Stellera chamaejasme and its constituents induce cutaneous wound healing and anti-inflammatory activities

Stellera chamaejasme L. (Thymelaeaceae) is a perennial herb that is widely used in traditional Chinese medicine to treat tumours, tuberculosis and psoriasis. S. chamaejasme extract (SCE) possesses anti-inflammatory, analgesic and wound healing activities; however, the effect of S. chamaejasme and its active compounds on cutaneous wound healing has not been investigated. We assessed full-thickness wounds of Sprague-Dawley (SD) rats and topically applied SCE for 2 weeks. In vitro studies were performed using HaCaT keratinocytes, Hs68 dermal fibroblasts and RAW 264.7 macrophages to determine cell viability (MTT assay), cell migration, collagen expression, nitric oxide (NO) production, prostaglandin E2 (PGE2) production, inflammatory cytokine expression and β-catenin activation. In vivo, wound size was reduced and epithelisation was improved in SCE-treated SD rats. In vitro, SCE and its active compounds induced keratinocyte migration by regulating the β-catenin, extracellular signal-regulated kinase and Akt signalling pathways. Furthermore, SCE and its active compounds increased mRNA expression of type I and III collagen in Hs68 fibroblasts. SCE and chamechromone inhibited NO and PGE2 release and mRNA expression of inflammatory mediators in RAW 264.7 macrophages. SCE enhances the motility of HaCaT keratinocytes and improves cutaneous wound healing in SD rats.


SCE and its active compounds enhance the motility and differentiation of HaCaT cells by activating the β-catenin, ERK and Akt signalling pathways.
To evaluate whether SCE and its constituents can influence keratinocyte migration and differentiation, we assessed the effect of SCE and its constituents on HaCaT cell motility and expression of keratinocyte differentiation markers. Treatment of HaCaT cell with SCE and its constituents for 24 hours increased the migration of keratinocytes ( Fig. 2A and B). In addition, protein expression of filaggrin, loricrin and involucrin that are important keratinocyte differentiation markers, was increased by SCE and its active compounds ( Fig. 2C and D).
Considering the correlation between wound healing and the β -catenin signalling pathway 3 , we investigated the effect of SCE and its constituents on β -catenin activation. SCE and its constituents increased nuclear translocation and protein expression of β -catenin ( Fig. 2E-H). The ERK and Akt signalling pathways enhance keratinocyte motility 14,15 . Therefore, we assessed the effects of SCE and its constituents on phosphorylation of ERK and Akt. Treatment with SCE and its constituents increased the phosphorylation of ERK and Akt ( Fig. 2G and H).

SCE and its active compounds induce collagen expression in Hs68 cells.
Fibroblast-tomyofibroblast transition plays a critical role in cutaneous wound healing 33 . mRNA levels of COL1A1 and COL3A1 and production of type І procollagen were markedly augmented by SCE and several compounds (especially daphnin and daphnetin-8-O-glucoside) in Hs68 cells, as shown by RT-PCR and ELISA analysis (Fig. 3A-D).

SCE and chamechromone inhibit NO and PGE 2 production and expression of inflammatory mediators in RAW 264.7 cells.
The production of NO and PGE 2 in LPS-stimulated RAW 264.7 cells was assayed after treatment with SCE or isolated compounds. SCE and chamechromone significantly suppressed LPS-induced NO production ( Fig. 4A and B). In addition, SCE and chamechromone pretreatment reduced LPS-induced PGE 2 production in a dose-dependent manner in RAW 264.7 cells (Fig. 4C and D). These data indicate that SCE and chamechromone significantly inhibit production of the inflammatory mediators NO and PGE 2 in LPS-stimulated macrophages. Macrophages initiate an immune response by identifying and phagocytosing pathogens, inducing the secretion of inflammatory mediators such as iNOS, COX-2, TNF-α , IL-1β and p65 34 . Thus, we examined the expression of proinflammatory mediators that have a nuclear factor-κ B-binding site in their promoter region 35 . To investigate the effects of SCE and chamechromone on the LPS-induced mRNA expression of proinflammatory cytokines, the mRNA levels of iNOS, COX-2, TNF-α , IL-1β and p65 in RAW 264.7 cells were measured using RT-PCR. Treatment with SCE and chamechromone significantly inhibited the production of iNOS, COX-2, TNF-α , IL-1β and p65 ( Fig. 4E and F). These results suggest that SCE and chamechromone suppress the LPS-induced expression of proinflammatory cytokines.

SCE promotes cutaneous wound healing.
We assessed the effect of SCE on cutaneous wound healing.
After topical application of SCE (1%, 2% or 3%) to the wound areas of SD rats, SCE significantly reduced the wound size and re-epithelialised skin lesions (Fig. 5A, Tables 1 and 2). Histological analysis (H&E staining) also revealed restoration of normal tissue structure after SCE treatment (Fig. 5B).

Discussion
The proliferative phase of wound healing is associated with the re-epithelialisation process including collagen production and ECM remodelling 36 . During wound re-epithelialisation stage, skin lesions closes the wound and remodels the cytoskeleton by enhancing the proliferation and migration of keratinocytes 37 . In this study, SCE (B) Chromatogram of SCE analyzed by HPLC detected at 330 nm and its assigned peaks; daphnin (peak 1), daphnetin-8-glucoside (peak 2), daphnetin (peak 3), isoquercitrin (peak 4), rutarensin (peak 5), chamechromone (peak 6), and daphnoretin (peak 7). The gradient profile was as follows: 0-5 min, initial mobile phase of 0.1% formic acid prepared in acetonitrile and 0.1% formic acid prepared in water (10:90); 5-45 min, linear gradient of 70:30; and 45-50 min, isocratic 70:30. The flow rate was 1.0 ml/min, and detection was at 254 nm. The sample concentration was 10 mg/min in MeOH, and the injection volume was 10 μ l. and its constituents augmented the expression of keratinocyte differentiation markers, which are influenced by the β -catenin signalling pathway 38 , in HaCaT keratinocytes. The β -catenin signalling pathway enhances the proliferation and motility of keratinocytes 10,11 . Although dysregulation of the β -catenin signalling pathway induces hypertrophic keloids and scars 39 , adequate activation of this pathway in wounds is crucial to improve wound healing. It was reported that the proliferation of keratinocytes is regulated by inhibition of apoptosis during early wound healing 10 . Our results indicate that SCE and its constituents enhance the proliferation of keratinocytes by activating ERK and Akt signalling.
During the early stage of wound healing, collagen production is promoted to increase scar formation, and then it is gradually decreased to normal levels in the final stage of wound healing 40 . Several studies verified that TGF-β 1 plays a significant role in the re-epithelisation process by increasing ECM production and inhibiting inflammation 41 . Furthermore, the TGF-β 1 signalling pathway also interacts with the β -catenin signalling pathway during the wound healing process 42,43 . It was reported that Wnt3a treatment increases the levels of differentiation markers in myofibroblasts by activating the TGF-β 1 signalling pathway 44 . Our results suggest that SCE and its constituents increased the expression of COL1A1, COL3A1 and TGF-β 1 in dermal fibroblasts by activating the β -catenin signalling pathway. Many bioactive mediators, such as eicosanoids, cytokines and growth factors, regulate each stage of the wound healing process. During infection and inflammation, activated macrophages that have infiltrated the site of inflammation produce a large amount of NO around the wounded tissues 45 . NO is a highly reactive radical that is generated by the activation of iNOS and contributes to various biological processes including inflammation. NO is thought to be a key disruptive factor in the wound healing process 46 , although it was reported that small amounts of NO may enhance wound healing during the early stage 47 . Excessive generation of PGE 2 by COX-2 is a crucial physiological factor accelerating inflammation 48 . PGE 2 is related to keratinocyte proliferation 49 , angiogenesis 50 and mediation of the inflammatory response 51 . Our results suggest that SCE and chamechromone inhibit LPS-induced NO and PGE 2 generation through inhibition of iNOS and COX-2 mRNA expression in RAW 264.7 macrophages. However, further investigation of the underlying mechanism is necessary. Macrophages are key factors in inflammation and reduce various harmful stimuli. Furthermore, they trigger the LPS-induced inflammatory response by producing proinflammatory mediators such as TNF-α , IL-1β and p65 52 . Overproduction of these mediators leads to excessive inflammatory responses 53 . Therefore, suppression of proinflammatory mediator production may be helpful in reducing the inflammatory response. In this study, SCE and chamechromone suppressed production of TNF-α , IL-1β and p65 in RAW 264.7 macrophages stimulated by LPS. Our results demonstrate that SCE and chamechromone suppress production of inflammatory mediators in RAW 264.7 cells.
We found that SCE promoted the epithelialization and wound closure rate in SD rat model. CAE, which is a famous traditional medicines proven to be effective in wound healing treatment 54,55 , was used as a positive control. Based on various studies, the biologically active constituents of CAE are known to be madecassic acid, asiatic acid, madecassoside and asiaticoside 56,57 . In our study, SCE group (SCE 3%) showed the enhanced cutaneous wound healing activity compared with CAE group (CAE 3%).
According to our data, all of eight tested constituents of SCE play a role at different molecular targets for exerting various biological effects in the wound healing system. For example, most of the constituents increase the keratinocyte migration, proliferation, and differentiation and β -catenin activation in HaCaT keratinocytes. In addition, daphnin and daphnetin-8-O-glucoside augmented collagen production and expression in Hs68 fibroblasts. Chamechromone inhibited the production of inflammatory mediators in Raw 264.7 macrophages. Therefore, in addition to the results that shows the positive effects of SCE on overall stages of wound healing, our results indicate that individual constituents of SCE exert their beneficial activity on different stages of wound healing process by regulating inflammatory, proliferative and remodelling phases.
In conclusion, our results suggest that SCE and its constituents induce cutaneous wound healing in SD rats, enhance keratinocyte motility and mRNA expression of type I and III collagen and inhibit PGE 2 production and inflammatory mediator expression. SCE enhanced re-epithelialisation in wounds at an early stage of wound healing. Many wound healing drugs, including chemical molecules and growth factors, often have serious side effects 58 . However, SCE can be used to treat cutaneous wounds without any side effects. Therefore, SCE and its constituents may be beneficial for the treatment of wounds.     Compound 7 (daphnoretin) was isolated from fraction 3 on Silica gel, eluting with Hexane-EtOAc (2:1 → 1:1), and recrystallised with CHCl 3 -MeOH to obtain white crystals.
The chemical structures of compounds 1-7 (Fig. 1A) were determined by 1 H and 13 C nuclear magnetic resonance data and compared with reported data 59-65 . High-performance liquid chromatography (HPLC) analysis. The high-performance liquid chromatography (HPLC) system consisted of an Agilent infinity series 1260 liquid chromatography system with a G1311B quaternary pump, G1329B autosampler, G1316A column oven and G1315D DAD detector, connected to Agilent ChemStation software (Agilent, Waldbronn, Germany). The separation was conducted with a YMC-PACK ODS column (4.6 mm × 250 mm, 5 μ m). The gradient profile was as follows: 0-5 min, initial mobile phase of 0.1% formic acid prepared in acetonitrile and 0.1% formic acid prepared in water (10:90); 5-45 min, linear gradient of 70:30; and 45-50 min, isocratic 70:30. The flow rate was 1.0 ml/min, and detection was at 254 nm. The sample concentration was 10 mg/min in MeOH, and the injection volume was 10 μ l. The HPLC chromatogram of SCE is shown in Fig. 1B.  Cell migration assay. HaCaT cells were seeded into 24-well plates (1 × 10 5 cells/well). After 24 h of incubation, the monolayers were scratched with sterile pipette tips and treated with or without SCE (5 or 10 μ g/ml) and its constituents (20 μ M). After 48 h, the cells were fixed in 4% formalin for 20 min and stained with 2% crystal violet. The wound closure rate was evaluated using an Eclipse TE2000U inverted microscope with twin CCD cameras (magnification, × 200; Nikon, Tokyo, Japan; n = 3).
Reporter assay. Luciferase transactivation was measured as described previously 66 . TOPflash and FOPflash assays were performed in HaCaT cells. Cells were seeded in 24-well plates (6 × 10 4 cells/well). After 24 h, cells were co-transfected with luciferase reporter constructs (TOPflash or FOPFlash) and the pRL-CMV-Renilla reporter plasmid using Fugene 6 (Roche Applied Science, Indianapolis, IN, USA). After 24 h, SCE (5 or 10 μ g/ml) and its constituents (10 or 20 μ M) were added to the HaCaT cells for another 24 h. The cells were then lysed with lysis reagent (Promega, Madison, WI, USA), and the luciferase assay substrate (Promega) was added using a dual-luciferase assay system according to the manufacturer's instructions. Transcriptional activity values were measured using a luminometer (Bio-Tek Instruments).
Enzyme-linked immunosorbent assay (ELISA) and NO assay. RAW 264.7 cells were incubated with SCE (50, 100 or 200 μ g/ml) and its constituents (20 or 40 μ M) for 1 h and stimulated with LPS (1 μ g/ml) for 24 h. Cell culture medium was collected, and the secretion of PGE 2 was determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Nitrite (indicator of NO production) was measured in culture medium by adding Griess reagent. The culture medium was mixed with Griess reagent for 20 min, and then the absorbance at 540 nm was measured by spectrophotometry (Bio-Tek Instruments).
Hs68 cells were incubated with serum-free DMEM containing SCE (1, 5 or 10 μ g/ml) and its constituents (20 μ M). Cell culture medium was collected after 24 h, type I procollagen production were quantified using a procollagen type I C-peptide enzyme immunoassay kit (MK101; Takara, Shiga, Japan).
Animal study. Seven-week-old male Sprague-Dawley (SD) rats (Central Laboratory Animal Inc., Seoul, Korea) were housed in a controlled environment (25 ± 2 °C, 55% ± 5% relative humidity, 12 h light-dark cycle) and allowed to acclimatise for 1 week. All mouse experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology in Seoul, Korea (2015-017). One day after dorsal hair removal, full-thickness incision wounds were made. SCE (1%, 2% or 3%) was topically applied to the wounds every day (n = 10), and wound sizes were measured three times per week. At the end of experiments, animals were anaesthetised with a Zoletil-Rompun mixture and skin lesions were removed and stored at − 80 °C until analysis.
Histological analysis. Skin lesions were embedded in paraffin, sectioned (4 mm thick), deparaffinised in xylene and rehydrated in a gradient of alcohol solutions. The sections were stained with haematoxylin-eosin (H&E). The H&E-stained slides were visualised using an Eclipse TE2000U inverted microscope with twin CCD cameras (magnification, × 200; Nikon).

Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was isolated from the skin lesion using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by spectrophotometry at 260 nm. An equal amount of total RNA was used to synthesise cDNA with reverse transcriptase premix (Elpis-Biotech. Inc., Taejeon, Korea). Reverse transcription was performed at 42 °C for 55 min and was terminated by incubation at 94 °C for 5 min. PCR amplification of the cDNA products (3 μ l) was performed with PCR premix (Elpis-Biotech) and the primer pairs (Bioneer, Daejeon, Korea) shown in Table 1. Prior to PCR amplification, primers were denatured at 94 °C for 5 min. PCR was performed on a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA). PCR products were separated by 1.5% agarose gel electrophoresis and visualised with ethidium bromide. β -Actin was used as an internal control for normalising data.

Western blot analysis.
HaCaT and Hs68 cells were lysed in RIPA buffer containing protease inhibitors and then incubated on ice for 10 min. The protein concentrations of the lysates were normalised by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20 μ g) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). The membrane was further incubated with specific primary antibodies for 16 h at 4 °C, followed by appropriate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Proteins were developed with ECL Western detection reagents (Amersham Biosciences, Little Chalfont, UK) and visualised by enhanced chemiluminescence (GE Healthcare, Hatfield, UK).
Scientific RepoRts | 7:42490 | DOI: 10.1038/srep42490 Statistical analysis. Each experiment was performed at least three times. All data are expressed as mean ± standard deviation. Differences between groups were analysed using a one-way analysis of variance followed by Scheffe's test (SPSS 17.0, Chicago, IL, USA). ## p < 0.01, * p < 0.05 and ** p < 0.01 were considered statistically significant.