Ethanolic Extracts of Artemisia apiacea Hance Improved Atopic Dermatitis-Like Skin Lesions In Vivo and Suppressed TNF-Alpha/IFN-Gamma–Induced Proinflammatory Chemokine Production In Vitro

Artemisia apiacea Hance is a traditional herbal medicine used for treating eczema and jaundice in Eastern Asia including China, Korea, and Japan. However, the biological and pharmacological actions of Artemisia apiacea Hance in atopic dermatitis (AD) are not fully understood. An ethanolic extract of Artemisia apiacea Hance (EAH) was tested in vitro and in vivo to investigate its anti-inflammatory activity and anti-atopic dermatitis effects. The results showed that EAH dose-dependence inhibited production of regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-6, IL-8, and thymus and activation-regulated chemokine (TARC). EAH inhibited the activation of p38, extracellular signal-regulated kinases (ERK), and STAT-1 and suppressed the degradation of inhibited both nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκB-α) in TNF-α/IFN-γ–stimulated HaCaT cells. EAH also suppressed the translocation of inflammation transcription factors such as NF-κB p65 in TNF-α/IFN-γ–stimulated HaCaT cells. In addition, EAH reduced 2,4-dinitrochlorobenzene (DNCB)-induced ear thickness and dorsal skin thickness in a dose-dependent manner. EAH appeared to regulate chemokine formation by inhibiting activation of and ERK as well as the NK-κB pathways. Furthermore, EAH significantly improved the skin p38 conditions in a DNCB-induced AD-like mouse model.


Introduction
Atopic dermatitis (AD) is a chronic skin disease that affects millions of individuals worldwide [1]. The current drugs for AD such as corticosteroids may be effective but have serious side effects. Therefore, alternatives are urgently needed. Accordingly, alternative medicines such as herbal extracts with potent efficacy and minimal side effects have been considered in the development of AD treatments [2,3]. Current treatments to reduce inflammation and itching mainly involve the application of topical ointments and administration of oral medications [4].
Keratinocytes are the major cell types in the epidermis and maintain the biochemical and physical condition of the skin. They are also involved in the progression of various inflammatory skin diseases. Epidermal keratinocytes release inflammatory mediators such as pro-inflammatory cytokines and

Chromatographic Conditions and Sample Preparation
The dried form of EAH was accurately weighed (25 mg) and dissolved in 1 mL of 100% methanol and then it was extracted by using ultra sonication for 30 min. The standard compound stock solutions were precisely weighed and dissolved in 100% methanol (1000 µg/mL). All working solutions (330 µg/mL, each) were filtered through 0.2 mm syringe membrane filters from Whatman Ltd. (Maidstone, UK) before being injected into the HPLC analysis system.
HPLC-UV/VIS diode array detector (DAD) studies were performed by using a Dionex Liquid Chromatography system (Dionex Co., Sunnyvale, CA, USA) fitted with an Ultimate 3000 Series binary pump, auto-sampler, column oven, and DAD. Chromatographic separation was achieved on a Waters Bridge C 18 column (Waters Corporation, Milford, MA, USA). The column temperature was 40 • C, injection volume was 10 µL, and the flow rate was 1 mL/min for the total run time. The mobile phase comprised 0.1% trifluoroacetic acid in water (v/v) (A) and acetonitrile (B) [12]. The HPLC gradient was 10% to 60% B over 0-70 min. The detection wavelength was 254 nm. Data acquisition was performed by using the Dionex Chromelon (Dionex Co., Sunnyvale, CA, USA).

MTT Assay
Cell viability was determined by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) agent. HaCaT cells (1 × 10 4 cells/well) were seeded into 96-well plates and an EAH extract was added to the wells at concentrations ranging between 1 µg/mL and 200 µg/mL. After 24 h, MTT solutions were then added to each well and the cells were incubated for an additional 2 h. The resulting formazan was dissolved by DMSO and the optical density at 570 nm was determined by using an ELISA reader (Infinite M200; Tecan, Männedorf, Switzerland).

Cytokine and Chemokine Analysis
The cells (1 × 10 6 cells/well) in 6 well plates were pretreated with EAH at indicated concentrations (1 µg/mL, 10 µg/mL, 50 µg/mL, and 100 µg/mL) for 1 h and stimulated with TNF-α/IFN-γ (each 10 ng/mL) for 24 h at 37 • C in a 5% CO 2 atmosphere. After stimulation, the culture medium was then harvested and the levels of chemokines such as RANTES, IL-8, IL-6, and TARC were measured using ELISA kits, according to the manufacturer's instructions. Recombinant human TNF-α, IFN-γ, and enzyme-linked immunosorbent assay (ELISA) kits for RANTES, IL-8, IL-6, and TARC were obtained from the BioLegend (San Diego, CA, USA). The plates were read at 450 nm and an inhibitory effect of EAH was determined from a standard curve.

Immunofluorescence Assay
The cells were seeded into a confocal dishes, incubated without or with EAH for 1 h, and stimulated with TNF-α/IFN-γ (each 10 ng/mL) for 20 min [15]. After treatment, cells were fixed with 4% paraformaldehyde in PBS. Fixed cells were blocked using 3% goat serum for 1 h at room temperature. After an overnight incubation at 4 • C with the anti-p65 and anti-STAT-1 antibodies at 4 • C, the cells were incubated with secondary Alexa-Fluor-488 and 568-labeled antibodies for 2 h at room temperature. The nuclei were stained with a nuclear marker, 4 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA), for 10 min at room temperature. All samples were then observed using a confocal microscope (FV3000 FLUOVIEW; Olympus, Tokyo, Japan).

Induction of AD and Drug Treatment
The AD-like mouse model involved induction, which was described previously [16]. DNCB dissolved in an acetone:olive oil mixture (3:1 v/v), which was applied to the dorsal skin and both ears of BALB/c mice to induce AD-like symptoms and skin lesions. Test drugs were administered before applying DNCD. EAH dissolved in saline (10 mL/kg body weight) was orally administered by gavage 50 mg/kg or 100 mg/kg 3 times a week for 4 weeks (days 0-24) and dexamethasone (Sigma-Aldrich, 1 mg/kg) was used as a control drug.

Ear Thickness Measurements and Histopathological Analysis
For each mouse, ear thickness was measured and recorded with a micrometer (Mitutoyo, Kawasaki, Japan). To minimize variation, a single investigator performed all measurements. The dorsal skin lesions of each mouse were removed and fixed with 4% paraformaldehyde overnight at room temperature. For hematoxylin and eosin staining, fixed samples were analyzed by a commercial services company (Garam Meditech, Garam, South Korea). All samples were observed using a microscope and data were representative of five observations (Nikon Eclipse Ti; Nikon, Tokyo, Japan).

Statistical Analysis
The data were analyzed by using the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). The results are expressed as the means ± SEM and SD, which were evaluated by using Student's t-test or the analysis of variance (one-way ANOVA, Dunnett, to compare control groups). A p-value of < 0.05 was considered for indicating statistical significance.

Measurement of the Representative Component in EAH by HPLC Analysis
The three standard compounds of A. apiacea Hance were determined by HPLC. The wavelength that provided the maximum UV absorption of each compound was determined by using a DAD coupled to the HPLC system. The retention times and UV spectra of the peaks were compared with those of the respective standard compounds [17]. As shown in Figure 1A, the retention times of quercetin-3-beta-D-glucoside, scoparone, and quercetin in the standard solution were 14.917 min, 20.960 min, and 26.353 min, respectively. In the samples, the retention time of peaks 1, 2, and 3 were 14.940 min, 20.973 min, and 26.410 min, respectively, which closely matched those of the standard compounds ( Figure 1B).

Measurement of the Representative Component in EAH by HPLC Analysis
The three standard compounds of A. apiacea Hance were determined by HPLC. The wavelength that provided the maximum UV absorption of each compound was determined by using a DAD coupled to the HPLC system. The retention times and UV spectra of the peaks were compared with those of the respective standard compounds [17]. As shown in Figure 1A, the retention times of quercetin-3-beta-D-glucoside, scoparone, and quercetin in the standard solution were 14.917 min, 20.960 min, and 26.353 min, respectively. In the samples, the retention time of peaks 1, 2, and 3 were 14.940 min, 20.973 min, and 26.410 min, respectively, which closely matched those of the standard compounds ( Figure 1B).

Effects of EAH on HaCaT Cell Viability
EAH was tested for cytotoxicity in human keratinocyte HaCaT cells by exposing them to various concentrations for 24 h. Figure 1C showed no toxic effects of EAH on cell viability up to a concentration of 200 µ g/mL. Therefore, the cells were treated with doses lower than 200 µ g/mL in subsequent experiments.

Effects of EAH on TNF-α/IFN-γ-Induced Production of Proinflammatory Cytokines and Chemokines in HaCaT Cells
To investigate the anti-AD effect of EAH on pro-inflammatory cytokines and chemokines production upon TNF-α/IFN-γ co-stimulation, HaCaT cells were pretreated with EAH for 1 h followed by TNF-α/IFN-γ for 24 h and the supernatant was collected for cytokine level measurement by ELISA ( Figure 2B,D). The results showed that EAH inhibited the production of RANTES, IL-6, IL-8, and TARC.

Effects of EAH on HaCaT Cell Viability
EAH was tested for cytotoxicity in human keratinocyte HaCaT cells by exposing them to various concentrations for 24 h. Figure 1C showed no toxic effects of EAH on cell viability up to a concentration of 200 µg/mL. Therefore, the cells were treated with doses lower than 200 µg/mL in subsequent experiments.

Effects of EAH on TNF-α/IFN-γ-Induced Production of Proinflammatory Cytokines and Chemokines in HaCaT Cells
To investigate the anti-AD effect of EAH on pro-inflammatory cytokines and chemokines production upon TNF-α/IFN-γ co-stimulation, HaCaT cells were pretreated with EAH for 1 h followed

Effects of EAH on Phosphorylation of Mitogen Activated Protein Kinases (MAPK), STAT-1, and NFκB-p65 in TNF-α/IFN-γ-Stimulated HaCaT Cells
We investigated the effects of EAH on the expression of inflammation-related factors such as MAPK, NFκB-p65, and STAT-1 in TNF-α/IFN-γ-stimulated HaCaT cells. Figure 3A shows the effects of EAH at 1-100 μg/mL on MAPK activities such as p38, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). The relative abundances of proteins were calculated for the p-ERK/ERK, p-p38/p38, and p-JNK/JNK ratios ( Figure 3B). As a result, EAH strongly inhibited the activation of p38 and ERK induced by TNF-α/IFN-γ without affecting the total protein level. In addition, we determined whether EAH affects transcription factors such as NFκB-p65 and STAT-1 in HaCaT cells. To investigate the effect of EAH on the NF-κB and STAT-1 signaling pathway, both the degree of IκB-α degradation and phosphorylation of STAT-1 were evaluated ( Figure 3C). As shown in Figure 3C,D, treatment with EAH inhibited degradation of the nuclear factor-kappa B (NF-κB) inhibitory protein IκBα but does not inhibit phosphorylation of STAT-1.

Effects of EAH on Phosphorylation of Mitogen Activated Protein Kinases (MAPK), STAT-1, and NFκB-p65 in TNF-α/IFN-γ-Stimulated HaCaT Cells
We investigated the effects of EAH on the expression of inflammation-related factors such as MAPK, NFκB-p65, and STAT-1 in TNF-α/IFN-γ-stimulated HaCaT cells. Figure 3A shows the effects of EAH at 1-100 µg/mL on MAPK activities such as p38, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). The relative abundances of proteins were calculated for the p-ERK/ERK, p-p38/p38, and p-JNK/JNK ratios ( Figure 3B). As a result, EAH strongly inhibited the activation of p38 and ERK induced by TNF-α/IFN-γ without affecting the total protein level. In addition, we determined whether EAH affects transcription factors such as NFκB-p65 and STAT-1 in HaCaT cells. To investigate the effect of EAH on the NF-κB and STAT-1 signaling pathway, both the degree of IκB-α degradation and phosphorylation of STAT-1 were evaluated ( Figure 3C). As shown in Figure 3C,D, treatment with EAH inhibited degradation of the nuclear factor-kappa B (NF-κB) inhibitory protein IκBα but does not inhibit phosphorylation of STAT-1. ERK/ERK, p-p38/p38, and p-JNK/JNK ratios ( Figure 3B). As a result, EAH strongly inhibited the activation of p38 and ERK induced by TNF-α/IFN-γ without affecting the total protein level. In addition, we determined whether EAH affects transcription factors such as NFκB-p65 and STAT-1 in HaCaT cells. To investigate the effect of EAH on the NF-κB and STAT-1 signaling pathway, both the degree of IκB-α degradation and phosphorylation of STAT-1 were evaluated ( Figure 3C). As shown in Figure 3C,D, treatment with EAH inhibited degradation of the nuclear factor-kappa B (NF-κB) inhibitory protein IκBα but does not inhibit phosphorylation of STAT-1.

Effects of EAH on NFκB-p65 and STAT-1 Translocation in TNF-α/IFN-γ-Stimulated HaCaT Cells
NFκB-p65 and STAT-1 are transcription factors that are critically involved in atopic dermatitisrelated signaling in TNF-α/IFN-γ-stimulated HaCaT cells. Consequently, we confirmed the effect of EAH on nuclear translocation of NF-κB and STAT-1 in TNF-α/IFN-γ-stimulated HaCaT cells. Similar to Figure 3C results, treatment with EAH inhibited translocation of p65 from the cytoplasm to the nucleus but does not inhibit STAT-1 (Figure 4).

Effects of EAH on NFκB-p65 and STAT-1 Translocation in TNF-α/IFN-γ-Stimulated HaCaT Cells
NFκB-p65 and STAT-1 are transcription factors that are critically involved in atopic dermatitis-related signaling in TNF-α/IFN-γ-stimulated HaCaT cells. Consequently, we confirmed the effect of EAH on nuclear translocation of NF-κB and STAT-1 in TNF-α/IFN-γ-stimulated HaCaT cells. Similar to Figure 3C results, treatment with EAH inhibited translocation of p65 from the cytoplasm to the nucleus but does not inhibit STAT-1 (Figure 4).

Effects of EAH on NFκB-p65 and STAT-1 Translocation in TNF-α/IFN-γ-Stimulated HaCaT Cells
NFκB-p65 and STAT-1 are transcription factors that are critically involved in atopic dermatitisrelated signaling in TNF-α/IFN-γ-stimulated HaCaT cells. Consequently, we confirmed the effect of EAH on nuclear translocation of NF-κB and STAT-1 in TNF-α/IFN-γ-stimulated HaCaT cells. Similar to Figure 3C results, treatment with EAH inhibited translocation of p65 from the cytoplasm to the nucleus but does not inhibit STAT-1 (Figure 4).

Effects of EAH on Development of DNCB-Induced AD Mouse Skin Lesions
Repeated cutaneous application of DNCB induces AD-like dermatitis in BALB/c mice. We investigated EAH following induction using DNCB of AD-like mouse skin lesions. As shown in Figure 5A, the skin conditions significantly prevented the EAH-administered groups relative to those in the control group. In addition, EAH prevented DNCB-induced ear thickness and dorsal skin thickness in a dose-dependent manner.

Effects of EAH on Development of DNCB-Induced AD Mouse Skin Lesions
Repeated cutaneous application of DNCB induces AD-like dermatitis in BALB/c mice. We investigated EAH following induction using DNCB of AD-like mouse skin lesions. As shown in Figure 5A, the skin conditions significantly prevented the EAH-administered groups relative to those in the control group. In addition, EAH prevented DNCB-induced ear thickness and dorsal skin thickness in a dose-dependent manner. The results shown are representative of (A,C). Vehicle, acetone:olive oil mixture (3:1 vol/vol), Control, DNCB + Vehicle, EAH, DNCB + EAH treated group (50, 100 mg/kg), Dexa, DNCB + 1 mg/kg dexamethasone treated group. Data are represented as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Discussion
AD is a chronic allergic inflammatory skin disease characterized by pruritic eczema and scratching behavior that can induce skin injury. Patients with AD exhibit elevated serum IgE levels and markedly increased levels of inflammatory cells including eosinophils, mast cells, and lymphocytes. Various cells involved in the allergic reaction infiltrate the lesions. Among the infiltrating cells, T helper 2 (Th2) cells are one of the most important cell types involved in AD development [18]. Although Th2 cells are the principal type of cells involved in the acute reaction of AD and Th1 cells are highly expressed during the chronic AD phase. Until now, local or systemic

Discussion
AD is a chronic allergic inflammatory skin disease characterized by pruritic eczema and scratching behavior that can induce skin injury. Patients with AD exhibit elevated serum IgE levels and markedly increased levels of inflammatory cells including eosinophils, mast cells, and lymphocytes. Various cells involved in the allergic reaction infiltrate the lesions. Among the infiltrating cells, T helper 2 (Th2) cells are one of the most important cell types involved in AD development [18]. Although Th2 cells are the principal type of cells involved in the acute reaction of AD and Th1 cells are highly expressed during the chronic AD phase. Until now, local or systemic glucocorticosteroids as well as emollients are the main therapeutic modalities for control of AD [19], but their long-term use is controversial because these steroids can produce side effects in AD patients. Therefore, many researchers are trying to develop more effective and safer treatments for AD.
HaCaT cells are one of the cell lines used to mimic AD symptoms in response to inflammatory stimuli such as TNF-α/IFN-γ [20]. Stimulation of keratinocytes by TNF-α/IFN-γ leads to activation of various signaling pathways that involve STAT-1, NF-κB, and MAPKs, which subsequently increase expressions of inflammatory mediators [21]. In this study, we activated HaCaT cells by treating them with TNF-α/IFN-γ for investigating the anti-inflammatory effect of EAH. The results showed that EAH inhibited the production of chemokines and pro-inflammatory cytokines such as RANTES, IL-8, IL-6, and TARC ( Figure 2). We also investigated how EAH inhibits the secretion of inflammatory cytokines and chemokines. As a result, EAH inhibited the activation of p38, ERK without JNK inhibition.
NF-κB and STAT-1 are protein transcription factors that are required for the transcription of a wide array of pro-inflammatory molecules in AD. In the resting state, NF-κB dimers are inactive in the cytoplasm of cells and are associated with the IκB. Upon stimulation with TNF-α/IFN-γ, the IκB-kinase complex is activated and phosphorylates IκB, which leads to the substrate's ubiquitination and subsequent degradation. The resulting free NF-κB is translocated to the nucleus where it can activate target genes by binding to regulatory elements in the target gene's promoter. Similarly, STAT-1 is phosphorylated and activated when stimulated with TNF-α/IFN-γ and translocated to the nucleus where it can activate target genes by binding to regulatory elements in the target gene's promoter [22]. As shown in Figure 3, EAH inhibited degradation of the nuclear factor-kappa B (NF-κB) inhibitory protein IκBα but did not inhibit phosphorylation of STAT-1. Furthermore, we observed the effect of EAH on transcription factors such as NF-κB and STAT-1. We confirmed that EAH inhibited the translocation of NF-κB p65.
Recently, many types of mouse models similar to our AD-like model have been developed. In one model, the ears or dorsal skin of a BALB/c mouse is repeatedly exposed to chemicals DNCB, trinitrochloridebenzene, and 1-fluoro-2,4-dinitrobenzene. Another model is the NC/Nga mice that have developed AD-like symptoms under conventional but not specific pathogen-free conditions [30]. Repeated cutaneous application of DNCB induces AD-like dermatitis in BALB/c mice. In AD-like skin by DNCB, many changes such as a thick epidermis of the epidermis and dermis, significant keratosis, infiltration of inflammatory cells, ulceration, and hemorrhage are observed [31]. We found that EAH restrained edema of the ear skin and restored epidermal thickness through the histological analysis ( Figure 5).
In this study, we demonstrated the mechanism underlying the anti-inflammatory activity of EAH in HaCaT cells and investigated the anti-AD effects in an AD-like mouse model. We found that EAH regulated expression of pro-inflammatory cytokines and chemokines via the p38/NF-κB pathway in allergic inflammation. In addition, EAH inhibited the degradation of IκBα and the translocation of NF-κB p65. Moreover, EAH improved the skin lesions and reduced the thickness of dorsal skin and ear skin thickness in a DNCB-induced AD-like model. These results indicate that EAH can therapeutically reduce AD-like symptoms in mice. Our results suggest that EAH is a promising adjuvant treatment for AD. However, many questions remain to be addressed and additional studies such as serum IgE, cytokine, chemokine levels, and histological analysis are needed.