Resveratrol Exerts Therapeutic Effects on Mice With Atopic Dermatitis

Introduction. Atopic dermatitis (AD) is a chronic inflammatory skin disease with a high prevalence in children worldwide. Resveratrol exerts various pharmacologic effects, and application of resveratrol has been suggested as an alternative treatment for microorganism infection and skin pathologies. Objective. The present study examines the effect of resveratrol on AD using an in vivo murine model. Materials and Methods. Atopic dermatitis was induced in 30 BALB/c mice by topical application of 2,4-dinitrochlorobenzene (DNCB) prior to treatment with 0 mg/kg, 5 mg/kg, or 25 mg/kg resveratrol. Histologic data changes were evaluated, and the levels of thymus- and activation-regulated chemokine; type 2 helper T cytokines interleukin (IL) 4, IL-5, and IL-13; and type 1 helper T cytokines IL-12 and interferon γ were examined by enzyme-linked immunosorbent assay. Messenger ribonucleic acid expression was evaluated with quantitative polymerase chain reaction. Filaggrin (FLG), envoplakin (EVPL), transglutaminase (TG), and kallikrein 7 (KLK7) protein expression were evaluated with Western blot. Results. Resveratrol ameliorated the onset of AD-like skin lesions and significantly improved the DNCB-induced dermal destruction in mice. In addition, resveratrol reduced the levels of the above chemokines, downregulated the expression of the proinflammatory cytokine KLK7, and upregulated the expression of several cytokines, such as EVPL, FLG, and TG. Conclusions. These results suggest resveratrol has therapeutic effects in the treatment of AD.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 15% to 30% of children worldwide.¹ It encompasses immunologic responses, susceptibility factors, and compromised skin-barrier function as a part of the atopic triad of eczema, bronchial asthma, and allergic rhinoconjunctivitis, with a higher incidence in Western or developed nations.¹ Patients with AD exhibit characteristic dry skin with pruritus and associated inflammatory skin changes. Atopic dermatitis exerts negative effects on a child’s quality of life, including physical impairment, psychological distress, and psychosocial problems, and the disease also poses a burden to the well-being of patients and their families.² Current treatments for AD include antibiotics, corticosteroids, immunomodulators, immunosuppressive agents, skin care, and lifestyle changes.³ However, current therapy is not effective, and finding an ideal therapy for AD is necessary to improve the prognosis of patients and minimize the burden to patients and their families. 

Resveratrol is a naturally occurring polyphenol and phytoalexin found in various types of fruits and vegetables, most notably in the skin of red grapes. Several studies have indicated that it exerts various pharmacologic effects, such as anticancer, antioxidant, antiangiogenic, and anti-inflammatory properties.^4,5 Resveratrol is synthesized in plant cells when stress or nutrient depletion occurs.⁶ It has been reported that resveratrol inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin-cancer model.⁷ In addition, resveratrol has been reported to have cancer chemopreventive activity in assays representing 3 major stages of carcinogenesis, and it has potent cancer chemoprotective activity with remarkable ability to inhibit COX1 and COX2.^7,8 It has been reported that resveratrol mediates anti-inflammatory effects, inhibits cyclooxygenase and hydroperoxidase functions (antipromotion activity), exerts antioxidant and antimutagen effects, induces phase II drug-metabolizing enzymes (anti-initiation activity), and induces human promyelocytic leukemia cell differentiation (antiprogression activity).^7,9 The study reported herein evaluates the effectiveness of resveratrol in the treatment of symptoms of mice with AD induced by DNCB. 

The animal experiments were approved by the Animal Care and Use Committee of Fudan University (Shanghai, China). 

Induction of AD-like skin lesions in DNCB-treated mice
Male BALB/c mice (6 weeks old, weighing 18g-22 g; N = 30, n = 6 per group) were purchased from Shanghai Animal Center (Shanghai, China). All mice were housed in an air-conditioned room at a temperature of 23°C ± 2°C, and food and water were supplied ad libitum. After a 2-week adaptation period, the mice were randomized into 5 groups: the normal group (normal animals without DNCB application), the blank group (AD-like skin lesions induced by DNCB), the 0 mg/kg resveratrol group (AD-like skin lesions treated by dimethyl sulfoxide [DMSO]), the 5 mg/kg resveratrol group (AD-like skin lesions treated by resveratrol at 5 mg/kg/day), and the 25 mg/kg resveratrol group (AD-like skin lesions treated by resveratrol at 25 mg/kg/day).

Atopic dermatitis was induced according to previously published methods.^10,11 In the first week, 100 mL of 0.5% DNCB (dissolved in a mixture of acetone and olive oil at a ratio of 4:1) was applied to the shaved backs of mice. Then, 100 mL of 0.2% DNCB was applied twice per week for 4 weeks, and AD-like lesions developed at the end of the fifth week. During the sixth week, DNCB was applied once to maintain inflammation. Resveratrol (dissolved in DMSO) was given at different doses (0 mg/kg resveratrol, 5 mg/kg resveratrol, 25 mg/kg resveratrol) according to assigned experimental group for 7 days during the sixth week. All drugs were administered via the orogastric route. Animals were euthanized by carbon dioxide inhalation at the end of week 6, and dorsal skin tissues were obtained for further analysis.

Measurement of skin histologic changes
Skin thickness, water content, and moisture loss were evaluated. Dorsal skin samples were fixed in 10% buffered neutral formaldehyde for 24 hours and embedded in paraffin wax. These 5-µm-thick sections of dorsal skin were stained with hematoxylin and eosin and observed by optical microscopy. Epidermis and dermis thickness were measured.

A skin moisture tester (TP-S806; Shenzhen City Topping Technology Co. Ltd, Shenzhen, China) was used to measure the skin moisture content.¹² A Tewameter TM 300 (Courage + Khazaka Electronic, Cologne, Germany) was used to measure skin moisture loss as per previously published methods.¹³ Each test was performed 5 times, and all measurements were taken in the same conditions.

Enzyme-linked immunosorbent assay
Mouse enzyme-linked immunosorbent assay kits (ELISA; BD Biosciences, Singapore) were used according to manufacturer instructions to detect the cytokines in the dorsal skin samples. An amount of sample (0.1 g) in each group was weighed and homogenized in 1 mL of T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA). Protein concentrations were quantified using a Protein Assay Reagent (Bio-Rad, Hercules, CA). The quantitation was performed with a BioTek EL808 ELISA plate reader (BioTek Instruments; Winooski, VT) at 450 nm, and 6 samples in each group were measured.

Quantitative real-time PCR
Total ribonucleic acid (RNA) in the tissue was isolated using the GenElute Mammalian Total RNA Kit (Sigma-Aldrich, St Louis, MO) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) according to instructions. Complementary deoxyribonucleic acid (cDNA) was amplified with the IQ SYBR Green Supermix (Bio-Rad) using the MiniOpticon Real-Time PCR Detection System (Bio-Rad). Fold change in gene expression was obtained by quantitative real-time polymerase chain reaction (PCR) using the 2^−ΔΔCt method and were normalized to β-actin. The primer sets are shown in the Table.

Western blot analysis
Western blot was performed to assess protein expression, including filaggrin (FLG), envoplakin (EVPL), transglutaminase (TG), and kallikrein 7 (KLK7), as described previously.¹⁴ Skin tissues were homogenized in radioimmunoprecipitation assay buffer containing the protease inhibitor cocktail and then centrifuged at 12 000 r/m for 10 minutes at 4°C. The supernatant was used to quantify protein concentrations. The protein was denatured and separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with primary antibodies; after incubation, the membranes were washed with tris-buffered saline and polysorbate 3 times for 5 minutes each wash and followed by a secondary antibody incubation for 1 hour at room temperature. The proteins were visualized using an enhanced chemiluminescence with a commercially available enhanced chemiluminescence kit (Thermo Fisher Scientific). The quantification of Western blot bands was performed using ImageJ software, and the relative protein expression was normalized to β-actin.

Effects of resveratrol on the skin of AD mice
The changes in skin thickness, moisture content, and skin moisture loss were measured to evaluate the effects of resveratrol on the skin of AD mice. As shown in Figure 1, the skin lesions of mice in the blank and DMSO groups presented with hyperplasia and hyperkeratosis compared with the normal group (P < .05). Resveratrol suppressed the increased thickness of the epidermis and dermis induced by DNCB significantly compared with the blank and DMSO groups (P < .05). 

There were no significant differences between the groups (blank and DMSO) in terms of skin thickness, moisture content, and skin moisture loss; however, the values of skin thickness and moisture content in resveratrol groups were higher than that of the control groups (blank and DMSO) (P < .05). The skin moisture loss in the resveratrol groups was lower than that in the control groups (P < .05), indicating the application of resveratrol ameliorates DNCB-induced AD-like lesions.

Inhibitory effects of resveratrol on cytokines
Several cytokine levels were determined to evaluate the effect of resveratrol on cytokine production. As shown in Figure 2, DNCB treatment increased cytokine concentration, such as thymus- and activation-regulated chemokine (TARC); type 2 helper T (T_H2) cytokines interleukin (IL) 4, IL-5, and IL-13; and type 1 helper T (T_H1) cytokines IL-12 and interferon (IFN) γ in the skin lesions, while resveratrol treatments significantly decreased the level of those cytokine compared with DNCB-induced mice (P < .05; Figure 2).

Quantitative real-time PCR
To determine whether resveratrol exerted therapeutic effects on AD, quantitative real-time PCR was used to evaluate the efficacy of resveratrol on the mRNA expressions of some related genes: FLG, EVPL, TG, and KLK7. As shown in Figure 3, DNCB decreased the mRNA expression of FLG, EVPL, and TG significantly, while resveratrol treatment significantly increased the gene levels of mRNA expression compared with the control groups. 

Western blot analysis
Western blot analysis was used to assess the effect of resveratrol of the related protein expression of AD mice. As shown in Figure 4, resveratrol treatment significantly increased the protein expression of FLG, EVPL, and TG and decreased KLK7 expression, compared with the control groups. 

Human skin diseases are numerous, especially for chronic inflammatory disorders, which include psoriasis, AD, and certain forms of ichthyosis. Atopic dermatitis is a chronic inflammatory skin disease characterized by intense pruritus and a waxing and waning course, which often presents in infancy and childhood and can persist throughout adulthood.¹⁵ The disease imposes burdens on patients, such as infection, behavioral problems, sleep disturbance, and growth impairment, and it poses a burden to patients’ families as well as health care systems. Treatment can be effective in alleviating symptoms of AD. Resveratrol has been exploited as an ideal chemopreventive agent for a variety of skin disorders, as it has been reported to exert anti-inflammatory, antioxidant, and immunomodulatory effects.¹⁶ Lee et al¹⁷ showed resveratrol inhibits the protein kinase B pathway by inducing Sirt1, leading to cell death, and they indicated resveratrol-mediated activation of Sirt1 histone deacetylase may be a potential therapeutic target for skin diseases, including psoriasis. Farris et al¹⁸ reported that a topical cream containing retinol 0.5% in combination with niacinamide 4.4%, resveratrol 1%, and hexylresorcinol 1.1% in a moisturizing base is efficacious and tolerable for skin brightening and antiaging when used with a complementary skin care regimen including sun protection factor 30. These results suggest resveratrol plays a positive role in skin disorders or diseases. In the study reported herein, the authors investigated the therapeutic effects of resveratrol in an murine model of AD.

In the current study, DNCB, an electrophilic and cytotoxic benzene derivative, was used to induce stable clinical AD-like skin lesions in BALB/c mice in order to closely mimic human AD. After 5 weeks of exposure to DNCB, BALB/c mice presented with erythema, lichenification with edema, and skin hemorrhaging. To assess the effect of resveratrol on these skin lesions, histologic examination was performed. Increased skin thickness is an obvious and histologically visible symptom of the skin lesion, which may be caused by hyperplasia, lichenification, disturbed epidermal differentiation, or increased proliferation. In this study, compared with the control group, the thickness of epidermis and dermis in the experimental groups decreased after the application of resveratrol, indicating resveratrol might suppress the hyperplasia and abnormal epidermal differentiation.

Skin moisture content and transepidermal water loss (TEWL), another index of epidermal barrier function, were determined in this experiment to evaluate the therapeutic effect of resveratrol on skin lesions. Matsumoto et al¹⁹ found patients with AD who have dry skin exhibit higher TEWL values than those without AD. The skin exposed to DNCB decreased in skin water content, and there was more skin water content and decreased TEWL in the resveratrol group than in the control group.

In addition, the authors found resveratrol treatment decreased levels of the chemokine TARC; T_H2 cytokines IL-4, IL-5, and IL-13; and T_H1 cytokines IL-12 and IFN-γ in the skin lesions. Serum TARC levels are associated with disease activity in patients with AD and sensitively reflect short-term changes in skin conditions.²⁰ The T_H2 cells have been reported to drive the cutaneous inflammation AD process and T_H2-related molecules, such as IL-4 and IL-13, dominate the immune infiltrate, and the levels of T_H1-associated factors IL-12 and IFN-γ increased with AD severity.²¹ The improvements in the current AD murine model may be associated with a reduction of the aforementioned chemokines. 

An impaired epidermal barrier allows for enhanced penetration of external antigens and readily induces skin inflammation. Filaggrin and its metabolites are key components in maintaining normal skin-barrier function, and it has been found to be reduced in the skin of patients with AD.^22,23 Plakins form vital connections between cell junctions and the cytoskeleton. The plakin protein EVPL is highly expressed in epithelial cells of the skin, and it plays a vital role in initiating cornified envelope formation, which is critical for epidermal permeability and barrier function.²⁴ Transglutaminase is a calcium-dependent enzyme that catalyzes an intermolecular isopeptide bond formation between proteins. The extensive cross-linking activity of TG during terminal epidermal differentiation contributes to skin-barrier function formation, and disorder of TG activity leads to an irregular phenotype in the skin.^25,26 Kallikrein 7 is differentially expressed in lesion biopsy specimens from patients with atopic eczema relative to normal skin, and the polymorphism of the KLK7 gene is reported to result in a gain of function of the protease that causes premature degradation of corneodesmosomes and consequently defective skin-barrier function.²⁷ In this study, the authors found that resveratrol treatment significantly increased the mRNA and protein expression of FLG, EVPL, and TG, which play a critical role in skin-barrier health,²⁸ and decreased KLK7 expression compared with the control groups, indicating resveratrol treatment improved skin-barrier function.²⁹ In addition, the inflammatory response is a normal part of the wound healing process, yet excessive inflammation may be unfavorable to subsequent wound healing, so the anti-inflammatory effects of resveratrol may have a positive role in regulating wound healing.

Poor bioavailability of resveratrol is a potential limitation for resveratrol treatment. Also, the exact mechanism of the therapeutic activity of resveratrol on mice with AD remains to be further determined.

The authors reported that resveratrol ameliorated the onset of AD-like skin lesions and improved the DNCB-induced dermal destruction in mice significantly, including increasing skin thickness, reducing skin moisture loss, and maintaining the water content of the stratum corneum. In addition, resveratrol reduced levels of chemokines. The present results suggest resveratrol has therapeutic effects in the treatment of AD. 

Authors: Yanyun Shen, MD; and Jinhua Xu, MD

Affiliation: Hospital, Fudan University, Shanghai, China

Correspondence: Jinhua Xu, PhD, Department of Dermatology, Huashan Hospital, Fudan University, 12 Central Urumqi Road, Shanghai 200040, China; yanyunshen@126.com; drjinhuaxu@126.com

Disclosure: The authors disclose no financial or other conflicts of interest.