JOURNAL OF MILITAftV PHRRMACO-MCDICINC 7-2013
STUDY ON INHIBITORY EFFECT OF NGOC LINH GINSENG STEM CELL EXTRACT ON COLLAGEN DEGRADATION IN
FIBROBLASTS BY 2D-PAGE
Nguyen Van Thinh*; Vu Tuan Anh"; Nguyen Huu Tuan Dung**
Pham Xuan Phong***; Nguyen Van Long*
S U M M A R Y
The ability of NgocHnh ginseng (NLG) stem cell extract in controlling the skin aging process In human fibroblasts was investigated. To evaluate the cosmeceutical effects of the extracts on the aging process, MTT assays were conducted with various concentrations of NLG stem cells. Wo extracts showed any cytotoxic activities on ^broblasts at concentrations of up to 10 mg/ml. An effect on the degradation of collagen matrix was observed when the dose concentration was 0.5 mg/ml.
Proteome profiling analysis showed that the extract promoted the expression of proteins related to collagen production and suggested the skin aging effect of the extract. The extract could be considered as an attractive, anti-wrinkle ingredient for cosmetic applications.
* Key words: NgocHnh ginseng; Collagen; 2-D PAGE; Fibroblasts.
I N T R O D U C T I O N
T h e mechanism of collagen degradation is primarily attributable to t h e activation of growth factor receptors on the surfece of fibroblasts and keratinocytes caused by UV irradiation, resulting in signal transduction through a protein kinase cascade and subsequent activation of A P - 1 in the nucleus. The signal transduction could also be strongly up-regulated by R O S induced by UV irradiation. This then stimulates M M P production in both dermis and epidermis and leads to the degradation of collagen and elastic fibers.
Ngoclinh ginseng (Vietnamese ginseng, Panax vietnamensis Ha et Grushv. Araliaceae)
is a wild Panax species that has been used as a herbal medicine in Central Vietnam. In recent years, NLG has been cultured in bioreactors for production of ginseng saponin and ginsenosides for application In pharniaceutical field [1]. In this study, w e extracted active compounds from NLG stem cells by water and prepared the extracts at various concentrations. A m o n g the various effects of NLG reported, a strong regulation of proteins related to collagen degradation was observed via proteome analysis.
Proteome analysis based o n 2-dimensional electrophoresis (2D-PAGE) w a s applied for the profiling of protein factors involved in t h ^ observed effects on collagen degradation.
* Vietnam Military Medical University
** University of Dalat
***Military Institute of Traditional Medicine
Address correspondence to Nguyen Van Thinh: Vietnam Military Medical Univensity E.mail: [email protected]
roUHNBl Of IHIUTflHV PH«BMiaCO-IVI«DLCINt 7-2013 MATERIALS AND METHODS
1. Materials and equipment.
* Materials:
Human fibroblasts were purchased from ATCC (USA). After thawing at 37°C, the cells were cultured in media containing DMEM, 1% antibiotic and anti-fungi, and 10% FBS. Then keeping in incubator with 5%C02, at37°Cfor2days.
Other chemicals for protein electrophoresis were obtained from GE Healthcare- USA.
• Equipment:
Electrophoresis system containing:
EttanlPGphor 3 (GE Healthcare, USA) for lEF, EttanDALTsix (GE Healthcare, USA) for gel electrophoresis, and Processor Plus (GE Healthcare, USA) for gel staining.
2. Methods.
* Preparation of NLG stem cell extract:
After washing the cell by water twice, then dry in vacuum. The dry cells were grinded with water at various ratios for 5 minutes. Filter through 0.45 pm membrane then store the extracts at 4°C before testing.
* Cytotoxicity test:
Human fibroblasts were cultured in 96 well-plate at 10" cell/well at 37°C in incubator with 5% CO2. After 24 hours, refresh the culture media containing the cell extracts at various concentrations. Keep in the incubator for 48 hours. Then add 50 pL MTT 1% into each well. Keep in the incubator for 4 hours. Dimethyl sulfoxide (DMSO) was added in each well and the absorbance was recorded in a microplate reader (BioTel< In-struments, Inc., Wlnoosi<i.
VT) at wavelength of 595 nm S«.100 T. =
Of which,
Jx- Cytotoxicity of the extract at concentration of X mg/ml.
Sxi Absorption value of the well containing fibroblasts treated with the extract at concentration of x mg/ml.
SB: Absorption value of control (treated with water).
* Protein extraction:
Fibroblasts were seeded in petri dish at 5 x 10^ cells/dish In DMEM with 10% FBS and 1% AA. After 24 hours of incubating in 5% CO2 incubator, the media was refreshed by media containing NLG stem cell extract at certain concentration, then the cells were incubated in the incubator. After 48 hours, the cells were trypsin harvested. Centrlfugatlon for collecting, the cells were performed followed by adding lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1 % DTT, 2% earner ampholyte, 4% PIC, 0.002% bromophenol blue), Incubating at 37°C in 5 hour, centrifuging at 1,380 rpm in 1 minute and taking the supernatant.
* Isoelectric focusing (IBF):
Linear strips immobilized pH gradient strips (pH 3 - 10) were rehydrated by a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 2% carrier ampholyte, 10% glycerol, 0.002% bromophenol blue.
The strip was kept with rehydration solution for 15 hours before loading protein. Each strip was loaded 100 pg protein, lysis buffer, 2% DNAse, 5% protein markers.
Equilibration: Prepare SDS equilibration buffer containingT M Urea, 2 M thiourea, 2% SDS, 50 mM Tris-HCI, 30% glycerol, 0.002% bromophenol blue. Prior to use, add 100 mg DTT or 500 mg iodoacetamide pet
JOUANALOFMIUTAAV PHAAMACO-MCDICINC 7-2013 10 ml SDS equilibration buffer. Equilibrate
for 15 minutes.
' Gel- electrophorBsis:
Prepare second dimension gels: The percentage of acrylamide was 12.5% (25 x 20 cm). The homogenous gels containing 42% aery lam ide/bis-acrylamide (30%/0.2%
v/v), 25.49% 1.5 M Tris-HCI, pH 8.8; 1.8%
APS; 0.18% TEMED.
After completing equilibration, the strips were transferred to 12.5% acrylamide homogenous gels (25 x 20 cm). A current at 40 nrWgel was connected to the running
buffer in the buffer tank. T^e electrophoresis was finished after 6 hours.
* Protein staining: Gel staining was conducted by nitrate silver staining method.
' Protein analysis:
After staining, gels were scanned and analyzed using Image Master (Healthcare, USA). Areas and pixel intensities of spots were compared, giving up and down- regulation protein data for the cells treated with NLG stem cell extract. Marker proteins were used to identify pi values and molecular weight of proteins in samples.
RESULTS AND DISCUSSION 1. Cytotoxicity of the stem cell extract on fibroblasts.
Cytotoxicity of NLG stem cell extract was evaluated using five determinations per concentration following the cytotoxicity test method. The absorption values of wells after mixing with DMSO were shown in table 1,
Table 1: Cytotoxicity of NLG stem cell extract on fibroblasts (n = 5).
SAMPLE
1 2 3 4 5 X(%)
SD RSD (%)
ABSORPTION VALUES 0
(mg/ml) 0.426 0.42S 0.420 0 426 0.442 100
0.05 (mg/ml)
0.407 0.366 0.395 0.412 0.429 93.90 4.50 4.80
0.10 (mg/ml)
0.425 0.392 0.372 0.398 0.413 93 49 4 0 4 4.32
0.50 (mg/ml)
0.388 0.407 0.415 0.392 0.397 93.50 3.71 3.96
1.00 (mg/ml)
0.417 0,392 0.377 0.366 0.414 91.89 4 46 4.85
5.00 (mg/ml)
0.411 . 0.388
0.369 0.387 0.397 91.26 3.20 3.61
10.00 (mg/ml)'
0.382 0.378 0.402 0.373 0.399 90.43 3.12 3.45
C* Calculated as final concentrations In the culture media).
The result showed that no toxicity of NLG at concentration of up to 10 mg/ml was observed. In our research, a concentration of 0.5 mg/ml was prepared to evaluate effects of NLG stem cell extract on collagen degradation.
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JOURNRl OF MILITRRV PHRRMflCO-MCDICINe 7-2013 2. Results of study on effects of NLG stem cell extract using 2D-PAGE.
Proteomics study was used as an effective method for investigating the regulation of anti-wrinkle related proteins in skin cells. Results of regulation of proteins related to formation and degradation of collagen were showed in table 2.
Fig 1: Gel images (18 x 24 cm) containing proteins on fibroblasts treated with (a) and
without (b) NLG stem cell extract.
By ImageMaster 7.0, 860 protein were detected. 1114 spots (557 proteins) were matched, 258 were up-regulated and 299 spots were down-regulated, 8 proteins related to anti-aging were identified by UniProt database. Those proteins were interleukin-1 beta (pi 7.0; 17 kDa), MMP-13 (pi 5,32; 63,82 kDa), MMP-1 (p| 6,47; 54 12
kDa), collagen type 1 (pi 5,6; 138,94 kDa), capthepsin D precursor (pi 6,1; 46 kDa), alpha-actinin (pi 5,4; 105,5 kDa), elastinase (pi 10,4; 68,5 kDa), chymottypsin-like eslastase 2B (pi 6,48; 28,81 kDa).
Table 2: Regulation of proteins on fibroblasts treated with NLS extract (n = 4).
PROTEIN Type 1 collagen Inlegnn beta-1 Alpha-sdinin Fibronectin Cathepsin D precursor Interieukin 1-beta Chymotfypsin-IIke elastase 2B MMP-8 MMP-1 MMP-13
UniParc ID P02452 P05556 P35609 P02751 P07339
0 4 3 6 4 5 P08218
P228g4 P03956 P45452
CONTROL
(%)
100 100 100 100
100
100
100
100 100 100
SAMPLE (%) ( X i S D ) 99.89 ± 7 35 96.15 ±8.45 110.09 ±7.94 171.64 ± 9 17
50.08 ± 6.75 156.01 ±7.47
97.50 ±5.13 87.42 ± 8.92 109.55 ±4.06 29.79 ± 6 1 8
The results in table 2 showed the up- regulatlon of Interleukin 1- beta and fibronectin and down-regulation of capthepsin and MMP-13. Fibronectin is the factor of collagen matrix formation. Interleukine 1-beta, ! factor acting as a fibroblast growth factor, leads to increase growrth rate of fibroblasts, and supports to remove wrinkle on skin. The results indicated that NLG stem cell extract down-regulated MMP-13, a factor degrading collagen I, decreasing the degradation of collagen and elastic fibers [2, 3].
Up-regulatlon of capthesin precursor and MMP-13 usually occurs when skin exposes to UV light As a result, ROS was induced.
The ROS Includes superoxide anion, peroxide and singlet oxygen [4, 5].
JOUANAl OF MIUTdAV PHnRMACO-MCDICINC 7-2013
C O N C L U S I O N
Based on the results, NLG stem cell extract showed no toxicity on fibroblasts at concentration of up to 10 mg/ml. Proteome analysis of protein expression in fibroblasts treated by NLG stem cell extract proved anti-wrinkle activity through up~regulafing growth factor interleukine 1-beta, fibronectin and inhibition of collagen degradation via down-regulating degradation factors n a m e d capthepsin precursor and MMP-13,
R E F E R E N C E S
1. Vu Binh Duong et al. Study on callus induction procedure of Ngoclinh ginseng ceils.
Journal of Military Pharmaco-medicine. 2006.
2. Gail Jenkins. Molecular mechanisms of skin ageing. The Biology of Ageing. 2002, 123, pp.801-810.
3. J. VaranI, G.J. Fisher, S. Kang, J.
Voorhees. Molecular mechanisms of intrinsic skin ageing and retinoid induced repair and reversal. J Invest Dermatol Symp Proc.1998, 3, pp.57-60.
4. Hitoshi MasakI Role of antioxidants in Uie skin: Anti-aging effects. Journal of Dermatological Science. 2010, 58, pp.85-90.
5. V\/est MD. The cellular and molecular biology of skin ageing. Archives of Dem^atoiogy.
1994,130, pp.87-89.
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