research article ...downloads.hindawi.com/journals/omcl/2012/819623.pdf166 gumi-ro, bundang-gu,...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2012, Article ID 819623, 7 pagesdoi:10.1155/2012/819623

Research Article

Redox Status Is Critical for Stemness in Skin Equivalents

Hye-Ryung Choi, Youn-A Kang, Jung-Won Shin, Jung-Im Na,Chang-Hun Huh, and Kyoung-Chan Park

Department of Dermatology, Bundang Seoul National University Hospital, Seoul National University College of Medicine,166 Gumi-ro, Bundang-gu, Seongnam-si 463-707, Republic of Korea

Correspondence should be addressed to Kyoung-Chan Park, [email protected]

Received 1 June 2012; Accepted 30 July 2012

Academic Editor: Jing Yi

Copyright © 2012 Hye-Ryung Choi et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The skin is constantly exposed to environmental oxidative stress. Skin equivalent (SE) models are three-dimensional systemsin which cell-cell or cell-matrix interactions can be investigated. In this study, the effects of vitamin C or plant extracts withhigh antioxidant activities were tested. There was no significant difference in the epidermal thickness, but the basal cells becamecuboidal when vitamin C or plant extracts were supplemented. Furthermore, immunohistochemical staining showed linear andintense staining of α6 and β1 integrin along the basement membrane in vitamin C or plant extract treated models. The p63 andPCNA were also stained. Results showed that the number of p63 and PCNA positive cells was higher in the vitamin C or plantextract treated models than in the control SEs. Although the relationship between oxidative stress and stem cells is not known,our results suggest that redox status affects the stemness and the proliferative potential of epidermal basal cells by modulatingmicroenvironment to epidermal basal stem cells.

1. Introduction

Most in vitro studies have been done in two-dimensionalmonocultures, while accumulating evidences suggest thatcells behave differently when they were grown within a three-dimensional (3D) extracellular matrix and also interact withother cells [1]. The development of 3D skin equivalent (SE)models has enabled the field to investigate cell-matrix andcell-cell interactions between different cell types [1]. So far,in vitro evaluation of skin sensitivity will be a most commonapplication of SE models [2]. Furthermore, SE models canbe used for an efficacy testing to prove antiageing activity[3] or hypopigmenting effects [4]. However, SEs will bemore useful experimental models in many aspects of biology.Vitamin C is an important low-molecular weight antioxidantmolecule that, in addition to stimulating collagen synthesis,also appears crucial for the healing process and woundrepair [5]. Furthermore, it has been reported that vitaminC promotes formation of the epidermal barrier [6], andwe also reported that vitamin C affects epidermalization inreconstructed human epidermis [7]. Recently, it is reported

that epidermal side population with stem cell-like character-istics exhibited less mitochondrial area, fewer peroxisomes,and produced lower levels of reactive oxygen species (ROS)than more differentiated keratinocytes [8]. These findingssuggest that redox status may be an important regulatorof epidermalization. Then, it will be interesting to examinevarious antioxidants for these effects. Ganoderma lucidum(G. lucidum), a highly nutritional and medicinal mushroom,is known to have strong antioxidant activities [9]. Rhodiolasachalinensis (R. sachalinensis), which had been widely usedas a traditional medicine, is known to have active compoundssuch as catechin, chlorogenic acid, p-Coumaric acid, and p-tyrosol [10]. In this study, the effects of vitamin C and plantsextracts from G. lucidum and R. sachalinensis were studiedin 3D SE models in terms of basement membrane formationand stem cell markers.

2. Material and Methods

2.1. Extraction Procedure. To obtain the extracts, the plantswere thoroughly dried and ground into powder, and then

2 Oxidative Medicine and Cellular Longevity

50% ethanol was used to extract the soluble components atroom temperature for 24 hrs. The extracts were then filteredthrough Whatman filter paper (Whatman Ltd, Maidstone,England) and concentrated as a stock solution (100 mg/mL).

2.2. DPPH Radical Scavenging Assay. A sample of each stocksolution (2 μL, 100x) was added to 80 μL of 0.25 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis,MO) and 118 μL of 70% ethanol to give a final DPPHconcentration of 0.1 mM. Next, the mixture was vigorouslyshaken and then left to stand for 10 min in the dark. Theabsorbance at 517 nm was then measured using an ELISAreader (TECAN, Salzburg, Austria) [11].

2.3. Primary Culture of Keratinocytes and Fibroblasts. Normalhuman keratinocytes and fibroblasts were isolated fromhuman foreskins obtained during circumcisions. All sampleswere obtained with informed consent. Keratinocytes werecultured in keratinocyte growth medium (KGM, Clonetics,San Diego, CA), and fibroblasts were cultured in Dulbecco’smodified Eagle’s medium (DMEM, LM001-05, WelGENE,Daegu, Republic of Korea) supplemented with 10% fetalbovine serum (FBS, Thermo Scientific HyClone, Logan, UT).

2.4. MTT Assay for Cell Cytotoxicity. Cells (4× 104 cells/well)were seeded into 24-well plates. After serum starvation for24 hrs, the cells were incubated with test samples for 24 hrs at37◦C under 5% CO2. MTT (Sigma-Aldrich) solution (100 μLof 5 mg/mL) was added, and the plates were incubated foran additional 4 hrs. The supernatant was removed, and theformazan crystals were dissolved in dimethylsulfoxide (1 mL,Sigma-Aldrich). Then, the optical density was determined at540 nm using an ELISA reader (TECAN) [12].

2.5. Reconstruction of Skin Equivalents. SEs were constructedfollowing our previous method [12]. Briefly, dermal sub-stitutes were prepared according to the reported methodwith some modifications [13]. Type I collagen was extractedfrom the tendons of rat-tails. Dermal substitutes were thenmade by mixing eight volumes of type I collagen with onevolume of 10x concentrated DMEM and one volume ofneutralization buffer (0.05 N NaOH, 0.26 mM NaHCO3, and200 mM HEPES) and then adding 5 × 105 fibroblasts. Aftergelling in a 30 mm polycarbonate filter chamber (3.0 μmMillicell; Millipore, Bedford, MA), human keratinocytes (1×106 cells) were then seeded onto the dermal substitute. After1 day in a submerged state, they were cultured at the air-liquid interface for 12 days. The growth medium consistedof DMEM and Ham’s nutrient mixture F12 at a ratio of3 : 1, supplemented with 5% FBS, 0.4 μg/mL hydrocortisone,1 μM isoproterenol, 25 μg/mL ascorbic acid, and 5 μg/mLinsulin. A low concentration of EGF (1 ng/mL, InvitrogenCo., Carlsbad, CA) was also added during the submergedculture, and a higher concentration of EGF (10 ng/mL) wasadded during the air-liquid interface culture. The mediumwas changed three times per week, and all experiments wererepeated at least twice under the same conditions. During theair exposure period, samples were added.

2.6. Histology and Immunohistochemistry. At the end of thegrowth period, the SEs were fixed with Carnoy’s fixativeand embedded in paraffin. Immunohistochemical stainingwas performed on deparaffinized sections using the avidin-biotin-peroxidase complex technique (DAKO, Carpinteria,CA). Antibodies against integrin α6 (#sc-6597), integrin β1(#sc-9970), and p63 (4A4, #sc-8431) were obtained fromSanta Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodyagainst PCNA (proliferating cell nuclear antigen, #M0879)was obtained from DAKO (Glostrup, Denmark). Results ofimmunohistochemistry were analyzed quantitatively usingMetaMorph Offline version 7.7.0.0 image analysis software(Molecular Devices, Downingtown, PA). The statistical anal-ysis was done using Microsoft Office Excel 2007’s TTESTfunction.

3. Results

3.1. Antioxidant Activity of Plant Extracts. G. lucidum and R.sachalinensis, which are frequently used in herbal medicine,were chosen because of their well-known antioxidant activ-ities [9, 10, 14]. After being extracted with 50% ethanol,the concentrated extracts (100x) were prepared, and theirscavenging effects on DPPH radicals were examined. Com-pared to vitamin C, the extracts from G. lucidum andR. sachalinensis showed relatively weak antioxidant activityat low concentrations (10 μg/mL) but good antioxidantactivities at high concentrations (100 μg/mL) (Figure 1).

3.2. Cytotoxicity of Plant Extracts. The cytotoxicity of theplant extracts was analyzed using an MTT assay. Culturednormal human keratinocytes were incubated with increasingconcentrations of plant extract up to 100 g/mL (Figure 2).The results showed that extracts from G. lucidum and R.sachalinensis were not toxic at any of the concentrationstested. Interestingly, high concentrations of extract from R.sachalinensis slightly increased the proliferation of normalhuman keratinocytes (Figure 2).

3.3. Histology of Normal Skin and SEs. Characteristic mul-tilayering and stratification of the epidermis were observedin all of the SE models. There was no significant differencein the thickness of the epidermis. However, the basal cellsbecame cuboidal when vitamin C or plant extracts wereadded (Figures 3(c), 3(d), 3(e), and inserts).

3.4. Immunohistochemical Staining for Integrins. Integrinα6 is a marker of extracellular adhesion receptors thatis distributed along the dermal epidermal junction andindicates the existence of hemidesmosomes [15]. Integrinβ1, which is expressed throughout the basal cell membrane,plays a role in cell-matrix and cell-cell interactions [16].Immunohistochemical staining revealed that linear andintense staining of α6 and β1 integrin was observed along thebasement membrane in vitamin C or plant extract treatedmodels (Figure 4).

Oxidative Medicine and Cellular Longevity 3

0

20

40

60

80

100

120

1 10 100

Sca

ven

gin

g ca

pabi

lity

(%)

−20

Vitamin C

R. sachalinensisG. lucidum

(μg/mL)

Figure 1: Scavenging effects of antioxidants for DPPH. Scavenging effects were assessed as described in Materials and Methods section. Datashown are the means ± SD of three independent experiments. Vit. C:, vitamin C; G. l:, G. lucidum; R. s:, R. Sachalinensis.

50

60

70

80

90

100

110

120

130

Cel

l via

bilit

y (%

of

CT

L)

CTL

0.1 100

1 μg/mL

μg/mL

10 μg/mL

μg/mL

R. sachalinensisG. lucidum

Figure 2: Effects of antioxidants on keratinocyte viability. Cells were serum-starved for 24 hrs and then treated with antioxidant plantextracts at 0.1–100 μg/mL for 24 hrs in serum-free media. Cell viabilities were determined using MTT assays. Each determination was madein triplicate, and the data shown are the means ± SD. G. l: G. lucidum; R. s: R. sachalinensis.

3.5. Immunohistochemical Staining for p63 and PCNA. Thep63 is a potential stem cell marker that belongs to a familythat includes two structurally related proteins, p53 and p73[17]. PCNA is known to be present throughout the cell cyclein proliferating cells [18]. Immunohistochemical staining forp63 and PCNA was performed, and the number of p63 andPCNA positive cells was counted in randomly selected 5different high-power fields. Results showed that the numberof p63 and PCNA positive cells was generally higher inthe vitamin C or plant extract treated models than in the

control SEs. However, the number of PCNA positive cells wasnot increased in low concentration of plant extract treatedmodels.

4. Discussion

The skin is constantly exposed to exogenous sources ofoxidative stress, including ultraviolet (UV) radiation, chem-ical oxidants, and aerobic microorganisms. Because the skinmust provide the first line of defense against environmental


(b) CTL

(c) Vitamin C

(a) Normal skin

1 μ

g/m

L10

μg/

mL

(d) G. lucidum (e) R. sachalinensis

Figure 3: Histologic findings of normal skin and SEs. SEs were constructed and incubated in the presence of vitamin C, G. lucidum, or R.sachalinensis at a concentration of 1 or 10 μg/mL. Sections of SEs were hematoxylin/eosin stained. (a) normal skin, (b) control SE, (c) SEtreated with vitamin C, (d) SE treated with G. l, (e) SE treated with R. s.

free radical stress, it has developed a complex antioxidantnetwork that includes enzymatic and nonenzymatic com-ponents [19]. Thus, various antioxidants are used to makeskin care products in order to provide protective effectsagainst environmental free radical. It is well known that thereare diverse actions of antioxidant in normal physiology andalso in pathologic conditions [20]. Then, various aspectsof research are absolutely needed to understand the role ofantioxidants.

SE models provide an investigative system in whichcell-cell or cell-matrix interactions can be tested [13]. Inthis study, the effects of vitamin C or plant extracts withhigh antioxidant activities were investigated using 3D SEmodels. First of all, histologic findings were compared.Although the thickness of the epidermis was similar in allof the models tested in this study, the basal layer appearedto be more compact in the models that were treated

with vitamin C or plant extracts (Figure 3). In normalskin, basal cells appear cuboidal in shape with their longaxes aligned perpendicular to the dermoepidermal junction(Figure 3(a), insert). Furthermore, cuboidal keratinocytesflatten as differentiation progresses (Figure 3(a)). Our studyalso showed that the basal cell looked cuboidal when vitaminC or plant extracts were added. Therefore, it can be saidthat cuboid-shaped basal cells are considered to be epidermalbasal cells with higher proliferative potential. Taken together,the results of this study suggest that antioxidants are animportant regulator in the maintenance of the proliferativepotential of epidermal basal cells.

In order to analyze the mechanism, expression of inte-grins was studied. Integrin α6 is distributed along the dermalepidermal junction [15]. Integrin β1 is expressed throughoutthe basal cell membrane [21, 22]. Our results showedthat there was increased expression of α6 and β1 integrin


CTL

Vitamin C R. sachalinensisG. lucidum

1 μg/mL 10 μg/mL 1 μg/mL 10 μg/mL 1 μg/mL 10 μg/mL

(b)

(b)

(a)

(a)

0

50

100

150

200

250

0

50

100

150

200

250

300

CTL

CTL

1 μg/mL 10 μg/mL

Integrin α6 (+) area Integrin β1 (+) area

P < 0.05∗

∗ ∗ ∗

,P < 0.01∗∗

∗∗∗∗

∗∗

(versus CTL)

CTL 1 μg/mL 10 μg/mL

P < 0.05∗

∗

∗

∗

,P < 0.01∗∗

∗∗

∗∗∗∗

(versus CTL)

Vitamin C R. sachalinensis

G. lucidum CTL


G. lucidum

Figure 4: Immunohistochemical staining for integrins. The SEs were constructed and incubated in the presence of vitamin C (Vit. C), G.lucidum, or R. sachalinensis at 1 or 10 μg/mL. Sections of SEs were stained for integrin α6 (a) or integrin β1 (b). Intensity of both integrinswas significantly increased by addition of Vit. C or plant extracts.

along the basement membrane when SEs were treated withvitamin C or plant extracts. It is reported that integrinexpression is reduced in standard SE models compared tonormal skin [7]. Thus, our findings suggest that vitaminC or these plant extracts improved the microenvironmentof epidermal basal cells by modulating integrin expression.Aging is usually defined as a phenomenon which exhibits adeviation from the normal program of extracellular matrixbiosynthesis [23]. In the skin, cell-ECM interactions alsoinfluence normal homeostasis, aging, wound healing, anddiseases [24]. Because basement membrane is an epidermalECM, changes of integrin expression can affect the fateof epidermal stem cells. The p63 is known as a putativestem cell marker of skin [17]. Our results also revealed thatvitamin C or plant extracts increased the number of p63positive cells compared to control model (Figure 5(a)). Thesefindings showed that vitamin C or plant extracts promotesthe survival of epidermal basal cells in SEs. Recently, it hasalso been reported that p63 regulates an adhesion programand cell survival in epithelial cells [25]. In order to foundthe relationship between the stemness and proliferative

potential, immunohistochemical staining for PCNA wasperformed. The number of PCNA positive cells increasedby the addition of vitamin C or high concentrations ofplant extracts (Figure 5(b)). These results showed that thenumber of proliferating cells is increased by the additionof vitamin C or plant extracts. In summary, it can be saidthat vitamin C or plant extracts increased the stemness andthe proliferative potential of epidermal basal cells. Althoughthe relationship between oxidative stress and stem cells isnot known, recent publication showed that epidermal sidepopulation with stem cell-like characteristics produced lowerlevels of ROS than more differentiated cells [8]. In addition,our results showed that addition of vitamin C or plantextracts increased the stemness of epidermal basal cells.Thus, these findings suggest that antioxidant increased thestemness and the proliferative potential of epidermal basalcells by providing favorable microenvironment. Althoughthe specific component of G. lucidum or R. sachalinensiscan show these effects, our results provide evidence thatredox status affects stemness and proliferative potential ofepidermal basal stem cells, but further study is necessary


CTL

Vitamin C R. sachalinensisG. lucidum

1 μg/mL 10 μg/mL 1 μg/mL 10 μg/mL 1 μg/mL 10 μg/mL

(b)

(b)

(a)

(a)

0

50

100

150

200

250

300

350

0

100

200

300

400

500

600

∗∗

∗

∗ ∗ ∗

∗

∗

∗∗

p63 (+) cells

CTL

CTL

1 μg/mL 10 μg/mL

P < 0.05∗ (versus CTL)


G. lucidum

CTL

CTL

1 μg/mL 10 μg/mL

P < 0.05∗ (versus CTL)


G. lucidum

PCNA (+) cells

Figure 5: Immunohistochemical staining for p63 and PCNA. The SEs were constructed and incubated in the presence of vitamin C (Vit.C),G. lucidum, or R. sachalinensis at 1 or 10 μg/mL. Sections of SEs were stained for p63 (a) or PCNA (b).

to explore the molecular mechanism of vitamin C or plantextract.

Acknowledgments

This study was supported by a grant of the Korea Healthcaretechnology R&D Project, Ministry of Health & Welfare,Republic of Korea (Grant no. A100179, A103017).

References

[1] L. Li, M. Fukunaga-Kalabis, and M. Herlyn, “The three-dimensional human skin reconstruct model: a tool to studynormal skin and melanoma progression,” Journal of VisualizedExperiments, no. 54, Article ID e2937, 2011.

[2] M. Nagasawa, H. Hayashi, and T. Nakayoshi, “In vitro eval-uation of skin sensitivity of povidone-iodine and otherantiseptics using a three-dimensional human skin model,”Dermatology, vol. 204, supplement 1, pp. 109–113, 2002.

[3] V. Frei, E. Perrier, I. Orly, A. Huc, C. Augustin, and O. Damour,“Activation of fibroblast metabolism in a dermal and skinequivalent model: a screening test for activity of peptides,”

International Journal of Cosmetic Science, vol. 20, no. 3, pp.159–173, 1998.

[4] Z. Wang, X. Li, Z. Yang, X. He, J. Tu, and T. V. Zhang, “Effectsof aloesin on melanogenesis in pigmented skin equivalents,”International Journal of Cosmetic Science, vol. 30, no. 2, pp.121–130, 2008.

[5] M. V. Catani, I. Savini, A. Rossi, G. Melino, and L. Avigliano,“Biological role of vitamin C in keratinocytes,” NutritionReviews, vol. 63, no. 3, pp. 81–90, 2005.

[6] S. T. Boyce, A. P. Supp, V. B. Swope, and G. D. Warden,“Vitamin C regulates keratinocyte viability, epidermal barrier,and basement membrane In Vitro, and reduces woundcontraction after grafting of cultured skin substitutes,” Journalof Investigative Dermatology, vol. 118, no. 4, pp. 565–572, 2002.

[7] S. Wha Kim, I. W. Lee, H. J. Cho et al., “Fibroblasts andascorbate regulate epidermalization in reconstructed humanepidermis,” Journal of Dermatological Science, vol. 30, no. 3,pp. 215–223, 2002.

[8] W. J. Carr, R. E. Oberley-Deegan, Y. Zhang, C. C. Oberley,L. W. Oberley, and M. Dunnwald, “Antioxidant proteins andreactive oxygen species are decreased in a murine epidermalside population with stem cell-like characteristics,” Histochem-istry and Cell Biology, vol. 135, no. 3, pp. 293–304, 2011.


[9] T. P. Smina, J. Mathew, K. K. Janardhanan, and T. P. A.Devasagayam, “Antioxidant activity and toxicity profile oftotal triterpenes isolated from Ganoderma lucidum (Fr.) P.Karst occurring in South India,” Environmental Toxicology andPharmacology, vol. 32, no. 3, pp. 438–446, 2011.

[10] S. H. Park, D. S. Kim, S. H. Park, J. W. Shin, S. W. Youn,and K. C. Park, “Inhibitory effect of p-coumaric acid byRhodiola sachalinensis on melanin synthesis in B16F10 cells,”Pharmazie, vol. 63, no. 4, pp. 290–295, 2008.

[11] L. M. McCune and T. Johns, “Antioxidant activity in medicinalplants associated with the symptoms of diabetes mellitus usedby the Indigenous Peoples of the North American borealforest,” Journal of Ethnopharmacology, vol. 82, no. 2-3, pp.197–205, 2002.

[12] D. S. Kim, H. J. Cho, H. R. Choi, S. B. Kwon, and K. C. Park,“Isolation of human epidermal stem cells by adherence andthe reconstruction of skin equivalents,” Cellular and MolecularLife Sciences, vol. 61, no. 21, pp. 2774–2781, 2004.

[13] E. Bell, B. Ivarsson, and C. Merrill, “Production of a tissue-like structure by contraction of collagen lattices by humanfibroblasts of different proliferative potential in vitro,” Proceed-ings of the National Academy of Sciences of the United States ofAmerica, vol. 76, no. 3, pp. 1274–1278, 1979.

[14] M. W. Lee, Y. A. Lee, H. M. Park et al., “Antioxidative PhenolicCompounds from the Roots of Rhodiola sachalinensis A. Bor,”Archives of Pharmacal Research, vol. 23, no. 5, pp. 455–458,2000.

[15] A. Sonnenberg, J. Calafat, H. Janssen et al., “Integrin α6/β4complex is located in hemidesmosomes, suggesting a majorrole in epidermal cell-basement membrane adhesion,” Journalof Cell Biology, vol. 113, no. 4, pp. 907–917, 1991.

[16] R. Grose, C. Hutter, W. Bloch et al., “A crucial role of β1integrins for keratinocyte migration in vitro and duringcutaneous wound repair,” Development, vol. 129, no. 9, pp.2303–2315, 2002.

[17] G. Pellegrini, E. Dellambra, O. Golisano et al., “p63 identifieskeratinocyte stem cells,” Proceedings of the National Academyof Sciences of the United States of America, vol. 98, no. 6, pp.3156–3161, 2001.

[18] R. Bravo, R. Frank, P. A. Blundell, and H. Macdonald-Bravo,“Cyclin/PCNA is the auxiliary protein of DNA polymerase-δ,”Nature, vol. 326, no. 6112, pp. 515–517, 1987.

[19] B. Poljsak and R. Dahmane, “Free radicals and extrinsic skinaging,” Dermatol Res Pract, vol. 2012, Article ID 135206, 4pages, 2012.

[20] I. Paur et al., “Antioxidants in herbs and spices: roles inoxidative stress and redox signaling,” in Herbal Medicine:Biomolecular and Clinical Aspects, I. F. F. Benzie and S.Wachtel-Galor, Eds., llc, Boca Raton, Fla, USA, 2011.

[21] H. Larjava, “Expression of β1 integrins in normal humankeratinocytes,” American Journal of the Medical Sciences, vol.301, no. 1, pp. 63–68, 1991.

[22] C. E. Klein, T. Steinmayer, J. M. Mattes, R. Kaufmann, andL. Weber, “Integrins of normal human epidermis: differentialexpression, synthesis and molecular structure,” British Journalof Dermatology, vol. 123, no. 2, pp. 171–178, 1990.

[23] J. Labat-Robert and L. Robert, “Aging of the extracellularmatrix and its pathology,” Experimental Gerontology, vol. 23,no. 1, pp. 5–18, 1988.

[24] F. M. Watt and H. Fujiwara, “Cell-extracellular matrix inter-actions in normal and diseased skin,” Cold Spring HarborPerspectives in Biology, vol. 3, no. 4, 2011.

[25] D. K. Carroll, J. S. Carroll, C. O. Leong et al., “p63 regulatesan adhesion programme and cell survival in epithelial cells,”Nature Cell Biology, vol. 8, no. 6, pp. 551–561, 2006.

Submit your manuscripts athttp://www.hindawi.com

Stem CellsInternational

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com

research article ...downloads.hindawi.com/journals/omcl/2012/819623.pdf166 gumi-ro, bundang-gu,...

Documents