Study on inhibitory effect of Ngoc Linh ginseng stem cell extract on collagen degradation in fibroblasts by 2D-PAGE

The ability of Ngoclinh 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. No extracts showed any cytotoxic activities on fibroblasts at concentrations of up to 10 mg/ml.

Journal of military pharmaco-medicine 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* * Vietnam Military Medical University ** University of Dalat ***Military Institute of Traditional Medicine SUMMARY The ability of Ngoclinh 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 No extracts showed any cytotoxic activities on fibroblasts 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: Ngoclinh ginseng; Collagen; 2-D PAGE; fibroblasts INTRODUCTION The mechanism of collagen degradation is primarily attributable to the activation of growth factor receptors on the surface of fibroblasts and keratinocytes caused by UV irradiation, resulting in signal transduction through a protein kinase cascade and subsequent activation of AP-1 in the nucleus The signal transduction could also be strongly up-regulated by ROS induced by UV irradiation This then stimulates MMP 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 pharmaceutical field [1] In this study, we extracted active compounds from NLG stem cells by water and prepared the extracts at various concentrations Among the various effects of NLG reported, a strong regulation of proteins related to collagen degradation was observed via proteome analysis Proteome analysis based on 2-dimensional electrophoresis (2DPAGE) was applied for the profiling of protein factors involved in the observed effects on collagen degradation MATERIALS AND METHODS Materials and equipment * Materials: Journal of military pharmaco-medicine 7-2013 Human fibroblasts were purchased from ATCC (USA) After thawing at 37oC, the cells were cultured in media containing DMEM, 1% antibiotic and anti-fungi, and 10% FBS Then were kept in incubator with 5% CO2, at 37oC for days Other chemicals for protein electrophoresis were obtained from GE Healthcare- USA * Equipment: Electrophoresis system containing: EttanIPGphor (GE Healthcare, USA) for IEF, EttanDALTsix (GE Healthcare, USA) for gel electrophoresis, and Processor Plus (GE Healthcare, USA) for gel staining 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 minutes Filter through 0.45 µm membrane then store the extracts at 4oC before testing * Cytotoxicity test: Human fibroblasts were cultured in 96 well-plate at 104 cell/well at 37oC 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 µL MTT 1% into each well Keep in the incubator for hours Dimethyl sulfoxide (DMSO) was added in each well and the absorbance was recorded in a microplate reader (BioTek In-struments, Inc., Winooski, VT) at wavelength of 595 nm Tx  SX 100 SB Of which, T x: Cytotoxicity of the extract at concentration of x mg/ml Sx: 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 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 Centrifugation for collecting the cells were performed followed by adding lysis buffer (7 M urea, M thiourea, 4% CHAPS, 1% DTT, 2% carrier ampholyte, 4% PIC, 0.002% bromophenol blue), incubating at 37oC in hour, centrifuging at 1,380 rpm in minute and taking the supernatant * Isoelectric focusing (IEF): Journal of military pharmaco-medicine 7-2013 Linear strips immobilized pH gradient strips (pH - 10) were rehydrated by a solution containing M urea, 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 µg protein, lysis buffer, 2% DNAse, 5% protein markers Equilibration: Prepare SDS equilibration buffer containing7 M Urea, M thiourea, 2% SDS, 50 mM Tris-HCl, 30% glycerol, 0.002% bromophenol blue Prior to use, add 100 mg DTT or 500 mg iodoacetamide per 10 ml SDS equilibration buffer Equilibrate for 15 minutes * Gel- electrophoresis: Prepare second dimension gels: The percentage of acrylamide was 12.5% (25 x 20 cm) The homogenous gels containing 42% acrylamide/bis-acrylamide (30%/0.2% v/v), 25.49% 1.5 M Tris-HCl, 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 mA/gel was connected to the running buffer in the buffer tank The electrophoresis was finished after 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 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 Table 1: Cytotoxicity of NLG stem cell extract on fibroblasts (n = 5) Absorption values Sample (mg/ml) 0.05 (mg/ml) 0.10 (mg/ml) 0.50 (mg/ml) 1.00 (mg/ml) 5.00 (mg/ml) 10.00 (mg/ml)* 0.426 0.407 0.425 0.388 0.417 0.411 0.382 0.425 0.366 0.392 0.407 0.392 0.388 0.378 0.420 0.395 0.372 0.415 0.377 0.369 0.402 0.426 0.412 0.398 0.392 0.366 0.387 0.373 Journal of military pharmaco-medicine 7-2013 0.442 0.429 0.413 0.397 0.414 0.397 0.399 (%) 100 93.90 93.49 93.50 91.89 91.26 90.43 SD 4.50 4.04 3.71 4.46 3.20 3.12 RSD (%) 4.80 4.32 3.96 4.85 3.51 3.45 (* 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 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 antiwrinkle related proteins in skin cells Results of regulation of proteins related to formation and degradation of collagen were showed in table a b 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, 850 protein were detected 1114 spots (557 proteins) were matched, 258 were up-regulated and 299 spots were down-regulated, 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; 53,82 kDa), MMP-1 (pI 6,47; 54 kDa), collagen type (pI 5,6; 138,94 kDa), capthepsin D precursor (pI 6,1; 45 kDa), alpha-actinin (pI 5,4; 105,5 kDa), elastinase (pI 10,4; 68,5 kDa), chymotrypsin-like eslastase 2B (pI 6,48; 28,81 kDa) Table 2: Regulation of proteins on fibroblasts treated with NLS extract (n=4) UniParc Protein Sample Control (%) ID Type collagen P02452 (%) 100 99.89 ± 7.35 Journal of military pharmaco-medicine 7-2013 Integrin beta-1 P05556 100 96.15 ± 8.45 Alpha-actinin P35609 100 110.09 ± 7.94 Fibronectin P02751 100 171.64 ± 9.17 Cathepsin D precursor P07339 100 50.08 ± 6.75 Interleukin 1-beta O43645 100 158.01 ± 7.47 Chymotrypsin-like elastase 2B P08218 100 97.50 ± 5.13 MMP-8 P22894 100 87.42 ± 8.92 MMP-1 P03956 100 109.55 ± 4.06 MMP-13 P45452 100 29.79 ± 6.18 The results in table showed the up-regulation 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, a factor acting as a fibroblast growth factor, leads to increase growth 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-regulation 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] CONCLUSION 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-regulating growth factor interleukine 1-beta, fibronectin and inhibition of collagen degradation via down-regulating degradation factors named capthepsin precursor and MMP-13 REFERENCES Vu Binh Duong et al Study on callus induction procedure of Ngoclinh ginseng cells Journal of Military Pharmaco-medicine 2006 Gail Jenkins Molecular mechanisms of skin ageing The Biology of Ageing 2002, 123, pp.801-810 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 Hitoshi Masaki Role of antioxidants in the skin: Anti-aging effects Journal of Dermatological Science 2010, 58, pp.85-90 West MD The cellular and molecular biology of skin ageing Archives of Dermatology 1994, 130, pp.87-89 ... evaluate effects of NLG stem cell extract on collagen degradation Results of study on effects of NLG stem cell extract using 2D-PAGE Proteomics study was used as an effective method for investigating... analysis of protein expression in fibroblasts treated by NLG stem cell extract proved anti-wrinkle activity through up-regulating growth factor interleukine 1-beta, fibronectin and inhibition of collagen. .. collagen degradation via down-regulating degradation factors named capthepsin precursor and MMP-13 REFERENCES Vu Binh Duong et al Study on callus induction procedure of Ngoclinh ginseng cells Journal