활성상태로존재하다가자외선 (ultraviolet, UV) 과같은외부자극을받으면활성화된후핵으로이동하여 COX2, RAGE, TNFA, IL6, IL8 등과같은염증반응을일으키는유전자의발현을촉진시킨다 (Baeuerle, 1998; Longley et al., 1988). 외인성노화의가장대표적원인중하나인자외선은그파장길이에따라 UVA 와 UVB 그리고 UVC 로나뉘는데, 이중 320 400 nm의장파장인 UVA 는 UVB보다에너지레벨자체는낮지만지구 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.
상에 20배가량더많이존재하고, 비교적파장이길어대기중에서거의소실되지않고, 진피층까지침투하는것으로알려져있다 (Kim et al., 2011). 이러한 UVA 의지속적인노출은진피섬유아세포에산화적스트레스를유발하여피부의 DNA, 지질, 단백질을손상시키고 (Cha & Kim, 2015), NFκB, p53과같은여러전사인자들을조절하여세포사멸과염증등을일으킨다 (Yoon et al., 2013). 특유의매운맛과향으로전세계적으로오랫동안사용되어온생강 (Zingiber officinale) 은특히항염증, 항산화, 항혈전, 항암그리고관절염치료에효과적인것으로알려져있다 (Kim et al., 2008; Ling et al., 2010). 이러한생강의효능을나타내는주요성분은 gingerols, shogaols, paradols 등이고, 이중신선한생강에가장많이함유되어있는 gingerols의가수분해산물이바로 shogaols이다 (Bak et al., 2012; Huang et al., 2014). 생강의신미성분중하나인 6-shogaol 의최근연구동향을살펴보면, 의약학, 생명과학, 식품분야에서의항암, 항염증, 항산화, 항균효능에관한것이대부분이고, 향장분야에서의연구는극히미미하며특히인간진피섬유아세포에서의 6-shogaol 의효능에대한연구는전무한실정이다. 그러므로본논문에서는자외선에의해노화된인간진피섬유아세포에서 6-shogaol 의세포보호효과와염증반응전사인자인 NFΚB의활성, COX2, RAGE, TNFΑ, IL6, IL8 유전자의발현을살펴보았다. 이를통해향후 6-shogaol 의노화예방화장품원료로서의활용가능성을살펴보고자한다. Methods 1. 세포배양및시료처리본연구에서는인간진피섬유아세포 (human dermal fibroblast, HDF; Lonza, Switzerland) 와 NFΚB reporter NIH3T3 stable cell line (Affymetrix, Thermo Fisher Scientific, USA) 이사용되었다. 세포배양은 Dulbecco s Modified Eagles Medium (DMEM; HyClone, GE Healthcare Life Sciences, UK) 에 10% fetal bovine serum (FBS; HyClone ), 1% antibiotics (penicillin 100 IU/mL, streptomycin 100 μg/ml; Invitrogen, Thermo Fisher Scientific) 를함유한배지가사용되었고, 37, 5% CO 2 가유지되는조건에서배양되었다. 6-Shogaol (Sigma-Aldrich, USA) 은순수정제 (>90%) 된가루형태로실험에사용될때는 dimethyl sulfoxide (DMSO; Sigma- Aldrich) 에적정농도로용해하여사용하였다. 세포배양접시에 HDF (2 10 5 cells/well) 를 24 h 배양한후적정농도의 6-shogaol 을배지에첨가하여 6 h 동안전처리한후, 세포에 UV-A lamp (UVP, USA) 를사용하여 UVA 를조사하였으며, UVA 파장은 Fiber Optic Spectrometer (USB2000; Ocean Optics, USA) 으로측정하였다. HDF 에 UVA 를조사하기위해세포배양접시의배지를제거 하고 ph 7.4의 phosphate buffered saline (PBS) 로 2회세척한후세포가건조되지않도록 PBS 1 ml을세척된 HDF 에넣은후세포배양접시의뚜껑을열고 UVA 를조사하였다. UVA 조사후 ph 7.4 의 PBS를제거하고다시새로운배지를첨가하여배양기에서 24 h 추가배양한후실험에사용하였다. 2. Cell viability 측정 6-Shogaol과 UVA 의세포독성을확인하기위해서 WST-1 assay 원리를이용하였다. 먼저, 6-shogaol 자체의세포독성은 96-well plate에 HDF 를 3 10 3 cells/well의농도로 100 μl씩접종하여 24 h 배양후 6-shogaol 을각각 5, 10, 20, 40, 80 μm 농도로 6 h 처리하여확인하였다. UVA 의세포독성은 HDF 를동일농도, 동일시간동안배양한후 UVA 조사한다음 24 h 동안배양하였다. 배양된 well에각각 EZ-Cytox cell viability assay kit reagent (ItsBio, Korea) 10 μl를첨가하여 1 h 배양후 microplate reader (Bio-Rad Laboratories, USA) 를이용하여 490 nm에서흡광도를측정하였다. 6-Shogaol 이 UVA 로손상된세포에미치는보호효과를확인하기위하여 WST-1 assay 방법을이용하여세포생존율을확인하였다. 96-Well plate에 HDF (3 10 3 cells/well) 의농도로 100 μl씩접종하여 24 h 배양후 6-shogaol 을각각 5, 10, 20, 40, 80 μm 농도로 6 h 처리하고, UVA 조사후 24 h 동안추가배양하였다. 배양된 well에동일한 cell viability assay kit reagent 를처리한다음흡광도를측정하였다. 3. RNA 추출과 cdna 제조세포배양이끝난세포를포집하여 TRIzol reagent (Invitrogen TM ) 를이용하여용해한후, 0.2 ml chloroform (Biopure, Canada) 를첨가하여상온에방치하였다. 12000 rpm, 4 조건으로 20 min 간원심분리하여단백질이포함된하등액과 mrna가포함된상등액을분리한후상등액은 0.5 ml isopropanol (Biopure) 을첨가하여 10 min 간상온에방치한다음 12000 rpm, 4 조건으로원심분리하여 RNA 를침전시키고, 75% ethanol (Biopure) 을이용하여세척후 ethanol 을제거하고상온에서건조시켰다. 건조된 mrna 는 diethylpyrocarbonate (DEPC; Biopure) water로녹여실험에사용하였으며, 추출된 RNA는 MaestroNano microspectrophotometer (Maestrogen, USA) 이용하여 260/280 nm의 ratio 1.8 이상의순도의 RNA 만을실험에사용하였다. 이렇게추출된 RNA 로부터 cdna 를제작하기위해서 PCR tube에 1 μg RNA, 0.5 ng oligo dt18, DEPC water를 total 10 μl로제조후 70 에서 10 min 간처리하여 RNA 변성을유도한다음 M-MLV reverse transcriptase (Enzynomics, Korea) 을이용하여 37 에서 1 h 반응시켜 cdna 를합성하였다. 368 http://dx.doi.org/10.20402/ajbc.2016.0158
인간진피섬유아세포에서 6-shogaol 의세포보호및항염증효과 Figure 1. The cellular toxicity of 6-shogaol and UVA in HDFs. Cell viability of 6-shogaol and UVA on HDFs (2 10 ⁵ cells/well) was measured by the WST-1 assay. (A) 6-Shogaol was non-toxic to HDF at the 5, 10, 20, and 40 µm concentrations. (B) Cell viability was decreased with increasing UVA intensity. The graph represents the M±S.D. of the relative cell viability in each sample from triplicate experiments. The student's t-test was conducted to determine statistical significance. * Significantly different from control (p<0.05). UVA, ultraviolet A; HDF, human dermal fibroblast; WST-1, water-soluble tetrazolium salt; M±S.D., mean±standard deviation. 4. qrt-pcr 6-Shogaol 에의한 HDF 내에서일어나는유전자발현패턴을정량적으로분석하기위하여 qrt-pcr 을이용하였다. qrt-pcr 은 PCR tube에 0.2 μm primers, 50 mm KCl, 20 mm Tris/HCl (ph 8.4), 0.8 mm dntp, 0.5 U Ex Taq DNA polymerase, 3 mm MgCl, 1 SYBR green (Invitrogen TM ) 을혼합하여반응액을제조하였으며, Line-Gene K (Hangzhou Bioer Technology, China) 를사용하여 94 에서 3 min 간 denaturation 후, denaturation (94, 30s), annealing (58, 30s), extension (72, 30s) 을 40 cycle 수행하여 PCR을진행하였다. PCR의유효성은 melting curve 로검증하였고, 각유전자의발현은 actin beta (ACTB) 의발현을표준화하여비교분석하였으며실험에사용된 primer 는 Table 1과같다. 5. NFKB luciferase assay 6-Shogaol 이 NFΚB의 promoter 활성에미치는영향을확인하기위하여 NFΚB promoter luciferase assay 를이용하였다. 본실험에서는 promoter 부분에 NFΚB promoter consensus sequence 를가진 reporter 유전자 (luciferase 유전자 ) 가내재되어있는 NF ΚB reporter NIH3T3 stable cell line을 60 mm 배양접시에 2 10 5 cells/well의농도로접종하여 24 h 배양후적절한조건으로 NIH3T3 세포를처치하고, 24 h 추가배양하였다. 배양된세포를수확하여 passive lysis buffer (Promega, USA) 를첨가하고 ice에서 10 min 간방치하여용해한다음 12000 rpm, 4 로 30 min 간원심분리하여상등액을회수하였다. 동량의단백질이포함된시료군을각각 80 μl씩 black 96-well plate에넣은후 luciferin (Promega) 을첨가하여혼합한직후 luminometer (Veritas Technologies, USA) 를이용하여 luciferin의발광정도를측정하였다. 6. 통계처리모든실험들은독립적으로 3회반복수행하였으며, 실험결과는평균 ± 표준편차로나타내었다. 실험결과는 Unpaired student s t-test로검정하였고, p-value 값이 0.05 이하인경우통계적으로유의하다고분석하였다. Table 1. List of primers used in this study Gene Forward primer (5 3 ) Reverse primer (5 3 ) COX2 CGCGGATCCGCGGTGAGAACCGTTTAC GCGAGGAAGCGGAAGAGTCTAGAGTCGACC RAGE CCTGGGAAGCCAGAAATT GCACAGGTCAAGGTCACA IL6 TAACAGTTCCTGCATGGGCGGC AGGACAGGCACAAACACGCACC IL8 CTCTCTTGGCAGCCTTCC CTCAATCACTCTCAGTTCTTTG TNFA CCCAGGGACCTCTCTCTAATC GGTTTGCTACAACATGGGCTACA ACTB GGATTCCTATGTGGGCGACGA CGCTCGGTGAGGATCTTCATG COX2, cyclooxygenase 2; RAGE, receptor for advanced glycation end product; IL6, interleukin 6; IL8, interleukin 8; TNFA, tumor necrosis factor alpha; ACTB, actin beta. http://www.e-ajbc.org 369
Figure 2. Effects of 6-shogaol on cell viability in UVAirradiated HDFs. Cellular toxicity was measured by the WST-1 assay. Cell viability by 6-shogaol was increased in a dose dependent manner up to 40 μm concentration in UVA-irradiated HDFs. The graph represents the M±S.D. of the relative cell viability in each sample from triplicate experiments. The student's t-test was conducted to determine statistical significance. * Significantly different from control (p<0.05). UVA, ultraviolet A; HDF, human dermal fibroblast; WST-1, water-soluble tetrazolium salt; M±S. D., mean±standard deviation. Results and Discussion 1. 6-Shogaol 및 UVA 의세포독성 인간진피섬유아세포에 6-shogaol 을 5, 10, 20, 40, 80 μm 농도로 처리하여 6-shogaol 자체의세포독성을확인한결과, 5 40 μm 농도 까지는각각 118%, 106%, 97%, 99% 의세포생존율을보였으나, 80 μ M 에서세포생존율이 78% 까지감소되었다. 그러므로이후실험에서 는세포독성이없다고판단되는 6-shogaol 40 μm 을최대사용농도 로적용하였다 (Figure 1A). 인간진피섬유아세포에서 UVA 의강도에따른세포생존율은 1 J/ cm² 에서 93%, 5 J/cm² 에서 84% 를보여실험적으로자연회복될가 능성이높아세포독성이미미하다고판단하였다. 또한 15 J/cm² 의 UVA 에서 59%, 20 J/cm² 에서 34% 의세포생존율을보여실험적으로 자연사멸할가능성이높아세포독성이너무강하다고판단하였다. 세포노화란세포가재생능력을잃고, 더이상의세포분열을안하는 것을말하며세포사멸을일으키는것보다조금약한 DNA 손상을주 는경우나타난다 (Campisi, 1998). 그러므로본실험에서는약 73% 의세포생존율을보이는 10 J/cm² 의 UVA 를세포노화의적정강도 로설정하였다 (Figure 1B). 2. 6-Shogaol 의세포보호효과 선행연구에따르면 UVA 는세포내활성산소를유도하여 DNA 손상및세포사멸을초래한다고하였으므로 (Jaszewska et al., 2013), 이에근거하여 10 J/cm² 의 UVA 에손상된인간진피섬유 Figure 3. 6-Shogaol regulates NFKB expression in UVAirradiated HDFs. NFΚB reporter NIH3T3 stable cell line (2 10 ⁵ cells/well) was seeded on 60 mm culture dish and then incubated for 24 h. The cells were pre-treated with various concentrations of 6-shogaol for 6 h. Then, the cells were washed by PBS and irradiated by 10 J/cm² UVA. After further incubation for 24 h, inhibition of NFΚB promoter activity by 6-shogaol was determined by the NFΚB promoter luciferase assay. 6-Shogaol pre-treatment downregulated the expression of NFΚB in a dose dependent manner. The graph is representative of three independent experiments (M±S.D.). The student's t-test was conducted to determine statistical significance. * Significantly different from control (p<0.05). pnfkb, NFΚB promoter; Luc, luciferase; UVA, ultraviolet A; HDF, human dermal fibroblast; PBS, phosphate buffered saline; M±S.D., mean±standard deviation. 아세포에서 6-shogaol 이이를얼마나회복시키는지를알아보 았다. 그결과 UVA 만조사된대조군의세포생존율 (69%) 에비해 6-shogaol 을 10 μm 전처리한경우 75%, 20 μm 에서 83%, 40 μm 에서 89% 의세포생존율을보여 6-shogaol 에의해농도의존적으 로세포생존율이회복되는것이확인되었다 (Figure 2). 이를통해 6-shogaol 은 UVA 에의해발생된세포손상으로부터인간진피섬유 아세포를보호하는효과가있음을알수있었다. 3. NFΚB 의 promoter 활성억제효과 6-Shogaol 이염증반응전사인자인 NFΚB 의 promoter 활성 에미치는영향력을알아보기위해 NFΚB promoter luciferase assay 를이용하여 luciferin 의발광정도를측정하였다. 그결과 10 J/cm² UVA 에의해 NFΚB 의 promoter 활성은 7.3 배증가되었 으나, 6-shogaol 각각 5, 10, 20 μm 전처리후 10 J/cm² UVA 조 사시 NFΚB 의 promoter 활성이 5.4, 2.3 1.6 배로감소되었다. 즉 6-shogaol 이농도의존적으로염증반응전사인자인 NFΚB 의 promoter 활성을감소시키는것이확인되었다 (Figure 3). NFκB 는 세포의성장및분화, 염증반응, 면역반응에중요한유전자들의발 현을조절하는전사인자로서특히자외선에의해활성화되어 TNF α, IL6, IL8 와같은염증성사이토카인과 COX2, RAGE 등의염증 관련유전자의발현을증가시키고, 이는궁극적으로피부노화를촉 370 http://dx.doi.org/10.20402/ajbc.2016.0158
인간진피섬유아세포에서 6-shogaol 의세포보호및항염증효과 Figure 4. Effects of 6-shogaol on expression of COX2, RAGE mrna in UVA-irradiated HDFs. HDF cells (2 10 ⁵ cells/well) were seeded on 60 mm culture dish and then incubated for 24 h. The cells were pre-treated with various concentrations of 6-shogaol. Then, the cells were washed by PBS and irradiated by 10 J/cm² UVA. After further incubation for 24 h, the expression level of COX2 (A), RAGE (B) mrna was measured by the qrt-pcr. 6-Shogaol pre-treatment downregulated the expression of COX2 and RAGE mrna. The graph is representative of three independent experiments (M±S.D.). The student's t-test was conducted to determine statistical significance. * Significantly different from control (p<0.05). COX2, cyclooxygenase 2; RAGE, receptor for advanced glycation end product; UVA, ultraviolet A; HDF, human dermal fibroblast; PBS, phosphate buffered saline; qrt-pcr, quantitative real-time polymerase chain reaction; M±S.D., mean±standard deviation. 진시킨다 (Oh et al., 2004). 그러므로 6-shogaol 은염증반응의첫단계인 NFΚB의활성을억제하여염증을직접적으로발생시키는데관여하는유전자의발현을차단하고궁극적으로피부노화를억제하는것으로보인다. 4. COX2 mrna, RAGE mrna 발현변화 UVA 에의해증가된염증유발유전자인 COX2 mrna, RAGE mrna가 6-shogaol 에의해어느정도억제되는지 qrt-pcr을통해확인하였다. 그결과아라키돈산 (arachidonic acid) 으로부터염증매개물질인 prostaglandin E2 (PGE2) 를생성하는것을촉매하는효소로일명 염증유발유전자 라고불리우는 COX2 (Mendes et al., 2009) 는 10 J/cm² UVA 에의해그발현이 0.37에서 1로약 2.7배증가되었으나, 6-shogaol 전처리후 UVA 조사시에는그발현이 0.84, 0.43, 0.33배로감소되었다. 즉, 6-shogaol 이농도의존적으로염증유발유전자인 COX2 mrna 발현을감소시키는것이확인되었다 (Figure 4A). 또다른염증관련유전자인 RAGE 는세포막표면에존재하는최종당화산물 (advanced glycation end product, AGE) 수용체로최종당화산물에의한세포손상을매개한다. 이는염증이있거나암세포존재시발현이증가하는유전자로다양한리간드와결합하여염증을발생시키고, 세포를손상시킨다 (Xie et al., 2013; Yang et al., 2014). 이러한 RAGE 유전자도 10 J/cm² UVA 에의해그발현이대조군대비 3.7배증가되었으나, 6-shogaol 5, 10, 20 μm 전처리후 UVA 조사시그발현이 0.87, 0.63, 0.55배로감소되었다. 즉 6-shogaol 이농도의존적으로 RAGE mrna 발현을감소시키는것이확인되었다 (Figure 4B). 그러므로 6-shogaol 은농도의존적으로 COX2 mrna와 RAGE mrna 의발현을감소시켜이로인해발생하는인간진피섬유아세포내염증반응및세포손상을감소시키는것으로보인다. 5. TNFΑ mrna, IL6 mrna, IL8 mrna 발현변화 UVA 에의해증가된염증성사이토카인인 TNFΑ mrna, IL6 mrna, IL8 mrna가 6-shogaol 에의해어느정도억제되는지 qrt-pcr을통해확인하였다. 그결과염증성질환의주요조절인자이자종양발생시에세포사멸 (necrosis) 을유도하여종양발생의감시기전으로사용되기도하는 (Agarwal et al., 1988) TNF Α mrna 의발현은 10 J/cm² UVA 에의해 6.9 배증가되었으나, 6-shogaol 전처리후 UVA 조사시그발현이 3.6, 2.3, 1.2배로감소되었다 (Figure 5A). 또한염증시과잉생산되어염증정도를나타내는마커로도이용되는사이토카인 (Frydas et al., 1996; Hirano et al., 1986) 인 IL6 mrna 의발현은 10 J/cm² UVA 에의해대조군대비 8.1배증가되었으나, 6-shogaol 전처리후 UVA 조사시그발현량이 6.7, 4.3, 1.8배로감소되었다 (Figure 5B). 염증부위에중성구가침윤되도록하는역할을하는 IL8 mrna의발현은 10 J/ cm² UVA 에의해대조군대비 7.6배증가되었으나, 6-shogaol 을각각 5, 10, 20 μm 전처리후 10 J/cm² UVA 조사시 IL8 mrna 의발현이 5.1, 2.3, 1.2배로감소되었다 (Figure 5C). 그러므로 6-shogaol 이농도의존적으로 UVA 에의해증가된염증성사이토카인인 TNFΑ mrna, IL6 mrna, IL8 mrna 발현을감소시켜인간진피섬유아세포내염증반응이감소될것으로보인다. http://www.e-ajbc.org 371
Figure 5. Effects of 6-shogaol on expression of TNFA, IL6, and IL8 mrna in UVA-irradiated HDFs. HDF cells (2 10 ⁵ cells/well) were seeded on 60 mm culture dish and then incubated for 24 h. The cells were pre-treated with various concentrations of 6-shogaol. Then, the cells were washed by PBS and irradiated by 10 J/cm² UVA. After further incubation for 24 h, the expression level of TNFΑ (A), IL6 (B), and IL8 (C) mrna was determined by the qrt-pcr. 6-Shogaol treatment downregulated the expression of TNFΑ, IL6, and IL8 mrna in a dose dependent manner. The graph is representative of three independent experiments (M±S.D.). The student's t-test was conducted to determine statistical significance. * Significantly different from control (p<0.05). TNFA, tumor necrosis factor alpha IL6, interleukin 6; IL8, interleukin 8; UVA, ultraviolet A; HDF, human dermal fibroblast; PBS, phosphate buffered saline; qrt-pcr, quantitative real-time polymerase chain reaction; M±S.D., mean±standard deviation. Conclusion 화장품산업은인류의오랜꿈가운데하나인미 ( 美 ) 에대한욕망을바탕으로지금까지꾸준히발전해왔다. 특히 21세기에들어서고령인구의증가로인한소비자층의확대와환경, 화장품원료의안전성에대한관심급증으로인해현재노화예방효능이우수하면서도안전한천연유래화장품원료개발이시급한실정이다. 6-Shogaol 은생강 (Zingiber officinale) 의매운맛을내게해주는주성분중하나로신선한생강에가장많이들어있는 gingerols의가수분해산물이다. 최근 6-shogaol 이 gingerol 보다항염증, 항산화, 항암효과가더뛰어나다는연구보고가잇따르고있으나이와관련한대부분의연구는식품이나의 약학, 생명과학분야에국한되어있다. 현재향장분야에서의 6-shogaol 에대한연구는극히미미하며, 특히인간진피섬유아세포에서의 6-shogaol 에대한연구는전무한실정이다. 본논문에서는항염증, 항산화, 항암효과가있다고알려진 6-shogaol 을인간진피섬유아세포에적용하여세포보호및항염증 효과를기초로한항노화화장품원료로서의가능성을알아보고자한다. 첫째, 인간진피섬유아세포에서 6-shogaol 자체의세포독성및 UVA 에대한세포보호효능을알아보았다. 그결과 6-shogaol 각각 5, 10, 20, 40 µm 농도에서는인간진피섬유아세포에대한독성을보이지않았으나 80 µm 농도에서세포생존율이 78% 까지감소되어세포독성이있는것으로판단되었고, 이후실험에서는 6-shogaol 40 μm을최대사용농도로적용하였다. 또한약 73% 의세포생존율을보인 10 J/cm² 의 UVA 가실험적으로세포노화의적정강도로설정되었으며 6-shogaol 의최대사용농도인 40 µm 이내에서는농도가증가될수록 10 J/cm² 의 UVA 에의해손상된인간진피섬유아세포의세포생존율이회복되는것이확인되었다. 이를통해 6-shogaol 은 UVA 에의해발생된세포노화로부터인간진피섬유아세포를보호하는효과가있음을알수있었다. 둘째, 6-shogaol 이 UVA 에의해손상된인간진피섬유아세포의염증억제에미치는영향을확인한결과, 10 J/cm² 의 UVA 는대표적인염증반응전사인자인 NFΚB의 promoter 활성을급격히 372 http://dx.doi.org/10.20402/ajbc.2016.0158
인간진피섬유아세포에서 6-shogaol 의세포보호및항염증효과 증가시켰지만 6-shogaol 은농도의존적으로이를감소시켰다. 즉, 6-shogaol 은 NFΚB가핵속으로이동하여활성화되는것을억제시켜각종염증관련물질의발현을감소시킨것으로판단되었다. 특히 NFΚB의활성화로인해발현이시작되는 COX2, RAGE, TNFΑ, IL6, IL8의경우도 6-shogaol 의농도가증가됨에따라그발현이감소되었다. 이상의실험결과들을통해 6-shogaol 은 UVA 에의해발생된인간진피섬유아세포의염증발생을억제시키는효능이있음이확인되었다. 결론적으로본실험을통해 6-shogaol 은농도의존적으로 UVA 에의해손상된인간진피섬유아세포를보호하고, 염증을억제하는효능을가진천연유래화장품원료로서활용이가능할것으로사료된다. This work is part of the Hyo Sun Han s Ph.D thesis at Konkuk University, Seoul, Korea. References Agarwal S, Drysdale BE, Shin HS. Tumor necrosis factormediated cytotoxicity involves ADP-ribosylation. The Journal of Immunology, 140: 4187-4192, 1988. Baeuerle PA. Pro-inflammatory signaling: last pieces in the NFkappaB puzzle? Current Biology, 8: 19-22, 1998. Bak MJ, Ok S, Jun M, Jeong WS. 6-Shogaol-rich extract from ginger up-regulates the antioxidant defense systems in cells and mice. Molecules, 17: 8037-8055, 2012. Campisi J. The role of cellular senescence in skin aging. Journal of Investigative Dermatology Symposium Proceedings, 3: 1-5, 1998. Cha HJ, Kim YJ. Procyanidin B1 regualtes matrix metallo protease 1 mrna expression using JNK-AP1-TRE axis in normal human dermal fibroblasts. Asian Journal of Beauty and Cosmetology, 13: 761-765, 2015. Frydas S, Karagouni E, Dotsika E, Reale M, Barbacane RC, Vlemmas I, Anogianakis G, Trakatellis A, Conti P. Generation of TNFα, IFNγ, IL-6, IL-4 and IL-10 in mouse serum from trichinellosis: effect of the anti-inflammatory compound 4-deoxypyridoxine (4-DPD). Immunology Letters, 49: 179-184, 1996. Gomez-Nicola D, Valle-Argos B, Nieto-Sampedro M. Blockade of IL-15 activity inhibits microglial activation through the NFκB, p38, and ERK1/2 pathways, reducing cytokine and chemokine release. Glia, 58: 264-276, 2010. Greaves MW, Søndergaard J. Pharmacologic agents released in ultraviolet inflammation studied by continuous skin perfusion. Journal of Investigative Dermatology, 54: 365-367, 1970. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, et al. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature, 324: 73-76, 1986. Hruza LL, Pentland AP. Mechanisms of UV-induced inflammation. Journal of Investigative Dermatology, 100: 35-41, 1993. Huang HC, Chang SJ, Wu CY, Ke HJ, Chang TM. [6]-Shogaol inhibits α-msh-induced melanogenesis through the acceleration of ERK and PI3K/Akt-mediated MITF degradation. BioMed Research International, 2014: 842569, 2014. Jaszewska E, Soin M, Filipek A, Naruszewicz M. UVA-induced ROS generation inhibition by Oenothera paradoxa defatted seeds extract and subsequent cell death in human dermal fibroblasts. Journal of Photochemistry and Photobiology B: Biology, 126: 42-46, 2013. Kim J, Lee CW, Kim EK, Lee SJ, Park NH, Kim HS, Kim HK, Char K, Jang YP, Kim JW. Inhibition effect of Gynura procumbens extract on UV-B-induced matrixmetalloproteinase expression in human dermal fibroblasts. Journal of Ethnopharmacology, 137: 427-433, 2011. Kim JS, Lee SI, Park HW, Yang JH, Shin TY, Kim YC, Baek NI, Kim SH, Choi SU, Kwon BM, et al. Cytotoxic components from the dried rhizomes of Zingiber officinale Roscoe. Archives of Pharmacal Research, 31: 415-418, 2008. Ling H, Yang H, Tan SH, Chui WK, Chew EH. 6-Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase-9 expression via blockade of nuclear factor-κb activation. British Journal of Pharmacology, 161: 1763-1777, 2010. Longley BJ, Braverman IM, Edelson RL. Immunology and the skin: current concepts. Annals of the New York Academy of Sciences, 548: 225-232, 1988. Mendes RA, Carvalho JF, Waal Iv. An overview on the expression of cyclooxygenase-2 in tumors of the head and neck. Oral Oncology, 45: 124-128, 2009. Nathan CF. Secretory products of macrophages. The Journal http://www.e-ajbc.org 373
of Clinical Investigation, 79: 319-326, 1987. Oh JH, Chung AS, Steinbrenner H, Sies H, Brenneisen P. Thioredoxin secreted upon ultraviolet A irradiation modulates activities of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in human dermal fibroblasts. Archives of Biochemistry and Biophysics, 423: 218 226, 2004. Xie J, Méndez JD, Méndez-Valenzuela V, Aguilar-Hernández MM. Cellular signalling of the receptor for advanced glycation end products (RAGE). Cellular Signalling, 25: 2185-2197, 2013. Yang WI, Lee D, Lee DL, Hong SY, Lee SH, Kang SM, Choi DH, Jang Y, Kim SH, Park S. Blocking the receptor for advanced glycation end product activation attenuates autoimmune myocarditis. Circulation Journal, 78: 1197-1205, 2014. Yoon Y, Bae S, An S, Choe YB, Ahn KJ, An IS. Effects of ultraviolet radiation on the skin and skin cell signaling pathways. Asian Journal of Beauty and Cosmetology, 11: 417-426, 2013. 374 http://dx.doi.org/10.20402/ajbc.2016.0158
인간진피섬유아세포에서 6-shogaol 의세포보호및항염증효과 국문초록 인간진피섬유아세포에서 6-Shogaol 의세포보호및항염증효과 이나경 1, 구정은 2, 한효선 3,4* 1 인천재능대학교화장품과, 인천, 한국 2 경인여자대학교피부미용과, 인천, 한국 3 한국피부과학연구원, 서울, 한국 4 건국대학교생물공학과, 서울, 한국 목적 : 6-Shogaol 은생강 (Zingiber officinale) 의매운맛을내는성분인 gingerols의가수분해산물로서, 본연구에서는 6-shogaol 의인간진피섬유아세포에서의세포보호와염증억제효능을검증함으로써천연유래화장품원료로서의사용가능성을알아보고자한다. 방법 : 6-Shogaol 의세포보호효과를검증하기위하여, water-soluble tetrazolium salt (WST-1) assay 를이용하여세포생존율을측정하였다. 6-Shogaol 의항염증효과를검증하기위하여, nuclear factor kappa-light-chain-enhancer of activated B cells (NFΚB) promoter luciferase assay 를이용하여염증반응전사인자인 NFΚB의발현을측정하였으며염증관련유전자들인 cyclooxygenase 2 (COX2), receptor for advanced glycation end product (RAGE), tumor necrosis factor alpha (TNFΑ), interleukin 6 (IL6), interleukin 8 (IL8) 발현량을 quantitative real-time polymerase chain reaction (qrt-pcr) 를이용하여측정하였다. 결과 : 6-Shogaol 은 5, 10, 20, 40 µm 각각의농도에서인간진피섬유아세포에대한독성을보이지않았으며농도의존적으로 10 J/cm² 의 ultraviolet A (UVA) 에의해손상된인간진피섬유아세포의생존율을회복시켰다. 대표적인염증반응전사인자인 NFΚB의 promoter 활성이 6-shogaol 에의해농도의존적으로감소되었으며, 염증유발유전자인 COX2, RAGE mrna 와각종염증관련사이토카인인 TNFΑ, IL6, IL8 mrna 의발현도 6-shogaol 에의해농도의존적으로감소되었다. 결론 : 6-Shogaol 은세포보호및항염증에대한확인된효능을통해피부노화를예방하는화장품원료로서의가능성을보여줄수있으리라사료된다. 핵심어 : 인간진피섬유아세포, 세포보호, 항염증, 6-Shogaol, 화장품 참고문헌 윤영민, 배승희, 안성관, 최용범, 안규중, 안인숙. 자외선 (Ultraviolet) 이피부및피부세포내신호전달체계에미치는영향. 아시안뷰티화장품학술지, 11: 417-426, 2013. 차화준, 김영주. 인간진피섬유아세포에서코코아주요성분인 Procyanidin B1이 JNK-AP1-TRE Axis를통한 Matrix- Metalloprotease 1 발현조절에미치는영향. 아시안뷰티화장품학술지, 13: 761-765, 2015. http://www.e-ajbc.org 375
中文摘要 6-Shogaol 对人皮肤成纤维细胞的细胞保护和抗炎功效 李奈耿 1, 具貞恩 2 3,4*, 韓孝仙 1 仁川才能大学化妆品学科, 仁川, 韩国 2 敬仁女子大学皮肤美容科, 仁川, 韩国 3 韩国皮肤科学研究院, 首尔, 韩国 4 建国大学生物工学科, 首尔, 韩国 目的 : 6-Shogaol 是生姜 (Zingiber officinale) 的辛辣成分 gingerols 的水解产物 探讨 6-shogaol 对人皮肤成纤维细胞的细胞保护和抗炎功效, 从而鉴定 6-shogaol 作为天然化妆品原料的应用可行性 方法 : 为确认 6-Shogaol 的细胞保护效果, 采用 water-soluble tetrazolium salt (WST-1) 法测定细胞生存率实验 为验证 6-Shogaol 的抗炎功效, 采用 nuclear factor kappa-light-chain-enhancer of activated B cells (NFΚB) promoter luciferase assay 测定炎症反应转录因子 NFΚB 的表达, 以及采用 quantitative real-time polymerase chain reaction (qrt-pcr) 法确定炎症相关遗传因子 cyclooxygenase 2 (COX2),receptor for advanced glycation end product (RAGE),tumor necrosis factor alpha (TNFΑ),interleukin 6 (IL6),interleukin 8 (IL8) 的表达量 结果 : 6-Shogaol 在 5 10 20 40 µm 的浓度下, 对人皮肤成纤维细胞没有毒性 6-Shogaol 对被 10 J/cm² ultraviolet A (UVA) 损伤的人皮肤成纤维细胞的生存率呈浓度依赖性恢复 代表性的炎症转录因子 NFΚB 的 promoter 活性被 6-shogaol 呈浓度依赖性降低 炎症诱发遗传因子 COX2,RAGE mrna 和各种炎症相关细胞因子 TNFΑ IL6 IL8 mrna 的表达被 6-shogaol 呈浓度依赖性降低 结论 : 通过以上研究确认了 6-Shogaol 具有细胞保护和抗炎功效, 因此作为预防抗衰老的化妆品原料充分具有可行性 关键词 : 人皮肤成纤维细胞, 细胞保护, 抗炎症,6-Shogaol, 化妆品 376 http://dx.doi.org/10.20402/ajbc.2016.0158