Atopic dermatitis (AD) is a chronic inflammatory skin disease that commonly occurs in children. The cause of AD is yet to be fully elucidated; however, the onset of AD is associated with genetic and environmental factors, skin architectural defects, and cell-mediated immune dysfunction [1
]. T-helper cell (Th2)-predominant inflammatory responses are believed to promote AD pathogenesis and immunoglobulin E (IgE)-mediated hypersensitivity [2
], and these are often associated with intractable chronic itchiness [4
]. The chronicity of itching and scratching is a common symptom of AD, which reduces the quality of life for patients.
Mast cells are multifunctional immune cells that link innate and adaptive immunity and play a major role in immunoglobulin E (IgE)-mediated hypersensitivity in AD [5
]. Activated mast cells produce various inflammatory mediators, including histamine; lipid mediators, such as prostaglandins; growth factors; cytokines; and chemokines, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-4, and IL-6, which are associated with the pathogenesis of AD [5
]. The number of mast cells increases in most patients with AD and skin lesions in mouse AD models, which implies that mast cells are involved in the incidence and severity of AD [6
The inflammatory chemokine C-C motif chemokine ligand 5 (CCL5), also known as regulated on activation, normal T cell expressed and secreted (RANTES), belongs to the C-C chemokine family and plays an active role in directing mast cells to inflammatory sites [7
]. CCL5 is overexpressed in the skin of patients with AD [8
], and CCL5 antagonism has shown therapeutic efficacy in models of contact skin inflammation [9
]. CCL5 expression is regulated in a cell-type- and stimulus-dependent manner by several transcription factors, including nuclear factor kappa B (NF-κB), Activator protein 1 (AP1), Nuclear factor for IL6 expression (NF-IL6), Specificity protein 1 (SP1), and Krüppel-like factor 13 [10
Chrysin (5,7-dihydroxyflavone, Figure 1
A) is a polyphenolic flavonoid compound that is abundant in honey, propolis, and carrots. It exhibits multiple biological properties, including anti-inflammatory and anticancer properties [12
]. Chrysin has been shown to inhibit allergen-induced [13
] and TNFα-induced NF-κB activity [14
] and alleviate AD-like skin lesions in a mouse model [15
]. However, the molecular mechanisms underlying the suppression of CCL5 expression and inhibition of NF-κB activity by chrysin remain unknown.
In this study, we found that chrysin is able to bind to the ATP-binding pocket of the inhibitor of κB (IκB) kinase (IKK), consequently inhibiting the IKK kinase activity and downregulating the NF-κB signaling pathway, thereby inhibiting the transcription of CCL5 at the gene promoter level.
3. Materials and Methods
Chrysin, 2,4-dinitrochlorobenzene (DNCB), toluidine blue (TB), and hematoxylin and eosin (H&E) staining kits were obtained from Sigma-Aldrich (St. Louis, MO, USA). Low-melting agarose was purchased from Lonza (Rockland, ME, USA). CCL5 was obtained from PeproTech (London, UK). TNFα was obtained from ProSpec-Tany TechnoGene, Ltd. (Ness-Ziona, Israel). A Firefly Luciferase Assay System was obtained from Promega (Madison, WI, USA). Anti-CCL5 antibody was obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA), and anti-IKKα/β, -IκB, -phospho-IκB (Ser32), -p65/RelA, and -phospho-p65/RelA (Ser536) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary antibody conjugated to rhodamine red-X was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
3.2. Cells and Cell Culture
Human keratinocyte HaCaT cells were obtained from Cell Lines Service (Eppelheim, Germany) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA).
3.3. Reverse Transcription-PCR (RT-PCR)
Total RNA was isolated using a TRIzol RNA extraction kit (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). RT-PCR was performed using a reverse transcriptase enzyme (Promega) and gene-specific PCR primers. The PCR primers and the thermal cycling conditions used in this study were as follows:
CCL5 forward, 5′-ACA GGT ACC ATG AAG GTC TC–3′,
CCL5 reverse, 5′–GCA AAT TTG TGT AAG TTC AGG–3,
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′–CCA AGG AGT AAG AAA CCC TGG AC–3′, and
GAPDH reverse, 5′–GGG CCG AGT TGG GAT AGG G–3′.
The PCR conditions were as follows: denaturation at 94 °C (5 min), followed by 30 cycles comprising denaturation at 94 °C (30 s), annealing at 58 °C (30 s), and elongation at 72 °C (1 min). The amplified products were electrophoresed in a 2% agarose gel containing ethidium bromide and observed under UV light.
3.4. Quantitative Real-Time PCR (Q-PCR)
The mRNA levels were quantified using an iCycler iQ system with an iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA). Validated commercial Q-PCR primers and SYBR Green-based fluorescent probes specific for CCL5 (id: qHsaCIP0028116) and GAPDH mRNA (id: qHsaCEP0041396) were obtained from Bio-Rad (Hercules, CA, USA). The thermal cycling conditions used for PCR were as follows: denaturation at 95 °C for 2 min, followed by 40 cycles performed using a step program (95 °C for 10 s and 60 °C for 45 s). The relative expression levels of CCL5 mRNA were normalized to those of GAPDH using the software provided by the manufacturer.
3.5. Agarose Spot Migration Assay
Chemotactic migration was analyzed in the agarose spot migration assay according to a process described previously [20
], with minor modifications introduced. Low-melting agarose (0.5%) in PBS was heated in a microwave and stirred for complete dissolution. When the agarose particles completely dissolved, the liquid agarose solution was cooled to 40 °C, followed by mixing with only PBS or PBS supplemented with CCL5 (final: 25 ng/mL) in a 1.5-mL Eppendorf tube. Next, 10-μL drops of agarose solution containing PBS or CCL5 were placed on each sterile glass coverslip in a 6-well plate using a cut pipette tip and were allowed to cool for 10 min at 4 °C to solidify the agar spots. A suspension of RBL2H3 cells in PBS was plated on the spot-containing coverslips and allowed to adhere for 4 h in an incubator at 37 °C. The cells were incubated overnight at 37 °C with a culture medium containing 0.1% fetal bovine serum. After 6 h, the motile cells that penetrated the agarose spot were analyzed under a microscope (EVOS FL Auto, Bothell, WA, USA).
3.6. Enzyme-Linked Immunosorbent Assay (ELISA)
CCL5 secreted in the culture medium was quantified using an ELISA kit (Biolegend, London, UK) according to the manufacturer’s instructions. In brief, the captured antibodies were coated on a Nunc C bottom immunoplate. The wells were washed three times with 50-mM Tris buffer (pH 8.0) containing 0.14-M NaCl and 0.05% Tween 20 (TBST). The collected culture supernatants and standard solutions were added to the wells, and the plates were incubated at 37 °C for 2 h. The wells were washed three times with TBST solution, 50 μL of biotin-conjugated anti-CCL5 antibody (1:200) was added, and the cultures were incubated at 37 °C for 1 h. After washing five times with TBST solution, horseradish peroxidase-conjugated tracer antibody was added to each well, and the cultures were incubated for an additional 1 h. An enzymatic reaction was initiated by adding tetramethylbenzidine substrate solution (containing 100-mM sodium acetate buffer (pH 6.0) and 0.006% H2O2), followed by incubation at 37 °C for 20 min. The reaction was terminated by adding an acidic solution (reaction stopper, 1-M H2SO4), and the absorbance was measured at 450 nm using an ELISA plate reader (SoftMax Pro; Molecular Devices, Sunnyvale, CA, USA). The final concentration of CCL5 was calculated using the standard curve.
3.7. Construction of Human CCL5 Promoter-Reporter Constructs
A CCL5 promoter fragment spanning nucleotides –1074 to +45 upstream of the transcription start site was synthesized from human genomic DNA (Promega, Madison, WI, USA) by PCR using the primers 5′-GAG GGC AAC TGG GTT CTG AT-3′ (forward –1074F) and 5′-GAG GTC CAC GTG CTG TCT TG-3′ (reverse, +45R). The amplified PCR products were ligated to a T&A vector (RBC Bioscience, Taipei County, Taiwan) and digested with KpnI and BglII. The products were ligated at the KpnI and BglII sites of the pGL4 basic vector (Promega), yielding pCCL5-Luc(–1074/+45). Several deletion constructs of the human CCL5 promoter fragments were synthesized by PCR using the pCCL5-Luc(–1074/+45) construct as a template plasmid. The forward primer sequences were 5′-TGA GTG TGC TCA CCT CCT TT-3′ (−500/+45) and 5′-TGT GCA ATT TCA CTT ATG AT-3′ (–100/+45). One reverse primer, +45R, was used to generate all the deletion constructs. The amplified PCR products were ligated into the T&A vector and then digested using KpnI and BglII. The products were ligated into the KpnI and BglII sites of the pGL4 basic vector. The insert sequence of each construct was verified by DNA sequencing (Macrogen, Seoul, Republic of Korea).
3.8. Luciferase Promoter-Reporter Assay
HaCaT cells were seeded onto 12-well plates and transfected with 0.1 µg of each CCL5 promoter-reporter construct using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. At 48 h post-transfection, the cells were treated with TNFα in the presence or absence of chrysin (20 and 40 μM). After 8–12 h, the cells were harvested, and the levels of firefly luciferase activity were measured using the Dual-GloTM Luciferase assay system (Promega). The relative level of luciferase activity in the untreated cells was designated 1. Luminescence was measured using a dual luminometer (Centro LB960; Berthold Tech, Bad Wildbad, Germany).
3.9. Immunoblot Analysis
HaCaT cells were lysed in ice-cold buffer containing 50-mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 500-mM NaCl, 1-mM ethylenediaminetetraacetic acid, 1 mM-Na3VO4, 1-mM NaF, 10-μg/mL leupeptin, and 1-mM phenylmethylsulfonyl fluoride. Proteins were separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After incubation with the appropriate primary and secondary antibodies, the blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA).
3.10. Molecular Docking
To elucidate the binding modes between chrysin and IKK1, in silico docking experiments were performed using the Sybyl 7.3 software (Tripos, St. Louis, MO, USA) built on an Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC. There are five three-dimensional (3D) structures of IKK1 deposited in the Protein Data Bank (PDB): 3brt, 5ebz, 5tqw, 5tqx, and 5tqy. One of them, 3brt.pdb, only includes residues between 732 and 745, owing to which, it could not be considered for constructing the 3D structure for use in the current study. The others include residues from 10–660. While the structures 5ebz, 5tqw, and 5tqx were determined by cryoEM, and their resolution was 5.60 Å, 5ebz.pdb was determined by X-ray crystallography, and its resolution was 4.50 Å. Therefore, 5ebz.pdb was selected for constructing the 3D structure in this study [21
]. Its gene originated in Homo sapiens
, and Spodoptera frugiperda
was used as the expression system. It consists of a trimer of IKK1 dimers; chain A (Gly10–Glu660) was selected for the docking experiment, and 2-azanyl-5-phenyl-3-(4-sulfamoylphenyl)benzamide was used as a reference ligand. The binding site was analyzed using the LigPlot program provided by the European Bioinformatics Institute (Cambridgeshire, UK) [22
]. The apo-protein without the ligand was prepared using Sybyl 7.3. The chrysin 3D structure deposited in PubChem (CID code 5281607, National Center for Biotechnology Information, Bethesda, MD, USA) was used after energy minimization using the molecular mechanics algorithms provided in Sybyl 7.3. All 3D images were constructed using PyMOL (The PyMOL Molecular Graphics System, Version 1.0r1, Schrödinger, LLC, New York, NY, USA).
BALB/c mice (7-week-old, male) were obtained from Orient Bio, Inc. (Seongnam, Korea). The mice were housed in a specific pathogen-free environment at a temperature of 20 ± 2 °C and relative humidity of 50% ± 10% and were maintained under a 12-h light-12-h dark cycle.
3.12. Induction of Atopic Dermatitis-like Skin Lesions in Mice
DNCB was dissolved in a 1:3 (v/v) mixture of acetone:olive oil. After the dorsal skin was shaved, BALB/c mice were randomly divided into three groups: group I, naïve, group II, DNCB + vehicle, and group III, DNCB + 8% chrysin (n = 5 each). All mice, except those in the naïve group, were treated with 4% SDS on the dorsal skin to disrupt the skin barrier. After 4 h, 100 μL of 1% DNCB was challenged once daily, and this was repeated for 3 days. After a 4-day rest period, a treatment with 100 μL of 4% SDS and 0.5% DNCB was repeated once daily, five times weekly, for 2 weeks (from days 8–21). Chrysin powder (250 mg) was dissolved in 1 mL of dimethyl sulfoxide to prepare a stock solution. Group III mice were applied with chrysin (25 mg/kg) from day 7 (once daily, five times weekly for 2 weeks). The animal experiments were conducted according to the guidelines for animal experiments and procedures approved by the Konkuk University Institutional Animal Care and Use Committee (IACUC, Seoul, Republic of Korea), and all experimental methods were confirmed to be in accordance with the relevant guidelines and regulations (approval number KU19129).
3.13. Tissue Preparation And Histopathological Analysis
Dorsal skin tissues of mice were removed, fixed with 100% acetone solution, and embedded in paraffin. Skin sections 5 μm in thickness were cut using a microtome (Leica Microsystems, Wetzlar, Germany). After dewaxing, the sections were stained with H&E. Images of each section were acquired using a light microscope (EVOS FL Auto, Bothell, WA, USA), and the epidermal and dermal thicknesses were measured from the digital images using ImageJ 1.52a (National Institutes of Health, Bethesda, MD, USA). The infiltrating mast cells were stained with 0.1% TB. The number of TB-positive cells in 2.5 cm2 was counted.
3.14. Immunohistochemical and Immunofluorescence Stainings
Paraffin-embedded dorsal skin sections were deparaffinized using xylene and rehydrated by treating with a graded ethanol series. Skin specimens were treated with hydrogen peroxide for 15 min to block endogenous peroxidase activity, followed by immersion in 1-mM EDTA (pH 8.0) at 70 °C for 20 min. After rinsing with PBS, the sections were placed in a blocking buffer containing 7% goat serum for 1 h and incubated with primary anti-p65/RelA antibody overnight at 4 °C. After washing with PBS, the sections were incubated with biotinylated anti-goat IgG secondary antibodies for 1 h and then incubated with an avidin/biotin complex for 30 min. Skin sections were stained with 3,3′-diaminobenzidine tetrahydrochloride for 5 min and counterstained with H&E.
For the fluorescent immunohistochemical analysis, each skin section was treated with a CCL5 antibody (1:100 dilution), followed by overnight incubation at 4 °C. After washing with PBS, the sections were incubated with rhodamine red-X-conjugated secondary antibody (1:300 dilution) at room temperature for 1 h. Nuclei were stained with Hoechst 33258 for 10 min. After washing with PBS, the sections were mounted on slides using a fluorescence mounting medium (ProLong Gold Antifade Reagent; Invitrogen). The fluorescence images were evaluated using an EVOS FL fluorescence microscope (Advanced Microscopy Group, Bothell, WA, USA).
3.15. Measurement of Serum IgE Levels
Mouse blood samples were collected before sacrifice, and the serum IgE levels were measured using an ELISA MAX™ Standard Set Mouse IgE Kit (BioLegend, San Diego, CA, USA), as described previously [23
]. Color development was quantified by measuring absorbance at 450 nm using an ELISA plate reader (SoftMax Pro; Molecular Devices, Sunnyvale, CA, USA).
3.16. Statistical Analysis
Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s or Sidak’s multiple comparisons tests using GraphPad Prism version 8.4.2 (GraphPad Software, Inc., La Jolla, CA, USA). In all analyses, a p-value < 0.05 was considered to indicate statistically significant differences.
The transcription factor NF-κB is a major regulator of inflammation that is responsible for the expression of multiple inflammatory cytokines and chemokines [24
]. The NF-κB family consists of five members: c-Rel, p65/RelA, RelB, p50/NF-κB1, and p52/NF-κB2 and mediates the transcription of various target genes as various homo- or heterodimers [25
]. Of these, p65/RelA is the most abundant form. In the resting state, NF-κB is localized in the cytoplasm and remains in an inactive form by binding to the inhibitor of κB (IκB) protein. This interaction prevents the translocation of NF-κB to the nucleus. Following cellular activation, IKK is activated, which consequently phosphorylates IκB, leading to the dissociation of IκB from p65 NF-κB and the eventual activation of NF-κB [26
]. The activated NF-κB complex is then translocated to the nucleus, where it binds to the NF-κB-binding sites in the promoter region of the regulated genes [28
]. Therefore, the phosphorylation of IκB by IKK is critical for the initiation of NF-κB activation [29
In this study, we observed that chrysin suppresses CCL5 expression at the transcriptional level by suppressing NF-κB in the inflammatory environment. Using an in silico molecular docking experiment, we predicted that chrysin could bind to the ATP-binding pocket of IKK, which prevents IκB degradation and NF-κB activation. The clinical efficacy of chrysin in targeting IKK was evaluated in DNCB-challenged skin lesions in BALB/c mice. It suppresses CCL5 expression, reduces the infiltration of mast cells into the inflammatory sites, and at least partially alleviates the inflammatory response of inflamed skin challenged with DNCB. Based on these findings, we suggest that chrysin might serve as an IKK inhibitor for the treatment of chronic inflammatory diseases, such as AD. To directly prove the binding between chrysin and IKK, more detailed studies, such as precipitation experiments using agarose-coupled chrysin and surface plasmon resonance experiments, are needed.
In conclusion, this study first demonstrated that chrysin inhibits NF-κB-dependent CCL5 expression by directly targeting IKK in the AD-like skin inflammatory microenvironment. Our findings contribute to a better understanding of the mechanistic insights into the biological effects of chrysin on anti-inflammatory activity.