Regulatory Effects of Lycium barbarum Extract and Isolated Scopoletin on Atopic Dermatitis-Like Skin Inflammation

Lycium barbarum and scopoletin are widely used in oriental Eastern medicine and are often consumed as teas. In this study, proinflammatory cytokines expressed in human keratinocytes (HaCaT) were induced by skin diseases caused by 2,4-dinitrochlorobenzene (DNCB) and tumor necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ). The inhibitory activity of L. barbarum EtOH extract (LBE) and scopoletin on proinflammatory cytokines and chemokines was investigated. In the DNCB-induced animal model, oral administration of LBE inhibited skin lesions and proinflammatory cytokines and chemokines and showed inhibitory effects in vitro. Additionally, as a result of examining the efficacy of scopoletin isolated from L. barbarum, scopoletin in HaCaT cells showed inhibitory effects on proinflammatory cytokines and chemokines. It shows promise in the treatment of chronic skin diseases.


Introduction
Lycium barbarum is the mature fruit of L. barbarum vine, a deciduous shrub belonging to the Solanaceae family [1]. It is a red fruit that looks a small pepper, is used medicinally in oriental medicine, and is also consumed in large amounts as a tea for the purpose of health promotion. L. barbarum has been reported to have many antioxidant and antiinflammatory effects [2], including inhibition of PC3 cell proliferation in vitro [3] and prevention of UVB-induced skin damage [4]. In addition, a component of L. barbarum attenuated type II collagen-induced arthritis [5]. Scopoletin (7-hydroxy-6-methoxycoumarin) is a coumarin derivative and is contained in many medicinal plants, such as L. barbarum, Erycibe obtusifolia, Aster tataricus, and Foeniculum vulgare [6]. A previous report showed that scopoletin had anti-inflammatory effects in the human mast cell line HMC-1 [7], anti-obesity and fatty liver inhibitory effects [8], hypoglycemic and hypolipidemic effects [1], and synergistic antifungal effects [9].
The main function of the skin is protection and defense against external stimuli. Persistent external skin irritation by irritants and allergens triggers the progression of atopic dermatitis toward skin inflammation [10]. Skin inflammation is largely divided into two types: acute and chronic inflammation. Acute inflammation is the initial reaction by the body that initiates healing, and chronic inflammation can cause various problems [11]. 2,4-Dinitrochlorobenzene (DNCB, a hapten)-induced atopic dermatitis is acute inflammation involving dendritic cells and macrophage activation resulting in dermal damage and edema [12]. An acute inflammatory state directly affects the course of dermatitis treatment [13], and appropriate treatment is important. Therefore, the development of natural drugs with high efficacy and few side effects is important.
Due to the pharmacological profile of L. barbarum extract and isolated scopoletin and its reported immunoregulatory properties, we hypothesized that L. barbarum extract and isolated scopoletin might have a therapeutic effect on atopic dermatitis, such as skin inflammation. The purpose of this study was, therefore, to test the therapeutic activity of L. barbarum extracts and scopoletin against skin inflammation and to elucidate the underlying therapeutic mechanisms of action, as part of our ongoing search for immunomodulatory medicines extracted from medicinal plants or edible plants.

Animals.
Female BALB/c mice (6 weeks) were purchased from Samtako (Osan, Korea). All animals had ad libitum access to standard rodent chow and filtered water during the study. The animals were housed (5 per cage) in a laminar air flow room maintained at a temperature of 22 ± 2°C and relative humidity of 55 ± 5%, with a 12 h light/dark cycle throughout the study. Care and treatment of the animals were conducted in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology.

Induction of DNCB-Induced Atopic Dermatitis-Like
Lesions. A total of 25 mice were divided into five groups (n = 5): PBS topically applied to ear and PBS-treated group, DNCB applied and PBS-treated group, DNCB applied and LBE (100 or 300 mg/kg) treated groups, and DNCB applied and dexamethasone (DX, 5 mg/kg) treated group. During the first week of induction, DNCB (2%, 20 μL/ear) was applied to each ear once for sensitization. Then, DNCB (1%, 20 μL/ear, once) was challenged to both BALB/c mouse ears for 3 weeks. LBE (100 or 300 mg/kg) or DX (5 mg/kg) was orally administered by gavage for five consecutive days per week at the time of the DNCB challenge.

Histological Assay.
Mouse ear tissues were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The fixed tissues were embedded in paraffin and cut into 4 μm thick sections, which were stained with hematoxylin and eosin (H&E).
2.9. Statistical Analysis. Statistical analysis was performed using Prism 5 software (GraphPad Software, San Diego, CA, USA). The data are presented as the mean ± SD of nine individual experiments. Statistical significance was determined by one-way ANOVA followed by Tukey's test for multiple comparisons.

Effect of LBE in DNCB-Induced Atopic Dermatitis.
To evaluate the antiatopic effects of LBE, a DNCB-induced atopic dermatitis animal model was used. DNCB is a powerful material for the induction of atopic lesions [12]. As shown in Figure 1(a), atopic skin lesions in the DNCBtreated group were significantly aggravated. The histopathological pathology analysis using H&E staining showed an increase in edema and immune cell infiltration. In addition, ear thickness was increased with DNCB treatment (Figure 1(b)). However, oral administration of LBE decreased the DNCB-induced atopic skin lesions (Figures 1(a) and 1(b)). IgE is an immunoglobulin associated with Th2 responses in atopic dermatitis [12]. The DNCB-treated group showed significantly increased serum IgE levels. However, oral administration of LBE decreased serum IgE levels (Figure 1(c)). In atopic dermatitis, damaged epithelial cells and keratinocytes produce proinflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, IL-8, CCL17, and CCL22 [15]. To assess the anti-inflammatory effect of LBE on atopic dermatitis, the gene expression of proinflammatory cytokines and chemokines was determined in ear tissues. The DNCB-treated group showed markedly elevated proinflammatory cytokine and chemokine levels, such as IL-1β, IL-6, IL-8, CCL17, and CCL22. However, oral administration of LBE alleviated gene expression levels ( Figure 2). Our data indicated that LBE attenuates Th2mediated atopic dermatitis-related skin lesions.

Effect of LBE on Proinflammatory Gene Expression in
HaCaT Cells. To determine the anti-inflammatory effect of LBE at the cellular level, proinflammatory cytokine and chemokine expressions under TNF-α/IFN-γ-induced inflammation in HaCaT human keratinocytes were analyzed.

Effect of LBE on Intracellular Signaling in HaCaT Cells.
To demonstrate the mechanism of LBE on anti-inflammatory efficacy, the TNF-α/IFN-γ-stimulated intracellular signaling pathway was evaluated. MAPKs, including p38, EKR, and JNK, have shown a wide spectrum of bioactivities, such as cell cycle, metabolism, and inflammation activities at the cellular level [16]. STAT1 drives to Th2 responses, leading to the development of atopic dermatitis [17]. NF-κB is a major transcription factor and plays critical roles in inflammatory gene expression [18]. Therefore, MAPKs, STAT1, and NF-κB are treatment targets for inflammatory responses. TNF-α/IFN-γstimulated HaCaT cells showed elevated phosphorylation of signaling molecules such as p38, ERK, JNK, STAT1, and NF-κB. However, pretreatment with LBE suppressed the TNF-α/IFN-γ-stimulated phosphorylation of signaling molecules ( Figure 5). β-Actin and Lamin B were used as loading controls in the whole and nuclear proteins, respectively. Our data indicated that LBE downregulated the inflammatory-associated signaling molecules, leading to anti-inflammatory efficacy. To verify the mode of action of LBE, we purified the LBE into fractions and single compounds. Scopoletin is an abundant molecule in natural products such as Lycium barbarum and has been reported various pharmaceutical activities [3,9,[19][20][21]. The HaCaT cells were pretreated with various concentrations of scopoletin (5, 10, and 50 μM) for 1 h before the stimulation. TNF-α/IFN-γ-stimulated HaCaT cells showed the increase of proinflammatory gene expression and production such as proinflammatory cytokines and chemokines. However, pretreatment of scopoletin suppressed the TNF-α/IFN-γ-stimulated proinflammatory gene expression and productions in a concentrationdependent manner (Figures 6 and 7). Moreover, the highest concentration of scopoletin showed an inhibitory efficacy similar to that of the positive control drug, cyclosporine A. Our data showed that scopoletin strongly regulated inflammatory gene expression in keratinocytes.

Effect of Scopoletin on Intracellular Signaling in HaCaT
Cells. To inspect the mechanism of action of scopoletin, the TNF-α/IFN-γ-stimulated intracellular signaling pathway was evaluated. TNF-α/IFN-γ-stimulated HaCaT cells showed an increase in the phosphorylation of signaling molecules. However, pretreatment with scopoletin suppressed the TNFα/IFN-γ-stimulated phosphorylation of signaling molecules ( Figure 8). Furthermore, the highest concentration of scopoletin significantly suppressed the phosphorylation of MAPKs, STAT1, and NF-κB. Our data indicated that scopoletin strongly regulates atopic skin lesions by controlling the inflammatory responses of keratinocytes.

Discussion
In this study, we investigated the inhibitory effects of L. barbarum and scopoletin on chronic inflammatory skin conditions. First, the anti-inflammatory activity of LBE was confirmed using an animal model of DNCB-induced dermatitis. LBE reduced DNCB-induced atopic like-skin lesions, such as increases in epidermal thickness and serum IgE levels. Additionally, LBE treatment inhibited the DNCBinduced gene expression of proinflammatory cytokines and chemokines. In HaCaT cells, TNF-α/IFN-γ-stimulated mRNA expression and protein secretion were verified for the anti-inflammatory activity of LBE on proinflammatory cytokine and chemokine levels, and the activity of scopoletin isolated from LBE showed similar activity. Moreover, the anti-inflammatory effects of LBE and scopoletin were demonstrated through the regulation of intracellular signaling pathways, including MAPKs, STAT1, and NF-κB.
Atopic dermatitis is a complicated skin barrier disease characterized by immune hypersensitivity reactions caused by infiltration and activation of immune cells (lymphocytes, eosinophils, mast cells, etc.), resulting in skin lesions (dry skin, itchiness, eczema, erythema, etc.), dermal damage, keratinocytes inflammation, and skin remodeling [22]. To evaluate the treatment effect of LBE on atopic dermatitislike skin inflammation, we used a DNCB-induced skin inflammation mouse model. The activation of dendritic cells and macrophages in the subcutaneous layer occurs after DNCB exposure, causing damage and inflammation of the skin layer through the secretion of proinflammatory cytokines and chemokines [23,24]. Many previous studies consider that inflammatory responses are a target of therapeutic targets and that drugs from natural products should be developed [23,25]. Therefore, verification of the efficacy of natural compounds for atopic dermatitis using the DNCB atopic model is appropriate for discovering new therapeutic substances. In this study, it was confirmed that after administration of LBE, changes in skin inflammatory properties were observed. Our data showed that oral administration of LBE, for 3 weeks, decreased the increase in epidermal thickness and the level of serum IgE in the DNCB-induced atopic dermatitis model. The gene expression of proinflammatory cytokines and chemokines was also suppressed by oral administration of LBE at the site of ear skin lesions.
Inflammatory stimuli such as TNF-α/IFN-γ are used in many skin disease studies because they can mimic the symptoms of atopic dermatitis such as skin inflammation in HaCaT cells [25]. In this study, LBE and scopoletin did not show toxicity on HaCaT cells in the experimental