Human dermal fibroblasts (Hs27) were obtained from the American Type Culture Collection (cat. no. ATCC CRL-1634) and cultured in the Dulbecco's modified essential medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum and maintained in an incubator with a 5% CO₂ humidified atmosphere at 37˚C. For all experiments, Hs27 cells were used between passage numbers five and ten. For time- and dose-dependent treatments, Hs27 cells were serum-starved for 24 h before exposure to the YIGSR peptide and emodin at specific doses and times. The synthetic YIGSR peptide was purchased from Anygen. emodin (cat. no. E7881), TGF-β (cat. no. T7039), and the inhibitors U0126 (cat. no. 662009) and compound c (cat. no. 171260) were purchased from Sigma-Aldrich (Merck KGaA).

Total RNA was extracted using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was reverse transcribed from 1 µg of total RNA using oligo (dT) primers and murine leukemia virus reverse transcriptase. The temperature protocol for reverse transcription was as follows: 25˚C for 10 min and 37˚C for 120 min, followed by heat inactivation at 85˚C for 5 min. A total volume of 20 µl PCR amplification mixtures were prepared by mixing 10 µl of 2X SYBR-Green I Premix ExTaq (Takara Bio, Inc.), 2 µl primer mix (1 µM forward and 1 µM reverse primers), and 8 µl diluted cDNA templates. Real-Time quantitative PCR was performed on the CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.) using the following conditions: 95˚C for 1 min, followed by 40 amplification cycles comprising 95˚C for 15 sec, 60˚C for 15 sec (annealing), and 72˚C for 30 sec. After amplification, melting curve analysis was performed according to manufacturer's instructions (Bio-Rad Laboratories, Inc.). Primer sequences for collagen were as follows: COL1A1 forward, 5'-gaacgcgtgtcatcccttgt-3'; COL1A1 reverse, 5'-gaacgaggtagtctttcagcaaca-3'; COL1A2 forward, 5'-cctggtgctaaaggagaaagagg-3'; COL1A2 reverse 5'-atcaccacgacttccagcagga-3'; COL2A1 forward, 5'-cctggcaaagatggtgagacag-3'; COL2A1 reverse, 5'-cctggttttccaccttcacctg-3'; COL3A1 forward, 5'-tggtctgcaaggaatgcctgga-3'; COL3A1 reverse, 5'-tctttccctgggacaccatcag-3'; COL4A3 forward, 5'-ggacaaaggagaaccaggtctc-3'; COL4A3 reverse, 5'-agtgctgcccaaatctcctctg-3'; COL5A1 forward, 5'-cacaacttgcctgatggaataaca-3', and COL5A1 reverse, 5'-gcagggtacagctgcttggt-3'. Collagen expression was measured using the 2^-ΔΔCq assay (32) and normalized against GAPDH.

For the MTT assay, Hs27 cells were seeded in 96-well culture plates (1x10⁴ cells/well) and cultured for 24 h. After serum starvation for 24 h, cells were exposed to emodin with the given dose (0.05, 0.1, 0.5, 1, 3 and 5 µM) and time conditions. After discarding the media, cells were treated with 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA) dissolved in serum-free media for 3 h in a 37˚C CO₂ incubator. After discarding the solution, 100 µl DMSO was added to each well, and the plate was vortexed for 10 min. The absorbance of the MTT solution was measured at a wavelength of 540 nm.

For apoptosis analysis, Hs27 cells were seeded in 6-well culture plates and exposed to a given dose of emodin (1 and 5 µM) for 24 h. After trypsinization, the cells were detached and harvested by centrifugation (400 x g for 5 min at 4˚C). Collected cells were stained with FITC-Annexin V (cat. no. 556419; BD Biosciences) and 7-ADD (cat. no. 559925; BD Biosciences) in the dark for 20 min. Living and apoptotic populations were detected using flow cytometry (FACS Aria; BD Biosciences).

The following antibodies were used for immunoblotting: COL1 (Rockland Immunochemicals; cat. no. 600-401-103-0.5), phospho-5' AMP-activated protein kinase (AMPK; Tyr¹⁷²; Cell Signaling Technology, Inc.; cat. no. 2535), phospho-Smad2 (Ser465/467; Cell Signaling Technology, Inc.; cat. no. 3108), phospho-ERK (Thr²⁰²/Tyr²⁰⁴; Abcam; cat. no. ab214362), MMP-1 (Cell Signaling Technology, Inc.; cat. no. 54376) and β-actin (MP Biomedicals; cat. no. 08691331). Immunoblotting was performed by harvesting the treated cells and by isolating the total proteins. To prepare total cell lysates, the cells were washed with cold phosphate-buffered saline and lysed in cold lysis buffer [in mM: 40 HEPES (pH 7.5), 120 NaCl, 1 EDTA, 10 pyrophosphate, 10 glycerophosphate, 50 NaF, 1.5 Na₃VO₄, 1 PMSF, 5 MgCl₂, 0.5% Triton X-100 and protease-inhibitor mixture). After determining protein concentration using a Bradford assay, protein lysates were subsequently denatured by boiling in Lammeli sample buffer for 10 min at 95˚C. A total of 15 µg protein was loaded and separated by 8% SDS-PAGE gel, and transferred onto nitrocellulose membranes. The membranes were subsequently blocked for 30 min at room temperature with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T; pH 7.6), followed by incubation with primary antibodies (1:1,000). After washing the membranes three times with TBS-T, blots were incubated with HRP-conjugated secondary antibody (SeraCare KPL; goat anti-rabbit; cat. no. 5220-0336; anti-mouse; cat. no. 5220-0342; each, 1:5,000) for 1 h at room temperature, washed three times with TBS-T and detected by enhanced chemiluminescence (ECL system; GE Healthcare Bio-Sciences). Densitometric analysis was performed using the ImageJ software (v.1.51; http://rsbweb.nih.gov/ij/index.html) and Multigauge software (Fujifilm). Data were obtained from three independent experiments.

All data were evaluated using the GraphPad software 5 (GraphPad Software, Inc.) and are expressed as mean ± SEM. The significant differences among groups were determined by one-way ANOVA with Dunnett's multiple comparison analysis. Comparison between two groups was analyzed by unpaired 2-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of emodin on type I collagen expression in dermal fibroblasts, Hs27 human dermal fibroblasts were initially exposed to emodin for 12 h (Fig. 1A). Hs27 cells were also treated with YIGSR, a peptide that induces collagen synthesis in Hs27 cells (33). As shown in Fig. 1A, YIGSR and emodin significantly increased protein levels of type I collagen, and these levels positively associate with emodin concentration (Fig. 1B). MMP-1 is a well-known protease that degrades collagen proteins. To investigate whether emodin affected MMP-1 expression, we analyzed MMP-1 levels following dose-dependent emodin treatment. As shown in Fig. 1B, no significant changes in MMP-1 levels were observed, indicating that the increase in type I collagen by emodin treatment was not caused by MMP-1 downregulation. Subsequently, the effect of emodin on mRNA expression was determined by qPCR analysis. Similar to protein levels, exposure to emodin increased transcript levels of type I collagen without any effects on types II, III and V collagen (Fig. 1C). Type IV collagen genes were not detectable in Hs27 cells (data not shown). This suggests that emodin specifically induces type I collagen expression in vitro.

To verify the effect of emodin on cell viability an MTT assay was performed. In particular, relatively high doses of emodin were used for the viability assay to exclude potential cytotoxicity with increased concentration. As a result, there was no significant changes in cell viability at varying concentrations of emodin (Fig. 2A). Light microscopy images further demonstrated that 24-h emodin treatment did not induce any changes in fibroblast cell morphology (Fig. 2B). Additionally, the number of apoptotic cells following emodin treatment was evaluated by flow cytometry, with Fig. 2C showing that the percentage of apoptotic cells was similar between vehicle- and emodin-treated groups. These results indicate that emodin increases type I collagen without affecting cell viability.

Several signaling pathways are involved in collagen synthesis, including the focal adhesion kinase (FAK), ERK, p38 mitogen-activated protein kinase and AKT pathways (34-37). Moreover, 5' AMP-activated protein kinase (AMPK) also appears to affect collagen expression (38,39). To investigate whether these pathways were responsible for emodin-induced type I collagen synthesis, the phosphorylation of each component was analyzed, and found that ERK and AMPK phosphorylation was increased by ~2.5-fold at 3 h after emodin treatment relative to untreated cells and without any effect on total protein levels (Fig. 3A). Phosphorylated p38, AKT and FAK were normalized with housekeeping protein, actin. In contrast to AMPK and ERK, no significant differences were observed in the phosphorylation of these proteins between control and emodin treatment at each time point. Total protein levels of p38, AKT and FAK were not examined, because there were no changes in the phosphorylation status. Since TGF-β signaling is an important regulatory pathway of collagen expression (40), the phosphorylation status of downstream molecules were also measured, which demonstrated that Smad2 phosphorylation was also unaffected by emodin treatment (Fig. 3B). Thus, the TGF-β pathway is not involved in emodin-induced collagen synthesis.

To determine which of the two pathways (ERK and AMPK) affected emodin-induced collagen expression, Hs27 cells were treated with the chemical inhibitor U0126 to suppress the ERK pathway. Although U0126 sufficiently decreased ERK phosphorylation, emodin-induced type I collagen expression remained similar to that in the vehicle-treated group (Fig. 4A). In marked contrast, collagen levels were largely inhibited by the AMPK inhibitor, compound c (Fig. 4B), suggesting AMPK as a crucial regulatory enzyme for emodin-induced type I collagen expression.