The spontaneously immortalized human keratinocyte HaCaT cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin. Culture conditions were maintained constant at 37°C in a 5% CO₂ humidified atmosphere.

P. americana was obtained from the Good Agricultural Practice (GAP) breeding base (Sichuan, China). P. americana (200 g) powder was extracted with 90% EtOH (1.2 liters) twice at 80°C. After solvent evaporation, the ethanol extract was recovered. The extract (20 g) was suspended in water (200 ml) at 80°C and filtered through a 0.22-mm filter membrane in appropriate concentrations and stored at −20°C until use. HPLC-diode array detector (HPLC-DAD) was used for the study of P. americana extract. Diamonsil C18 (250×4.6 mm; 5 μm) was selected as the chromatography column. The optimized mobile phase consisted of solvent A (3% v/v methanol in water containing 0.07% v/v acetic acid) and solvent B (methanol). The following gradient was used to represent time (min)/mobile phase A (%)/mobile phase B (%): 0.0/100/0, 10/100/0, 20/70/30, 21/50/50, 35/0/100, at a flow rate of 0.6 ml/min at 25°C, and 254 nm in 10 μl injection volume.

Proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in a volume of 200 μl (2,000 cells/well) on 96-well plates after cultivation with or without PAE. The culture medium containing serum was replaced by MTT every 24 h. A final MTT concentration of 0.5 mg/ml was added to the wells followed by incubation for 4 h at 37°C. The supernatant was discarded and replaced with dimethyl sulfoxide (DMSO) (150 μl/well). The optical densities (OD) were measured at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). The experiment was repeated in triplicate.

Cell migration was analyzed in a 24-well plate with Millicell Cell Culture Insert, using an 8-μm pore size polycarbonate membrane (Millipore Corporation, Billerica, MA, USA). After 48 h of cultivation in a medium with or without PAE (0.3125 mg/ml), the cells (2×10⁴/well) were transferred into the upper chamber with the non-coated membrane and suspended in 200 μl of serum-free DMEM. In the lower chamber, 600 μl of the medium supplemented with 10% fetal bovine serum was added. After incubation for 24 h, the chambers were fixed with 4% paraformaldehyde for 20 min, and stained with hematoxylin for 15 min. Images were captured with an optical microscope.

The wound healing assay was performed by seeding the cell cultures on a 6-well plate and grown to 90% confluency, followed by 48 h of starvation in serum-free medium with or without PAE (0.3125 mg/ml). The culture medium was removed and the monolayers were scratched using a 200-μl pipette to create a uniform cell-free wound area. Debris was removed by gently washing with sterile phosphate-buffered saline (PBS). Cell migration into the wound area was monitored and photographed at 0, 24 and 48 h using an optical microscope.

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer and phosphatase inhibitors. The protein concentration was measured using the bicinchoninic acid (BCA)-protein quantification assay (Beyotime Biotechnology, Shanghai, China). Normalized lysates (30 μg) were separated by electrophoresis on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore Corporation). The membrane was blocked for 1 h at room temperature and incubated overnight at 4°C with primary antibody. After three washes with TBST, the membrane was incubated with horseradish peroxidase-(HRP)-conjugated IgG. Signals were visualized with enhanced chemiluminescence (ECL; Millipore Corporation). Primary antibodies against Smad3 (#9523), phospho-Stat3 (#9145), Stat3 (#12640), JAK1 (#3344), JAK2 (#3230) (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA), phospho-Smad3 (ab52903), phospho-JAK2 (ab32101) (dilution, 1:1,000; Abcam, Cambridge, MA, USA), phospho-JAK1 (sc-101716; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.

Each group of HaCaT cells was washed with PBS followed by fixation with 4% paraformaldehyde (pH 7.4) in 6-well plates. Cells were incubated with 0.5% Triton X-100 for 30 min at room temperature, and treated with 5% BSA for 1 h. Cells were incubated with the following primary antibodies: Smad3 (#9523; dilution, 1:100), phospho-Stat3 (#9145; dilution, 1:100), Stat3 (#12640; dilution, 1:500) (all from Cell Signaling Technology), and phospho-Smad3 (ab52903; dilution, 1:100; Abcam) overnight at 4°C. The cells were incubated with the corresponding fluorescent dye-conjugated secondary antibodies (#4412; dilution, 1:200; Cell Signaling Technology) at 37°C for 1 h, and protected from light. The cells were visualized via fluorescence microscopy.

Healthy adult C57 male mice were purchased from the West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan, China. All the experiments were conducted according to the Guide for the Care and Use of Laboratory Animals at the Animal Experimental Center of Sichuan University. We obtained ethical approval of the Medical Ethics Committee of Sichuan University with approval no. K2016033. Thermal injuries were created with a solid aluminum bar measuring 10 mm in diameter, and pre-heated in boiling water at a temperature of 100°C. The bar was maintained symmetrically in contact with the skin on the dorsal flank for 15 sec (29). The right skin of the dorsal flank was wiped with PAE (original fluid, 5 mg/ml), while the left was treated with normal saline as the untreated control. The daily treatment regimen lasted for 21 days. After injury, to minimize the suffering of the animals, each mouse was housed individually and fed with sterilized food and tap water to prevent infection. The wound healing rates were measured on days 0, 7, 14 and 21 after injury, and the complete wound healing time was calculated as follows: Healing rate = (original wound area - non-healing wound area)/original wound area (30). Mice were sacrificed after 3 weeks.

All the immunohistochemical assays were conducted following the manufacturer's instructions. Briefly, the skin tissues were fixed in formalin and embedded in paraffin. Consecutive paraffin sections (4-μm) of the tissue samples were prepared and incubated overnight at 4°C with primary antibodies followed by incubation with peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (EnVision/HRP; Dako, Glostrup, Denmark). Primary antibodies against Smad3 (#9523), phospho-Stat3 (#9145), Stat3 (#12640), JAK1 (#3344), JAK2 (#3230) (dilution, 1:1,000; all from Cell Signaling Technology), phospho-Smad3 (ab52903), phospho-JAK2 (ab32101) (dilution, 1:1,000; both from Abcam), phospho-JAK1 (sc-101716; dilution 1:1,000; Santa Cruz Biotechnology, Inc.) were used.

Statistical analyses were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA) or Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Quantitative data were analyzed using a two-tailed Student's t-test, and one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post-test. Differences were considered statistically significant at P<0.05, P<0.05 and P<0.01.

PAE contained polyalcohols, amino acids, pyrimidines and proteoglycans. Analysis of PAE revealed the presence of uracil (retention time 7.626 min, 1.145 mg/g), hypoxanthine (retention time 11.707 min, 4.253 mg/g), and inosine (retention time 20.427 min, 8.156 mg/g) (Fig. 1).

The MTT assay was used to confirm the proliferation of HaCaT cells in the presence of PAE at three time-points. The low (0.3125 mg/ml) dose of the extract induced optimal growth in HaCaT cells at 24, 48 and 72 h, respectively (p<0.05) (Fig. 2A). Conversely, high doses of PAE (1.25 and 2.5 mg/ml) inhibited cell proliferation. Furthermore, the treatment with the extract caused obvious proliferation after 48 h of treatment. Transwell and wound healing assays were used to determine HaCaT cell migration in skin. Fig. 2B shows that compared with the control group, cell migration in the PAE-treated group increased significantly (p<0.01). Furthermore, consistent with the results from the Transwell assay, data from the wound healing assay also showed significantly improved migration of wound closure in the PAE treatment group compared with the control group in the HaCaT cells (p<0.01) (Fig. 2C).

To better understand the signaling pathway or mechanism underlying the PAE-mediated regulation of HaCaT cells, the signaling molecules associated with proliferation and migration were screened. After PAE treatment, the expression levels of p-JAK1, p-JAK2 and its downstream molecule p-STAT3 were markedly upregulated in the HaCaT cells, while the total protein levels (JAK1, JAK2 and STAT3) were unchanged (p<0.01) (Fig. 3A). In addition, phosphorylation of Smad3 (p-Smad3) was also significantly increased, while Smad3 was not affected (p<0.01) (Fig. 3B). However, nuclear factor-κB (NF-κB)/p65 and β-catenin expression was not distinctly different between the PAE treatment and the control groups of HaCaT cells (p>0.05) (Fig. 3C).

To further confirm that PAE increases cell proliferation and migration of human keratinocytes via JAK/STAT3 signaling, the inhibitor of STAT3 was used to verify the effects of PAE on cell proliferation and migration. A STAT3 inhibitor, stattic, was used to block STAT3 activation. Phosphorylation of STAT3 was decreased after treatment with stattic in a dose-dependent manner in the HaCaT cells. However, the total expression of STAT3 was not changed. Furthermore, when cells were treated with PAE, after inhibition of STAT3 phosphorylation by stattic, the p-STAT3 levels remained unchanged (Fig. 4A and B). Furthermore, as shown in Fig. 4C and D, PAE-induced cell growth and migration were abrogated by pretreatment with static in the HaCaT cells. The result confirmed that the PAE extract promoted HaCaT cell proliferation and migration by activating the JAK/STAT3 signaling pathway.

The distribution pattern of STAT3 and Smad3 was investigated using fluorescence immunostaining. The results demonstrated that PAE treatment increased STAT3 phosphorylation in the cytoplasm followed by nuclear translocation (Fig. 5A). By contrast, the total expression of STAT3 was constant irrespective of PAE stimulation in the HaCaT cells (Fig. 5B). Furthermore, phosphorylation of Smad3 (p-Smad3) was also significantly increased in PAE-treated cells, while total Smad3 remained unchanged (Fig. 5C and D). The altered phosphorylation of STAT3 and Smad3 suggested activation of the JAK/STAT3 and Smad3 signaling cascade by PAE in HaCaT cells.

To establish the in vivo effect of PAE on cutaneous wound healing, we established a mouse model of deep second-degree thermal burn (16). Skin lesions were not prominent initially, but eventually appeared and worsened. The results demonstrated that the burn wound in the PAE (original fluid, 50 mg/ml)-treated side (right side) showed significant fibroblast proliferation and fibrosis resulting in scar tissue formation compared with the control (left side) (Fig. 6A). Histopathological analysis revealed an increase in epithelial repair, follicle regeneration and additional fibrous tissues in the PAE-treated groups (Fig. 6B). The healing rates and times of skin wounds without and with PAE treatment were sequentially recorded and evaluated. The wound healing rates were measured on days 0, 7, 14 and 21 after burn, and the complete wound healing time was calculated. The healing rate of the treated group was significantly higher than that of the control group at different time-points (p<0.05) (Table I and Fig. 6C). The results suggest that PAE accelerates the healing of burn wounds.

To further confirm the signaling cascade and mechanism of PAE-induced regulation of wound healing, immunohistochemical (IHC) and western blot analysis were used to assay the JAK/STAT3 signaling and phosphorylation of Smad3 in burn wound specimens. Enhanced nuclear staining of p-JAK1, p-JAK2, p-STAT3 and p-Smad3 was detected in the PAE treatment group compared with the control group. By contrast, the nuclear expression of JAK1, JAK2, STAT3 and Smad3 proteins showed no significant difference between the control wounds and the treated wounds (Fig. 7A). Consistent with the IHC findings, the western blot analysis further validated the increased expression of p-JAK1, p-JAK2, p-STAT3, p-Smad3, but not JAK1, JAK2, STAT3 and Smad3 proteins (Fig. 7B).