Atopic dermatitis, also known as atopic eczema, is an inflammatory skin disorder that is characterized by intense pruritus, scratching, and cutaneous hypersensitivity to allergens [1
]. It is well established that genetic predisposition is a strong risk factor for the development of atopic dermatitis. Therefore, patients with atopic dermatitis often carry a personal or familial history of other allergic diseases such as asthma and allergic rhinitis, and many of them possess the genetic mutations in the genes associated with defective epidermal differentiation and skin barrier formation [2
]. In addition, preferential T helper 2 (Th2) cell-mediated pathway activation [3
], neuroimmune interactions [4
], and microbial pathogens [5
] trigger the development of atopic dermatitis. A hallmark of atopic dermatitis is a dry, itchy, and cracked skin caused by defects in the barrier function of keratinocytes, and current treatment for atopic dermatitis includes topical application of moisturizers, anti-inflammatory agents, and phototherapy [6
]. A number of experimental mouse models recapitulating atopic dermatitis have been developed and they are largely categorized into three groups [7
]: (i) mouse models that develop atopic dermatitis by cutaneous application of sensitizers, (ii) genetically modified mice that overexpress or lack selective molecules, and (iii) mouse models that spontaneously develop atopic dermatitis-like skin lesions.
Oxidative stress is defined as the formation of oxidants that exceeds the antioxidant defense capacity in cells [8
]. It is known that oxidative stress promotes inflammation in the skin by upregulating the pro-inflammatory genes, contributing to the pathogenesis of atopic dermatitis [9
]. NF-E2-related factor 2 (NRF2) is a redox-sensitive transcription factor, which is responsible for the induction of phase II cytoprotective enzymes and the detoxification of reactive oxygen species (ROS) by binding to the antioxidant response element (ARE), a cis
-DNA element existing in the promoter of phase II cytoprotective enzymes [10
]. Under basal condition, NRF2 is constantly polyubiqutinated in the cytosol by Kelch-like ECH-associated protein 1 (KEAP1), an adaptor for Cullin 3 (CUL3) E3 ubiquitin ligase. Exposure of oxidants and electrophiles inactivates KEAP1, allows NRF2 to translocate into the nucleus, and activates the expression of NRF2 target genes. Previous studies have demonstrated that NRF2 activators are useful for treatment of various oxidative stress-related diseases, and a number of NRF2 chemical activators are currently undergoing clinical trials for treatment of multiple sclerosis, cardiovascular disease, diabetes mellitus, and psoriasis [11
]. However, whether and, if so, how NRF2 activation could effective against atopic dermatitis is still elusive.
Polyphenols are one of the most abundant ingredients found in our diets and chalcones represent an important group in the polyphenolic family compounds [12
]. Cardamonin (Figure 1
A) is a naturally-occurring compound with a hydroxylated chalcone structure. The production of cardamonin in plants starts with the deamination of phenylalanine into cinnamic acid, which undergoes additional transformation into hydroxylated chalcone via condensation with malonyl-CoA [13
]. Previous studies have demonstrated that cardamonin possesses a number of beneficial pharmacological effects, such as anti-inflammatory, anti-neoplastic, vasodilative and anti-infectious effects [14
]. In the present study, we have identified that cardamonin inhibits oxazolone-induced atopic dermatitis in vivo by inhibiting the production of Th2 cytokines and subsequent oxidative damages through the induction of NRF2.
2. Materials and Methods
2.1. Cell Culture, Chemicals, and Reagents
Oxazolone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cardamonin was purchased from TOCRIS bioscience (Bristol, UK). Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin/streptomycin (Pen/Strep) were purchased from Welgene (Daegu, Korea). Polyclonal antibodies against NRF2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies against total actin and 8-hydroxydeoxyguanosine (8-OH-dG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies against 4-hydroxynonenal (4-HNE) were purchased from Abcam (Cambridge, MA, USA). Human keratinocyte HaCaT cells were acquired from American Type Culture Collection (Manassas, VA, USA).
2.2. Examination of the Effect of Cardamonin on Oxazolone-Induced Atopic Dermatitis
The animal experiment was carried under the Institutional Animal Care and Use Committee-approved Protocol (IACUC-2019-002-1) of Dongguk University (Seoul, Korea). Six-week-old Balb/c mice were purchased from Daehan Biolink (Eumseong, Korea), housed in sterile filter-capped microisolator cages, and provided with water and diet ad libitum. After a week of acclimation, 30 mice were distributed to control (n
= 8, Group 1), oxazolone (n
= 11, Group 2), and oxazolone + cardamonin (n
= 11, Group 3) groups (Figure 1
B). After distribution, mice were topically applied with oxazolone alone or in combination with cardamonin on to the ear, and the thickness of the ear was measured by a caliper during the course of experiment. At sacrifice, tissues were excised, weighed, and stored in a deep freezer for biochemical analysis or in 10% formalin solution for immunohistochemistry.
2.3. Tissue Dehydration and Paraffin Embedding
At sacrifice, the mouse ears were fixed in 10% formalin solution overnight. Tissue dehydration was performed by serially immersing the tissues into 75%, 80%, 85%, 90%, 95%, and 100% ethanol and xylene solution for 1 h at each step. Dehydrated tissues were embedded in the paraffin block.
2.4. Hematoxylin & Eosin (H&E) Staining
Paraffin-embedded tissues were sectioned at 5 μm, mounted on the slide, and deparaffinized. Tissues were stained with Mayer’s hematoxylin solution for 5 min at room temperature, then rinsed in tap water until the water becomes clear. In the bluing step, the tissues were stained with repeated cycles of eosin Y ethanol solution for 70 sec, 5 dips in 95% ethanol, and 5 dips in 100% ethanol at room temperature. The tissues were rinsed with distilled water and the images were taken on the microscope (Olympus, Tokyo, Japan).
2.5. Preparation of Primary Mouse Embryonic Fibroblasts (MEFs)
Nrf2 (−/−) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and bred in the animal facility of Dongguk University. Primary Nrf2 (+/+) and Nrf2 (−/−) mouse embryonic fibroblasts (MEFs) were generated from E12.5 embryos and the genotype was confirmed by PCR analysis. Primary MEFs separated from embryos were cultured in DMEM media containing 10% heat-inactivated FBS and 1× Pen/Strep at 37 °C in humidified 5% CO2 incubator.
2.6. Masson’s Trichrome Staining
Paraffin-embedded tissues were sectioned at 5 μm, mounted on the slide, and deparaffinized. Tissues were stained with Masson’s trichrome reagents as recommended by the manufacturer (Labcore, Seoul, Korea) and the images were taken on the microscope (Olympus, Tokyo, Japan).
2.7. Immunohistochemistry Staining with DAB
Tissues on the slide were incubated with 1% bovine serum albumin (BSA) blocking solution for 30 min. After washing three times with 1× PBS, the tissues were hybridized with primary antibodies overnight at 4 °C. The slides were washed with 1× PBS three times and incubated with anti-rabbit and anti-mouse UltraTEk HRP antibodies (ScyTek Inc., Logan, UT, USA). Development of the slides was performed with 3,3′-diaminobenzidine (DAB) (GBI Labs, Bothell, WA, USA). The slides were then sealed with mounting medium and the images were taken on the microscope (Olympus, Tokyo, Japan).
2.8. Measurement of ARE-Luciferase Activity in HaCaT-ARE-Luciferase Cells
Establishment of human keratinocyte HaCaT-ARE-GFP-luciferase cells was previously described [15
]. Established HaCaT-ARE-luciferase cells were seeded in six-well plates, cultured until 70% confluence, and exposed to cardamonin. Sulforaphane was used as a positive control. After 24 h, cells were lysed with a luciferase lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA) and the resulting luciferase activity was measured by the GLOMAX Multi-system (Promega, Madison, WI, USA). The data is depicted as a fold ratio of the firefly luciferase activity, compared with the control after normalization with protein concentration.
2.9. Western Blot Analysis
Tissues were ground by pestle and incubated with 200 μL RIPA buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors cocktail) for 1 h on ice. Lysates were collected by centrifugation and protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher, Pittsburgh, PA, USA). Equal amounts of lysates were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was incubated in blocking buffer (5% skim milk in 1× PBS-0.1% Tween-20, PBST) for 1 h and hybridized with the appropriate primary antibodies in 1× PBS containing 3% BSA overnight at 4 °C. After washing three times with 1× PBST for 30 min, the membrane was hybridized with appropriate HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature and washed three times with 1× PBST solution for 30 min. The membrane was visualized using an enhanced chemiluminescence (ECL) detection system. A β-actin blot was used as the control for equal loading of samples.
2.10. Real-Time RT-PCR Assay
Total RNA was isolated using a Hybrid-R RNA extraction kit (GeneAll, Seoul, Korea). Total RNA (1 μg) was subject to cDNA synthesis, using PrimeScript RT-PCR kit (TaKaRa Korea, Seoul, Korea). Real-time PCR was performed on a CFX96 instrument (Bio-Rad, Hercules, CA, USA) using EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). The primer sequences used for the quantitation of target genes are illustrated in Table 1
. GAPDH was used as an internal control.
2.11. Statistical Analysis
Statistical analysis was conducted using Student’s t-tests. Asterisks indicate a statistical significance of * p < 0.05, ** p < 0.01, and *** p < 0.001.
We have demonstrated that cardamonin suppresses oxazolone-induced atopic dermatitis in vivo (Figure 1
). Suppression of oxazolone-induced atopic dermatitis by cardamonin was associated with maintaining the integrity of the connective tissues (Figure 2
C) and inhibiting the production of Th2 cytokines in the skin (Figure 3
). We also provided evidence that NRF2 plays a significant role in suppressing oxazolone-induced production of Th2 cytokines (Figure 3
). Because production of various Th2 cytokines stimulates B cell proliferation, immunoglobulin class-switching to immunoglobulin E (IgE), and macrophage polarization to an M2-like phenotype [28
], suppressing the production of Th2 cytokines by NRF2 could explain, at least in part, how NRF2 modulates the immune system to exert anti-inflammatory responses (Figure 1
C). However, the detailed molecular mechanisms underlying how NRF2 affects Th2 cells to attenuate oxazolone-induced production of Th2 cytokines are largely unclear. In addition, we observed that topical administration of oxazolone onto the ear of mice caused splenomegaly (Figure 2
B). Considering that Nrf2 (−/−) mice phenotypically exhibit hypertrophy in the spleen [29
], it is possible to assume that NRF2 in the spleen might be implicated in allergic responses to oxazolone in keratinocytes. This hypothesis harmonizes with the observation that oxazolone caused a significant accumulation of mast cells in mouse skin (Figure 2
Cardamonin stimulates ARE-dependent gene expression (Figure 5
A) in HaCaT-ARE-GFP-luciferase cells, and significantly induces the expression of NRF2 (Figure 5
B) and its target genes (Figure 5
C) in mouse skin. However, the molecular mechanisms underlying the induction of NRF2 by cardamonin remain elusive. One possibility is that the activation of the intracellular kinase pathways by cardamonin such as mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3′-kinase (PI3K) might be responsible for the activation or induction of NRF2 [30
]. Alternatively, it can be assumed that the induction of NRF2 by cardamonin occurs via the blockade of poly-ubiquitination of NRF2. Indeed, many NRF2 inducers are known to be Michael acceptors that form direct adducts with cysteine residues in KEAP1 [31
]. In view of the structure, cardamonin can be classified as a Michael acceptor because it possesses the α,β-unsaturated lactone moiety (Figure 1
A). In addition, the role of E3 ubiquitin ligases other than CUL3/KEAP1 on the induction of NRF2 by cardamonin also merits further exploration, considering that there are multiple E3 ubiquitin ligases that target NRF2 for proteolysis [32