Herbal drugs are difficult to analyze due to their complexity and the presence of toxicity due to alkaloids. To avoid these complications, the effect of single active component of herbs is often explored, and this single active component represents the effects of the total herb; examples are shikonin for
Proteomics is a powerful tool and has been widely used to elucidate protein profile changes in response to drug treatment and to identify disease-relevant biomarkers. Using proteomics,
The HPLC system was equipped with BAS PM-80 pumps, a DGU-20A5 degasser, a CMA/170 autosampler, and a Varian (model 340) photodiode array detector. Chromatographic separation was performed using a Phenomenex Fusion RP-80 (2504.6 mm, 4 m). The mobile phases were acetonitrile (solvent A) and 2% acetic acid (solvent B). For the analysis of ferulic acid, the mobile phase involved the following linear gradient: from 25% A to 75% A in 0–15 min at a flow rate of 1.0 mL/min, and the detection wavelength was set on 280 nm. The sample injection volume was 20
Human embryonic skin fibroblast was obtained from Bioresource Collection and Research Center (BCRC) no. 60118 (Detroit 551). Cells were cultured in 10 cm culture dish (Corning) with minimum essential medium eagle (MEM) containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate (MEM alpha-modifications, Sigma), supplemented with 10% fetal bovine serum (SAFC Bioscience), 50 units/mL penicillin, 0.05 mg/mL streptomycin, and 0.1 mg/mL neomycin (Sigma); the cells were grown in a humidified atmosphere at 37°C and 5% CO2. For passage, when fibroblasts had grown to a confluence of approximately 90% in the 10 cm culture dish, medium was discarded and the cells washed with 5 mL PBS twice. They were then harvested using 2 mg/mL EDTA and 5 mg/mL trypsin in phosphate-buffered saline (PBS). After washing with 5 mL PBS twice, the cells were collected by centrifugation at 1300 rpm for 5 min. The supernatant was discarded and the cells pipetted into fresh culture medium. They were then aliquoted into several 10 cm culture dishes.
The cell viability assay was performed according to the procedure described in the manufacturer’s manual (Roche) with minor modifications. This is a colorimetric assay for the quantification of cell proliferation based on the cleavage of the water-soluble tetrazolium salt (WST-1) by mitochondrial dehydrogenases in viable cells. Drug-treated cells were seeded at a concentration of 4 × 104 cells/well (about 40% confluence) in 1 mL culture medium into a 24-well culture plate and incubated at 37°C in an incubator containing 5% CO2. After cells had attached, they were treated with 0.1% DMSO, 300
Fibroblasts were washed twice in phosphate-buffered saline (PBS) and lysed with 1 mL of NP-40 lysis buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% NP-40, 30 mM Na2P2O7, 30 mM NaF, 1 mM Na3VO4, 1% protease inhibitor, and 1% phosphatase inhibitor (Sigma). The whole cell lysate was centrifuged at 14000 rpm for 20 min at 4°C to remove insoluble material. The supernatant was transferred to a concentrator (GE Health, vivaspin 20, 100k MWCO) and centrifuged at 6000 ×g, 4°C, until volume less than 500
The 2D PAGE was performed according to the method described in the manufacturer’s manual (Amersham Biosciences) with minor modifications. For the first-dimension IEF, pH 4–7, IPG strips (18 cm) were rehydrated with 400
After 2D PAGE electrophoresis, the gel was removed, immersed in fix solution (40% methanol and 10% acetic acid in dH2O) for 10 min, washed with dH2O for 10 min twice, and then immersed in solution A (0.25 mM sodium thiosulfate) for 30 min, which was then replaced by dH2O for 10 min. Next the gel was immersed in solution B (3.5 mM silver nitrate) for 30 min. After rinsing with dH2O, a mixture of solution C (0.357 M) and solution D (4.37 mM) in dH2O were used to develop the protein spots on the 2D PAGE gel. The reaction was stopped by 5% acetic acid.
2D PAGE images were obtained, and the amount of protein in each spot was analyzed using ImageMaster 2D Elite software version 5.0 (Amersham Biosciences). The volume of a protein spot was defined as the sum of the intensities of the pixel units within the protein spot. To correct for quantitative variations in the intensity of protein spots, spot volumes were normalized as a percentage of the total volume of all the spots present in a given gel.
The process is described in a document from the Institute of Biological Chemistry, Academia Sinica Institute of Biological Chemistry (
A plot of the calibrated intensity for the expression value of each protein as measured by ImageMaster 2D Elite software version 5.0 (Amersham Biosciences, Sweden) among the different groups of samples was used in conjunction with an average linkage hierarchical clustering algorithm (UPGMA, Unweighted Pair Group Method with Arithmetic Mean); this was done using Hierarchical Clustering Explorer 3.5 [
Proteins extracts from fibroblast cells (Detroit 551) were separated by 12.5% SDS-PAGE and then transferred onto a nitrocellulose (NC) membrane. The NC membrane was blocked with 5% nonfat milk in TBST at room temperature for 1 hr and probed with various primary antibodies (anti-p-Erk1/2 (no. 9101), 1 : 2000; anti-Erk1/2 (no. 9102), 1 : 1000; anti-p-Akt (no. 9271), 1 : 1000 and anti-Akt (no. 9272), 1 : 1000, Cell Signaling; anti-TGF-
To measure the ROS content of the fibroblasts after DMSO, AS extract, or FA treatment, the intracellular content of H2O2, which is in proportion to ROS, was determined using the redox-sensitive fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Sigma). Briefly, cells were cultured to confluence and trypsinized. After centrifugation, discard supernatant and the cells were resuspended and incubated with 10
After cells were cultured to confluence, they were trypsinized, centrifuged, and resuspended in culture medium. A total of 2 × 104 fibroblasts were seeded in a Transwell (24 well, Corning) and treated with DMSO, AS extract, or FA; the Transwell was then inserted in culture medium without bubbles. After 6 hr, the Transwell was removed from the culture medium and the cells fixed using methanol for 10 min. The methanol was then discarded and the cells air-dried. Finally the cells were stained with 5% Giemsa (solved in dH2O) in room temperature overnight. The Transwell was washed with dH2O, and the inner cells on the Transwell were removed by scraping with a cotton swab. The number of cells migrated down through the Transwell was manually counted under microscope. Student’s
A total of 2.5 × 104 fibroblasts were seeded onto both sides of a culture insert (ibidi) in a 24-well plate to generate 500
The process is described in Sircol collagen assay general protocol (Biocolor). In brief, 3 mL cell medium was collected after drug treatment, and collagen was precipitated by 4 M NaCl to avoid FBS interference. The collagen pellet was collected by centrifugation at 15000 ×g in room temperature, and the pellet was dissolved in 0.5 mL of 0.5 M acetic acid. 1 mL of Sircol dye reagent was mixed with 100
In order to establish a model system to analyze the efficacy of total herbal extracts, we used
Effects of AS extract or FA on cell viability after 24 hr treatment in human fibroblast. Graph represents the ratio of cell viability compared with the DMSO control. (a) AS extract promotes cell growth in a dose dependent manner up to 400
To further investigate how AS extract and its active component FA can affect the processes of wound healing of the injured skin, two-dimensional (2D) PAGE was then carried out. In this study, 0.1% DMSO, 300
Differential effect of AS and FA treatment on human fibroblasts as visualized by 2D PAGE. 300
To further characterize these differentially expressed proteins, the concentration of total protein lysate was increased to more than 1 mg, which was followed by 2D PAGE and staining with Coomassie blue. Forty abundant differentially regulated protein spots were identifiable on this 2D PAGE gel, and these were excised and analyzed by LC-MS/MS (Q-TOF) mass spectrometry. In addition, eleven rare differentially regulated protein spots were excised from the earlier silver stained 2D PAGE gel and analyzed by the more sensitive LC-MS/MS (Orbitrap) mass spectrometry. The resulting peptides were screened using the Mascot database online search engine and SEQUEST to identify the proteins. The detailed results of the protein identification are presented in Table
Proteins showing altered abundance after treatment with either AS extract or FA compared to the DMSO control and identified by LC-MS/MS.
ID | Protein identity | Accession number | Abbreviation | MW/pI | Score | Match peptide | Sequence coverage (%) | AS/DMSO | FA/DMSO | ||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | Peroxiredoxin-6 | P30041 | PRDX6 | 25019/6.00 | 40 | 3 | 12 | ↑ | 1.91 | ↑ | 1.96 |
2 | Annexin A2 | P07355 | ANXA2 | 38580/7.57 | 110 | 3 | 9 | ↑ | 1.50 | ↑ | 1.51 |
3 | Peroxiredoxin-4 | Q13162 | PRDX4 | 30521/5.86 | 105 | 4 | 14 | ↑ | 2.02 | ↑ | 1.88 |
4 | Triosephosphate isomerase | P60174 | TPIS | 26713/6.45 | 236 | 7 | 27 | ↑ | 1.86 | ↑ | 1.52 |
5 | Parkinson disease protein 7 | Q99497 | PARK7 | 19878/6.33 | 67 | 2 | 12 | ↑ | 1.57 | ↑ | 1.77 |
6 | GTP-binding protein SAR1a | Q9NR31 | SAR1A | 22353/6.21 | 45 | 1 | 5 | ↑ | 1.72 | ↑ | 1.51 |
7 | Glutathione S-transferase P | P09211 | GSTP1 | 23341/5.43 | 214 | 7 | 43 | ↑ | 1.70 | ↑ | 1.50 |
8 | Glutathione S-transferase P | P09211 | GSTP1 | 23341/5.44 | 174 | 6 | 38 | ↑ | 2.54 | ↑ | 1.50 |
9 | Peroxiredoxin-2 | P32119 | PRDX2 | 21878/5.66 | 135 | 5 | 23 | ↑ | 1.77 | ↑ | 1.96 |
10 | Translationally controlled tumor protein | P13693 | TCTP | 195834.84 | 119 | 4 | 17 | ↑ | 1.57 | ↑ | 1.71 |
11 | Protein disulfide-isomerase A3 | P30101 | PDIA3 | 56747/5.98 | 1019 | 27 | 40 | 0.53 | 0.51 | ||
12 | Ubiquitin-conjugating enzyme E2 | P61088 | UBE2N | 17127/6.13 | 2.5 (criteria) | 4 | 26.3 | ↑ | 2.04 | ↑ | 1.77 |
13 | Nucleoside diphosphate kinase B | P22392 | NDKB | 17287/8.52 | 70 | 4 | 28 | 0.63 | 0.63 | ||
14 | Microtubule-associated protein RP/EB family member1 | Q15691 | MARE1 | 29980/5.02 | 117 | 4 | 20 | 0.49 | 0.62 | ||
15 | Protein SEC13 homolog | P55735 | SEC13 | 35518/5.22 | 130 | 4 | 9 | ↑ | 1.64 | ↑ | 1.60 |
16 | Serine-threonine kinase receptor-associated protein | Q9y3f4 | STRAP | 38414/4.98 | 2.5 (criteria) | 5 | 18.9 | ↑ | 1.97 | ↑ | 1.78 |
17 | Heat shock protein 60 | P10809 | HSP60 | 61016/5.70 | 379 | 15 | 19 | ↑ | 1.99 | ↑ | 1.56 |
18 | Isoform A of eukaryotic translation initiation factor 5A-1 | P63241-2 | IF5A1 | 20157/5.07 | 2.5 (criteria | 4 | 21.2 | 0.56 | |||
19 | L-Lactate dehydrogenase B chain | P07195 | LDHB | 36615/5.17 | 115 | 5 | 17 | ↑ | 2.01 | ||
20 | proteosome activator complex subunit 1 | Q06323 | PSME1 | 28705/5.78 | 289 | 7 | 27 | ↑ | 2.77 | ||
21 | Heat shock protein beta-1 | P04792 | HSPB1 | 22768/5.98 | 223 | 9 | 37 | ↑ | 1.65 | ||
22 | Phosphoglycerate kinase 1 | P00558 | PGK1 | 44586/8.30 | 94 | 1 | 4 | ↑ | 2.05 | ||
23 | Vimentin | P08670 | VIME | 53619/5.06 | 2.5 (criteria) | 10 | 23.8 | 0.64 | |||
24 | Vimentin | P08670 | VIME | 53619/5.07 | 2.5 (criteria | 10 | 23.8 | 0.43 | |||
25 | Annexin A5 | P08758 | ANXA5 | 35914/4.94 | 272 | 13 | 40 | 0.47 | |||
26 | Calpain small subunit 1 | P04632 | CAPNS1 | 28298/5.05 | 2.5 (criteria) | 8 | 36.2 | ↑ | 1.62 | ||
27 | Heat shock protein 6 | O14558 | HSP6 | 17125/5.95 | 75 | 4 | 38 | ↑ | 1.67 | ||
28 | Galectin-1 | P09382 | LEG1 | 14706/5.34 | 81 | 3 | 19 | 0.66 | |||
29 | Synaptic vesicle membrane protein VAT-1 homolog | Q99536 | VAT1 | 41893/5.88 | 2.5 (criteria | 8 | 28 | ↑ | 1.78 | ||
30 | Ferritin light chain | P02792 | FRIL | 20007/5.51 | 2.5 (criteria) | 3 | 20 | ↑ | 2.48 | ||
31 | Stathmin | P16949 | P08758 | 17292/5.76 | 134 | 6 | 36 | ↑ | 1.96 | ||
32 | Glutaredoxin | O76003 | GLRX3 | 37408/5.31 | 152 | 5 | 18 | ↑ | 1.66 | ||
33 | Actin-related protein 2/3 complex subunit 5 | O15511 | ARPC5 | 16310/5.47 | 179 | 6 | 27 | ↑ | 1.53 | ||
34 | Annexin A5 | P08758 | ANXA5 | 35914/4.94 | 2.5 (criteria) | 16 | 65 | ↑ | 1.53 |
Bioinformatic tools were used to categorize the differentially expressed proteins. All of the parameters of the proteins identified by mass spectrometry (Table
Hierarchical clustering and functional classification of the changes in protein expression between the DMSO control cells and AS/FA-treated cells. The expression pattern for each protein was categorized by UPGMA using Hierarchical Clustering Explorer 3.5. Proteins with a similar expression clustered into five different discrete groups (clusters A, B, C, D, and E) in a tree-like organization. Each row in the color heat map indicates a single protein and each column represents different groups of proteins from the DMSO, AS, and FA treatments. A high protein expression value for a specific protein spot is represented by a bright red color, and a low protein expression value is represented by a bright green color. A black color indicates that the protein spot was expressed at an average level. A gray color represents protein spots that were undetectable. Further information about the differentially expressed protein spots that were identified is given in Table
After clustering, we use NCBI PubMed and BGSSJ (Bulk Gene Search System for Java) with the Swiss-Port database to classify the biological functions of these proteins. The delineated protein functions included protein transport, protein metabolism, calcium ion binding, oxidoreductase activity, antiapoptosis, protein binding, metabolism, signal transduction, cell mobility, and cell growth.
Interestingly, seven oxidation-related proteins were found among these differentially expressed proteins. Among them, five oxidation-related proteins were found to be upregulated in fibroblasts when treated with either AS extract or FA. These proteins were peroxiredoxin (PRDX) 2, PRDX4, PRDX6, glutathione S-transferase Pi (GSTP1), and Parkinson’s disease protein 7 (PARK7) (Figure
Calcium ions are very important in the regulation of wound healing process, and its function is to modulate the expression of genes involved in cell growth, differentiation, attachment, motility, and collagen secretion [
A number of motility-associated proteins were also identified. Actin-related protein 2/3 complex subunit 5 (ARPC5) and stathmin (STMN1) were only upregulated in cells treated with FA (Figure
Western blot analysis was used to confirm the changes in protein expression detected by the proteomic approach. Human skin fibroblasts were seeded into plates and then treated with either AS extract or FA for 24 hr; then cell lysates were prepared as indicated in Materials and Methods. As shown in Figures
Analysis of the results of 2D PAGE by immunoblotting and ROS assay. According to hierarchical clustering and functional classification, the expressions of GSTP1 and TPIS were both increased in 300
The PRDX family of proteins and GSTP1 act as antioxidants [
Collagen secretion and crosslinking are the most important step when repairing wounded tissue and collagen synthesis is controlled by transforming growth factor beta (TGF-
Functional confirmation of the effects of AS and FA treatment on human fibroblasts. (a) Collagen secretion was significantly increased after AS extract treatment. After cells were seeded in a 24 well plate and treated with 0.1% DMSO, 300
The functions of STMN1, ARPC5, NDKB, and MARE1 are known to be associated with cell mobility and wound healing [
Only CAPNS1 is involved in cell growth based on the proteomic classification (Figure
The cell viability test revealed that AS extract was able to effectively promote human skin fibroblast proliferation with low levels of cytotoxicity even at high concentrations. It is well known that skin fibroblasts play an important role in the proliferation phase of wound healing [
ROS plays an important role in the early phase of wound healing [
TCTP (a translationally controlled tumor protein also named fortilin) and GSTP1 are upregulated by treatment with either AS extract or FA (Figure
Calcium ions play an important role in wound healing, cell proliferation, differentiation, adhesion, migration, and collagen secretion [
Cell mobility is an important part of the wound healing process [
CAPNS1 (calpain small subunit 1) is able to promote cell proliferation, migration, and adhesion [
The main energy resource of fibroblasts is glycolysis, and an increase in LDHB expression in these cells would promote propyl hydroxylase activity, which in turn would increase lactate production and collagen hydroxylation; these effects, in turn, would help cells tolerate an hypoxia environment and accelerate the wound healing process [
Taken together, our results revealed a possible pharmacological wound healing processes induced by the AS extract or FA treatment of fibroblasts (Figure
Summary of the effects of the AS and FA treatments on human skin fibroblasts.
This study was supported by grants from the National Research Program for Genomic Medicine (NRPGM), NSC 99-3112-B-010-006, and the National Science Council, NSC 98-2320-B-010-008-MY3.