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Asiaticoside enhances normal human skin cell migration, attachment and growth in vitro wound healing model.

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Keywords:

Cutaneous wound repair

Centella asiatica

Asiaticoside

Human dermal fibroblast

Human epidermal keratinocyte

ABSTRACT

Wound healing proceeds through a complex collaborative process involving many types of cells. Keratinocytes and fibroblasts of epidermal and dermal layers of the skin play prominent roles in this process. Asiaticoside, an active component of Centella asiatica, is known for beneficial effects on keloid and hypertrophic scar. However, the effects of this compound on normal human skin cells are not well known. Using in vitro systems, we observed the effects of asiaticoside on normal human skin cell behaviors related to healing. In a wound closure seeding model, asiaticoside increased migration rates of skin cells. By observing the numbers of cells attached and the area occupied by the cells, we concluded that asiaticoside also enhanced the initial skin cell adhesion. In cell proliferation assays, asiaticoside induced an increase in the number of normal human dermal fibroblasts. In conclusion, asiaticoside promotes skin cell behaviors involved in wound healing; and as a bioactive component of an artificial skin, may have therapeutic value.

[C]2012 Elsevier GmbH. All rights reserved.

Introduction

Skin is the largest organ in the body comprising approximately 15% of body weight, with epidermis, dermis and the subcutaneous layer included. Epidermis, the outermost layer of the skin, maintains a vital barrier against external insults. Dermis, the layer between the epidermis and subcutaneous tissue, endows the skin with firmness, elasticity and strength, and also regulates body temperature through control of blood flow and sweating. Keratinocytes comprise 95% of epidermis, as the major cellular constituent, and fibroblasts predominate among cell types in the dermis (Zouboulis 2000; Braun-falco et al. 2000).

Wound healing spans several complicated phases, including inflammation, granulation and re-epithelialization (new tissue formation), contraction and regeneration of tissues (Singer and Clark 1999). Soon after wounding, cytokines attract inflammatory cells to remove damaged tissues and foreign substances. As inflammation subsides, fibroblasts and keratinocytes migrate into the wound area, adhere and proliferate to form new tissue. In this phase, changes in gene expression and phenotype guide the proliferation, migration and differentiation of keratinocytes and fibroblasts. New tissue formation begins with the migration of keratinocytes over injured dermis followed by capillary formation to support fibroblasts and macrophages as these cells replace the fibrin clot with granulation tissue. A second wave of keratinocytes will use the granulation tissue as a substrate (Wong et al. 2007; Gurtner et al. 2008). Fibroblasts also compensate for lost tissue and protect the wound area from intrusion by debris. During re-epithelialization, keratinocytes proliferate and differentiate to restore function to the epithelium as a barrier against external conditions (Gurtner et al. 2008). Wound repair concludes through contraction of the wound site and tissue remodeling through differentiation and programmed cell death. Cutaneous wound healing is important for both medical and esthetic reasons and a large number of bioactive compounds have been tested for capacity to promote this process (Gupta et al. 2005; Phan et al. 2001; Kim and Mendis 2006).

Centella asiatica has a long history of use in Asia for treating skin and vascular disease. Active components derived from the leaves of this small flowering plant, include asiaticoside, asiatic acid, made-cassic acid and other compounds not yet identified. Among these components, asiaticoside displays the highest activity, as observed in the healing of gastric ulcer, leprosy and certain types of tuberculosis (Guo et al. 2004; Boiteau and Ratsimamanga 1956; Shukla etal. 1999). Asiaticoside may also inhibit proliferative activity related to keloid and hypertrophic scar. The effects of asiaticoside on skin disorders such as keloid and hypertrophic scar have been studied in vitro and in vivo (Xie et al. 2009; Tang et al. 2011; El-Hefnawi 1962); however, studies have not yet clearly defined the effects of asiaticoside on normal skin cells in wound healing. We studied the effects of asiaticoside on behaviors of normal human skin cell to evaluate asiaticoside as a natural pharmaceutical for wound healing.

Materials and methods

Cell culture conditions

Adult human dermal fibroblasts (aHDFs) were purchased from Lonza Group, Ltd. (Walkersville, MD, USA) and maintained in fibroblast basal medium-2 (FBM-2) supplemented with growth kit (10 ml of fetal bovine serum, 0.5 ml of insulin, 0.5 ml of gentamicin sulfate amphotericin-B (GA-1000) and 0.5 ml of r-human fibroblast growth factor-B, Lonza). Adult normal human epidermal keratinocytes (aNHEKs) were purchased from Lonza and maintained in keratinocyte basal media (KBM-Gold) supplemented with growth kit (2 ml of bovine pituitary extract, 0.5 ml of insulin, transferrin, hydrocortisone, GA-1000 and r-human epidermal growth factor, and 0.25 ml of epinephrine, Lonza). Cells were incubated at 37 C in a 5% CO2 atmosphere.

Asiaticoside treatment

Asiaticoside (C43 H78019, molecular weight = 959.12, purity <99%) determined by HPLC was purchased from Xi'an Bosheng Biomedical Technology (Xi'an, Shaanxi, China). To dissolve the water-insoluble asiaticoside, dimethyl sulfoxide (DMSO; Sigma-Aldrich Corporation, St. Louis, MO, USA) was used to prepare a stock solution of 200 mM asiaticoside for in vitro assays. The stock solution was diluted with serum free media without growth factors to concentrations of 0, 62.5, 125, 250, 500, and 1000 p.M asiaticoside and cells were treated with equal volumes at each concentration. The same amount of DMSO was used to make various concentrations of asiaticoside using the stock solution and to avoid the effect of DMSO on skin cells behaviors.

Cell viability assay

The cytotoxicity of asiaticoside was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MU) assay. The aHDFs were plated at a density of 1 x 105 cells/well in 48-well plates and aNHEKs were plated at 1 x 105 cells/well in 24-well plates. Cells were incubated for 24 h and treated with various concentrations of asiaticoside (62.5 [micro]M to 1 mM) as described above. After 24 h of asiaticoside treatment, cells were incubated with 0,5 mg/m1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-cliphenyltetrazolium bromide (Amresco, Inc., Solon, OH, USA) for 4 h in the dark. The formazan salts formed were dissolved in DMSO and the optical density was measured at 570 nm.

Cell migration assay (wound-healing assay)

A wound closure seeding model was constructed using silicon culture inserts (lbidi, LLC, Munchen, Germany) with two individual wells for cell seeding. Each insert was placed in a culture dish, and 8 x 103 cells of aHDF or 2 x 104 cells of aNHEK were plated in each well and grown to form a confluent and homogeneous layer. Twenty-four hours after cell seeding, the culture insert was removed and a cell-free area, the "wound" made by the culture insert, could be observed. The wound was approximately 500 [micro]m wide, The cells were treated with 10 [micro],g/m1 of mitomycin C in serum free media without growth factors for 2 h to suppress proliferation. Healing of the wound by migrating cells after asiaticoside treatment was observed over time by light microscopy (IX-70, Olympus) and analyzed using Image J software (NIH, USA).

Cell attachment assay

Cell attachment was determined at 4-6 h after seeding, depending on the cell type, using the MIT assay. Briefly, fibroblasts were seeded at an initial density of 5 x [10.sup.4]cells/well in 24-well plates with asiaticoside co-treatment and incubated for 4h in a C[O.sub.2]atmosphere. Keratinocytes were plated at the initial density of 5 x [10.sup.4]cells/well in 48-well plates with treatment of asiaticoside as described above, and then incubated for 6 h. Unattached cells were removed by gentle washing with phosphate buffered saline (PBS) and cell numbers attached were determined by MU assay as described above.

For cell morphometric analysis, cells were plated at the initial density of 5 x [10.sup.4]cells/well in 24-well plates with asiaticoside co-treatment and incubated for 4-6h in a C[O.sub.2]atmosphere as described above. Cells were then fixed with ice-cold 70% ethanol for 30 min. Actin cytoskeleton was visualized by staining with Alexa (488)-conjugated phalloidin (1 U/sample, Invitrogen, Carlsbad, CA, USA) and cell nuclei were counterstained with propidium iodide (Sigma-Aldrich Corporation, Steinheim, Germany). Six random pictures were taken using an IX-70 microscope equipped with a DP-71 digital camera (Olympus, Japan). Areas with 30 cells attached were measured for each group using Imagej software.

Cell proliferation assay

To assess proliferation, cells were plated at an initial density of 1 x [10.sup.4]cells/well in 48-well plates and asiaticoside was treated. After incubation for 1, 3 and 5 ci, cell numbers were determined by MTT assay for aHDFs as described above, or a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay (Roche Applied Science, Basel, Switzerland) for aNHEKs.

For the BrdU incorporation assay, a BrdU-labeling solution was added to cells at given times and cells were returned to incubation for 2 h at 37 C. Labeling medium was then removed and the cells were incubated with fixation solution for 30 min at room tern-perature. The substrate solution was added and absorbance was measured at 370 nm with a 492 nm reference using an automatic microplate reader (Spectra Max 340, Molecular Devices, Inc., Sunnyvale, CA, USA).

Statistical analysis

Treatment groups were compared using Student's t-test. Experimental values were expressed as mean values with standard deviation (SD). A p-value less than 0.05 was considered significant.

Results and discussion

Asiaticoside increased the migration rate of skin cells

In the cell viability assay, asiaticoside was not cytotoxic in aHDFs at any concentration from 61.5 to 1000 [micro]M or in aNHEKs at concentrations from 61.5 to 500 [micro]M. At 1000[micro]M, however, asiaticoside decreased viability in aNHEKs (data not shown).

Fig. 1 shows the effects of asiaticosicle on migration of normal human skin cells. Compared with a control group, asiaticoside-treated cells migrated faster, and the most effective concentration varied with cell type. In fibroblasts, asiaticoside at 250 [micro]M increased migration rate most effectively and improved wound healing by approximately 20% compared with a control group. In keratinocytes, 500[micro]M asiaticoside improved wound healing by about 20%. Winter has suggested that movement of epidermal cells on the surface of the wound influences epidermal repair in cutaneous wound healing (Winter 1964). New tissue formation during wound repair is characterized by migration and proliferation of different cell types including keratinocytes and fibroblasts. It is therefore plausible that asiaticoside may promote tissue repair by increasing skin cell migration rates.

In hypertrophic scars and keloids, asiaticoside may increase the expression of matrix metalloproteinase-1 (MMP-1), and this may induce an intermediate state of cell adhesiveness (cellular "de-adhesiveness") that may be involved in tissue adaptation and repair (Murphy-Ullrich 2001; Lu et al. 2005). Asiaticoside may also down-regulate the expression of tissue inhibitor of metalloprotease-1 (TIMP-1) (Zhang et al. 2006). These findings suggest that asiaticoside-induced cell migration might contribute to an adaptive morphogenic process such as healing.

Asiaticoside accelerates initial attachment of skin cells

To investigate the effect of asiaticoside on initial adhesion and spreading of skin cells, we treated cells with increasing concentrations of asiaticoside in serum-free media for 4-6h. In Fig. 2, (A) and (C) show the numbers of aHDFs and aNHEKs attached as assessed by MIT assays, and (B) and (D) show the cell attachment areas of aHDFs and aNHEKs, respectively, as investigated by fluorescent staining. As shown in Fig. 2 (A) and (C), asiatico-side treatment significantly enhanced the numbers of fibroblasts attached by approximately 40% and the numbers of keratinocytes, by more than 10%. At 1000M asiaticoside, aNHEKs appeared to decrease in number, which may indicate cell type-specific cytotoxicity. Asiaticoside also increased the areas occupied by attached skin cells (Fig. 2(B) and (D)).

Cell adhesion occurs in three stages, i.e., attachment, spreading, and the formation of focal adhesions and stress fibers (Murphy-Ullrich 2001). The spreading of cells may be involved in adhesion stability. Our findings indicate that asiaticoside may facilitate and stabilize skin cell adhesion.

The initial cell adhesion and spreading activities would be especially important in the use of artificial skin preparations such as wound dressings and scaffolds for tissue engineering (Min et at. 2004). In the case of a full-thickness skin wound with loss of both of epidermis and dermis, an artificial skin can be used to "stand in for" both skin layers and promote cutaneous wound repair. Currently, bioactive compounds may be introduced into artificial skins to promote healing. Merrell et al. incorporated curcumin isolated from the root of Curcumin longa L into poly(caprolactone) as a diabetic wound dressing with anti-oxidant and anti-inflammatory properties (Merrell et al. 2009). Film-and foam-like structures of N-carboxybutylchitosan and of agarose impregnated with quercetin and thymol show properties compatible with use in wound dressings (Dias et al. 2011). After transplantation of an artificial skin as substitute for dermis, a cultured or bio-modified artificial epidermis is often transplanted over the dermis substitute if the wound area is too broad to be healed naturally. Our findings for cell adhesion suggest that if asiaticoside is incorporated into an artificial dermis, the transplanted epidermis could attach more rapidly and securely.

Asiaticoside increases the proliferation rate in skin cells

In a dose-dependent manner, asiaticoside promoted the growth of aHDFs (Fig. 3). At 62.5 [micro]M asiaticoside, the cell number did not increase significantly compared with a control DMSO until the 5-d assessment; however, at 125 [micro]M and higher concentrations of asiaticoside the numbers of treated cells increased steadily from 1 to 5 d compared with the control group. In contrast, asiaticoside did not influence the growth rate of keratinocytes.

Asiaticoside is known to induce a dual specificity protein phosphatase (DUSP) and dual specificity phosphatase 3 (DUSP3) that negatively regulate the mitogen-activated protein (MAP) kinase superfamily, and the associated cellular proliferation and differentiation, in normal human dermal fibroblasts (Lu et al. 2004). Asiaticoside is also reported to have an antiproliferative effect on hypertrophic and keloid fibroblasts and keratinocytes (Tang et al. 2011; Sampson et al. 2001). In this study, asiaticoside enhanced proliferation in normal human dermal fibroblasts and did not affect the growth rate of normal human epidermal keratinocytes.

Migration, adhesion and proliferation are vital skin cell behaviors in wound repair (Singer and Clark 1999; Wong et al. 2007; Curtner et at. 2008; Min et at. 2004). In this study, asiaticoside increased the migration rates and initial attachment of skin cells and promoted normal human dermal fibroblast proliferation. Skin cell behaviors such as spreading and migration are major determinants of the wound closure rate (Albuquerque et at. 2000). The biological activities of asiaticoside support its use as a promoter of wound healing and potentially, as a bioactive component of an artificial skin.

Acknowledgment

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2011-0007747).

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* Corresponding author at: Department of Medical Engineering, Yonsei University College of Medicine, Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea. Tel.: +8222228 1917; fax: +8223639923.

E-mail address: parkjc@yuhs.ac (j.-C. Park).

0944-7113/$--see front matter [C] 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.08.002

Jeong-Hyun Lee (a), (b), Hye-Lee Kim (a), (b), Mi Hee Lee (a), Kyung Eun You (a), (b), Byeong-Ju Kwon (a), (b), Hyok Jin Seo (a), (b), Jong-Chul Parka (a), (b) *

(a) Department of Medical Engineering, Yonsei University College of Medicine, Shinclion-dong, Seodoemun-gu, Seoul 120-752, Republic of Korea

(b) Brain Korea 21 for Medical Science, Yonsei University College of Medicine, Shinclion-dong, Seodaermingu, Seoul 120-752, Republic of Korea
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Title Annotation:Short communication
Author:Lee, Jeong-Hyun; Kim, Hye-Lee; Lee, Mi Hee; You, Kyung Eun; Kwon, Byeong-Ju; Seo, Hyok Jin; Park, Jo
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9SOUT
Date:Oct 15, 2012
Words:3263
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