Quercitrin, the Main Compound in Wikstroemia indica, Mitigates Skin Lesions in a Mouse Model of 2,4-Dinitrochlorobenzene-Induced Contact Hypersensitivity

Hapten-induced contact hypersensitivity (CHS) is widely utilized to induce immune activation in animal models of allergic contact dermatitis. Our previous findings suggested that the 95% EtOH extract of Wikstroemia indica (L.) C. A. Mey. has antiallergic and anti-inflammatory effects in DNCB-treated CHS SKH-1 hairless mice. The aim of this study was to evaluate the protective effects of compounds isolated from the EtOAc fraction of W. indica in RBL-2H3 cells and 2,4-dinitrochlorobenzene- (DNCB-) induced CHS mice. Of eight compounds in W. indica, that is, umbelliferone, daphnoretin, wikstrocoumarin, (+)-syringaresinol, tricin, (+)-lariciresinol, erythro-guaiacylglycerol-β-coniferyl ether, and quercitrin, quercitrin exhibited the most antiallergic activity against antigen-induced β-hexosaminidase release and IL-4 mRNA expression, which are markers of degranulation in RBL-2H3 cells. After a 7-sensitizing period, 14 days of DNCB treatment with or without topical pimecrolimus (1%) or quercitrin (0.5%) treatment, quercitrin was found to suppress DNCB-induced increases in serum IL-4 and IgE concentrations and transepidermal water loss. These results indicate that quercitrin has therapeutic potential for treatment of allergies and allergy-related contact dermatitis.


Introduction
Contact hypersensitivity (CHS) is one of the most common skin inflammatory diseases and affects 15-20% of individuals worldwide [1]. Contact hypersensitivity is a type IV delayed hypersensitivity mediated by T cells and manifests as an eczematous skin condition that occurs when a substance (e.g., urushiol from poison ivy, a fragrance, or nickel) that acts as an allergen or hapten comes into contact with skin [2][3][4]. Contact hypersensitivity occurs in two phases, namely, a sensitization phase and an elicitation phase [5]. e sensitization phase occurs when a hapten applied to skin is first introduced to the immune system [6,7]. Langerhans cells recognize haptens and then mature, migrate to lymph nodes, and present processed haptens to naive T cells. ese sensitized naive T cells then migrate to epidermis and differentiate into effector T cells [4,8]. ese processes involve cytokines such as IL-1β, IL-12, and TNF-α [4,5]. Subsequently, when sensitized skin is reexposed to a hapten, the elicitation phase of CHS begins. is reexposure of hapten to "sensitized" T cells results in the explosive differentiation and proliferation of T cells [9], and cytokines secreted by these T cells stimulate cells in skin, which leads to the recruitment of T cells and the enhancement of inflammation at hapten-challenged sites [9,10]. e cytokines of INF-c, IL-4, and IL-17 play major roles during the elicitation phase [5,6].
Flavonoids are a group of aromatic ring compounds with a C6-C3-C6 composition and occur widely in plants (about 400 plant species have been identified) [11]. Flavonoids act as pigments that color fruits and flowers and are also involved in plant growth and defense [12]. Fruits, vegetables, and medicinal plants contain large quantities of flavonoids, and much research has shown they have many beneficial effects [13,14]. In particular, several studies have demonstrated the antiallergic, anti-inflammatory, and antioxidant effects of flavonoids [15,16]. Naturally occurring flavonoids reduce the productions of inflammatory cytokines secreted by mast cells, basophils, and cells in immunoglobulin E-(IgE-) mediated allergies [17][18][19][20][21]. Wikstroemia indica (L.) C. A. Mey. ( ymelaeaceae) is a plant native to Southeast Asia and China and is used in Chinese traditional medicine to treat coughs, syphilis, and arthritis [22]. W. indica contains diverse flavonoids, such as quercetin and epicatechin, with anti-inflammatory and antioxidant activities [23][24][25]. In a previous report, our findings supported the use of W. indica to treat types of allergic contact dermatitis like atopic dermatitis [26]. However, relatively few studies have indicated that flavonoids isolated from this plant are effective on skin inflammatory diseases. e aim of the present study was to isolate bioactive flavonoids from W. indica and to investigate their preventive and therapeutic potentials on CHS.

2
Evidence-Based Complementary and Alternative Medicine

2.6.
Release Activity of β-Hexosaminidase. RBL-2H3 cells were suspended in MEM media at 1 × 10 5 cells per well in a 24-well plate and sensitized with anti-DNP IgE for 24 hours. ey were then washed in Siraganian buffer (pH 7.5; 100 ng/ ml DNP-IgE, 119 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2 , 25 mM PIPES, and 40 mM NaOH) twice and incubated for 10 minutes in the same buffer. Cells were then treated with DMSO or compounds (10 μM and 30 μM) for 1 hour, with 1 μg/mL DNP-BSA antigen for 30 minutes to induce cell degranulation, and then supernatants obtained by microcentrifugation were transferred to 96-well plates and incubated with substrate solution (1 mM poly-N-acetyl glucosamine in 0.1 M sodium citrate buffer) for 3 hours at 37°C. Finally, stop solution was added and well absorbances were measured using a microplate reader at 405 nm.

Animal
Experiments. SKH-1 hairless mice (females, 6 weeks old) were procured from Orient Bio Inc. (Seongnam, Republic of Korea). All mice were housed under 12 h lightdark cycle and controlled conditions (RH 55 ± 5% and 25 ± 5°C) for 1 week before experimentation and fed a standard laboratory diet and water ad libitum. Mice care and experimental protocols were approved by the Institutional Animal Care and Use Committee of KIST (Certification No. KIST-2016-011) beforehand and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, 2011 revision). For CHS induction, SKH-1 hairless mice were acclimated for 1 week and then divided into 4 groups of 7 animals; a normal control group (animals were treated with distilled water), a negative control group (0.1% DNCB plus vehicle), a positive control group (0.1% DNCB and 1% pimecrolimus), and an experimental group (0.1% DNCB sensitized plus treatment with 1% quercitrin). On day 1, mice were sensitized by applying 1% DNCB (100 μL) dissolved in propylene glycol and ethanol (7 : 3) topically to dorsal skins for 7 days to induced CHS (day 1 to day 7). Mice were stimulated with 0.1% DNCB (100 μL) for 2 weeks (day 8 to 21) at 2-day intervals, and DNCB + quercitrin group and DNCB + pimecrolimus (Elidel®) group were treated with 0.5% quercitrin (100 μL) and 1% pimecrolimus cream twice daily (day 8 to 21). On day 22, animals were sacrificed, and dorsal skin tissue was removed for histological examination and blood was collected from abdominal aortas for serum IgE and IL-4 testing.

Measurement of Skin Conditions.
All experiments used to assess skin conditions were performed at 25 ± 5°C and 50 ± 5% RH. Transepidermal water loss (TEWL) was measured using a Tewameter TM210 (Courage and Khazaka, Cologne, Germany) with an open chamber system. Skin barrier damage was expressed in g/m 2 /h. A SKIN-O-MAT device (Cosmomed, Ruhr, Germany) was used to measure hydration levels of mouse skins.  2.11. Statistical Analysis. Statistical analysis was performed using one-way ANOVA followed by Tukey's HSD multiple comparison test. Results are presented as mean ± standard deviations, and statistical significance was accepted for p values < 0.05.

Effects of Quercitrin from WIE-EtOAc on DNCB-Induced CHS in SKH-1 Hairless Mice.
To investigate the effects of quercitrin from WIE-EtOAc on DNCB-induced skin symptoms, dermatitis levels were evaluated using skin images. e procedure used to establish the DNCB-induced CHS model is shown in Figure 4(a). DNCB treatment for 3 weeks resulted in severe CHS-like skin symptoms, that is, dried skin, cornification, exudation, and erythema, and these symptoms were markedly improved by 0.5% quercitrin treatment for 2 weeks and this improvement was similar to that observed in the 1% Elidel-treated group (the positive control group) (Figure 4(b)).

Effects of Quercitrin from WIE-EtOAc on Skin Histopathological Changes in DNCB-Induced CHS Mice.
To evaluate the histological changes induced by quercitrin in the dorsal skins of DNCB-induced CHS mice, we used H&E and toluidine blue staining. H&E results showed that epidermis thickness was significantly greater in the DNCB group than in the CON group. However, epidermis thickness in the 0.5% quercitrin group was 77.8% lower than that in the DNCB group (Figures 5(a) and 5(c)). Toluidine blue staining showed mast cell numbers were greater in the DNCB group than in the CON group and that mast cell numbers were 66.7% lower in the quercitrin group (Figures 5(b) and 5(d)), which was a greater reduction in numbers than that observed in the Elidel group.   Evidence-Based Complementary and Alternative Medicine DNCB group than in the CON group. However, 0.5% quercitrin treatment significantly decreased the serum IgE and IL-4 level increases induced by DNCB to 52.4% and 62.5%, respectively ( Figure 6).

Effects of Quercitrin from WIE-EtOAc on Skin Barrier
Function in DNCB-Induced CHS Mice. Changes in skin barrier function after treatment with 0.5% quercitrin for 2 weeks were evaluated by measuring TEWL and skin hydration.    Evidence-Based Complementary and Alternative Medicine e results showed that TEWL was 2.7-fold higher and skin hydration was 0.3-fold lower in the DNCB group than in the CON group. However, 0.5% quercitrin treatment significantly reduced this TEWL DNCB-induced increase to 50% and improved skin hydration to 32.4% (Figure 7).

Discussion
e skin is made up of several tightly connected layers that are in combination function as a primary barrier, which protects the body from harm, prevents the entry of exogenous substances, and reduces water loss [27]. If the skin barrier is damaged, external antigens are likely to penetrate, and this causes T-helper 2 ( 2) cell-mediated immune response and subsequent inflammation [28]. Furthermore, excessive, prolonged inflammatory reactions cause tissue damage, which in turn detrimentally affects the skin barrier and establishes a vicious cycle [29]. Skin barrier dysfunction and immune system dysregulation are the main causes of skin inflammatory diseases [30]. Because skin barrier dysfunction plays an important role in the development of allergic contact dermatitis, strengthening this barrier is considered a primary strategy for preventing and treating contact dermatitis [31,32]. Several methods can be used to assess skin barrier dysfunction, and TEWL provides a simple and noninvasive means of doing so [33]. e TEWL values of patients with allergic contact dermatitis are higher than those of healthy individuals [34].
Our previous study of WIE revealed that its topical application on the dorsal skins of mice decreased the severity of DNCB-induced allergic contact dermatitis by reducing edema, erythema, and inflammation [26]. e therapeutic     effects of WIE against hapten-induced CHS are believed to be due to skin barrier recovery and the subsequent inhibition of IL-4. Accordingly, attempts have been made to develop a procedure for separating biologically active components from WIE. In the present study, flavonoids, lignans, and coumarins were isolated from an EtOAc fraction of WIE using various chromatographic methods, and of these isolated compounds, quercitrin (a glycoside of quercetin) exhibited strong in vitro inhibitory activities against DNP/IgE-induced β-hexosaminidase release and PI-induced IL-4 mRNA expression. In vivo testing using DNCB-induced CHS mouse model was performed to assess the therapeutic potential of quercitrin for the treatment of contact dermatitis. Murine CHS models are often used to investigate the pathogeneses of human and animal allergic CHS [1]. Typical haptens, such as 2-chloro-1,3,5-trinitrobenzene (TNCB), 2,4-dinitro-1-fluorobenzene (DNFB), 2,4-dinitrochlorobenzene (DNCB), and oxazolone, are known to induce rapid CHS in animals [1,35]. In the present study, the severe CHS reaction induced by DNCB was clearly suppressed by treatment with 0.5% quercitrin for 2 weeks. Furthermore, barrier functional damage caused by DNCB was largely prevented by quercitrin. According to the previous literature, 2 cytokine IL-4 enhances expression of kallikrein 7 (KLK7) and downregulates expression of filaggrin, a key epidermal barrier protein in allergic skin disease [36]. e inhibitory effects of quercitrin on IL-4 production may provide an important clue for the mechanism underlying its skin barrier protective effect. RBL-2H3 cells are an excellent experimental model for studying the effects of drug involved in 2 immune responses and have characteristics resembling those of mast cells, for example, they secrete cytokines and histamine, which induce IgE receptor expression and immune responses on cell surfaces [37,38]. In the present study, the antiallergic and anti-inflammatory effects of compounds isolated from WIE were determined using procedures previously used in RBL-2H3 cells. Out of the eight compounds isolated, quercitrin most effectively suppressed β-hexosaminidase release and IL-4 mRNA expression in RBL-2H3 cells. is data suggests that quercitrin partially inhibits the differentiation of 0 cells into 1 cells and decreases IL-4 release in DNCB-stimulated mice. Cytokines produced by T cells, such as IL-4, IL-10, and IFN-c, play keys roles during the pathogenesis of CHS [4]. In particular, IL-4 is a pleiotropic cytokine of 2 cells that regulates immunoglobulin isotype switching to IgG4 and IgE [39]. In a previous study, it was suggested that total loss of endogenously produced IL-4 in BALB/c mice was closely associated with impairment of DNCB-induced CHS [35]. is result supports the importance of IL-4 as an immune response regulator during the early phase of skin CHS. We observed the productions and expressions of IL-4 and IgE were significantly increased in DNCB-sensitized mice in-line with CHS induction and that dermal application of quercitrin, a major constituent of W. indica, greatly reduced IL-4 expression, which suggested quercitrin suppresses 2 immunity in mice CHS.

Conclusion
Our study shows that quercitrin isolated from W. indica has a novel protective effect against CHS in the DNCB-induced SKH-1 hairless mouse model. Furthermore, quercitrin significantly attenuated DNP/IgE-induced β-hexosaminidase release and PI-induced IL-4 mRNA expression in RBL-2H3 cells. e inhibitory effects of quercitrin on IL-4 and IgE in CHS mice suggest that it might be useful for treating allergic skin disorders. Furthermore, the observed improvement of skin barrier function by quercitrin would seem to have considerable pharmacological potential for the treatment of contact dermatitis.   Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors report no conflicts of interest.