Studies have been directed to discover bioactive ingredients with physiological activities such as phytochemicals from various natural resources. Natural substances exert fewer side-effects than conventional synthetic materials [1
], and can effectively inhibit the overproduction of reactive oxygen species (ROS) such as unstable and highly reactive free radicals, thereby preventing mutation and cytotoxicity [2
]. Oxidative stress has been reported to promote diseases or aging by inducing damage to cells and tissues. Hence, increasing attention has been diverted to the development of products that reduce oxidative stress [3
]. In this direction, potent bioactive substances have been discovered from plants and have been actively applied to functional foods, pharmaceuticals, and cosmetics [6
Inflammation caused by infections or tissue injury is associated with various human diseases. Neutrophils produce ROS, reactive nitrogen species (RNS), and nitric oxide (NO), all of which are inflammation-inducing agents [8
]. The incidence of atopic dermatitis (AD), of all the skin diseases, is gradually increasing. AD is caused by intrinsic and extrinsic factors related to the skin. Acute or chronic skin damage allows easy access of various allergens to the skin, resulting in an increase in allergic inflammatory responses [9
]. Among various inflammatory mediators, interleukin (IL)-4 and IL-13 produced following the activation of type-2 helper T cells (Th2) are known to be overexpressed and induce immunoglobulin E (IgE) production in patients with allergic dermatitis and AD [12
]. Cells that play the most important role in inducing allergic reactions are mast cells and basophils, which mediate allergic reactions by secreting β-hexosaminidase. IgE binds to the IgE-binding subunit of the high-affinity IgE receptor (FcεRI), a heterotetrameric receptor (one α, one β, and two γ subunits), on mast cells and basophils and promotes degranulation and cytokine secretion, leading to an allergic reaction [15
]. IgE binding to the α-subunit results in the activation of the β- and γ-subunits of the FcεRI, consequently recruiting Lyn and Syk and inducing phosphorylation of protein tyrosine kinases (PTKs). Activated Syk is shown to be involved in the phosphorylation and activation of phospholipase C (PLC)-γ [15
]. Mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 are also activated by FcεRI-IgE crosslinking. Phosphorylation and activation of these MAPKs mediate the expression of tumor necrosis factor-α (TNF-α) and IL-2 [18
]. Macrophages play an important role in immune response and regulate various inflammatory mediators such as NO, prostaglandin (PG), and preinflammatory cytokines [23
]. In mammals, there are three types of nitric oxide synthases (NOS), of which type III inducible NOS (iNOS) is expressed only in response to stimuli such as lipopolysaccharide (LPS), cytokines, and bacterial toxins in some cells [24
]. NO is mainly produced by iNOS and promotes inflammatory responses by inducing the expression of inflammatory mediators [26
]. Another inflammatory factor, cyclooxygenase (COX), is an enzyme that converts arachidonic acid into PG. There are two types of COX, COX-1, and COX-2, that exhibit different expression patterns in various cells. COX-2 is primarily expressed during the inflammatory response and induces the production of prostaglandin E2 (PGE2), an inflammatory mediator associated with pain and fever [27
]. In addition, one of the cytokines, TNF-α, plays an important role in the induction of inflammatory reactions through the activation of T cells and macrophages and enhancement of other pro-inflammatory cytokines [28
], leading to an inflammatory response. IL-6 is also an important inflammatory factor secreted by macrophages upon LPS exposure [29
The skin comprises the epidermis, dermis, and subcutaneous tissue (hypodermis) and acts as a barrier to maintain moisture. It prevents the invasion of external infectious agents through the production of antimicrobial peptides [30
]. In particular, keratinocytes constituting the epidermis produce involucrin, loricrin, and filaggrin to aggregate keratin filaments and form a cornified cell envelope [32
]. In addition, hyaluronic acid (HA) synthesized by hyaluronic acid synthase (HAS) in keratinocytes and fibroblasts also functions as a moisturizing barrier in the skin [33
]. Aquaporins (AQPs) are small hydrophobic integral membrane proteins that regulate the water retention rate of skin and other organs. To date, 13 types of AQPs have been identified in mammals [34
], of which AQP3 is involved in the transport of water and glycerol. Human epidermal keratinocytes express AQP3 on their membranes [35
]. In mice lacking AQP3 expression, the water transport capacity of the collecting duct was found to be reduced by about three times, consistent with the occurrence of polyuria and delayed wound healing owing to increased skin dryness [36
]. Keratinocytes also produce antimicrobial peptides such as defensin (human β-defensin (HBD)-1, HBD-2, HBD-3), cathelicidin, secretory leukocyte proteinase inhibitor, dermcidin, and adrenomedullin for defense against infectious agents. Chronic skin disease in response to high bacterial infection rate has been reported to be associated with the decreased expression of these antimicrobial peptides [40
]. The abnormalities of these skin cell components and antimicrobial peptides are also presumed to be the cause of AD along with other factors such as genetic and environmental factors and imbalance in the immune response [44
While screening various natural resources, we found that barley sprout exhibits excellent anti-inflammatory and anti-allergic activities [47
]. Barley (Hordeum vulgare
L.) is a major crop belonging to the Poaceae (Gramineae) family. In particular, barley leaves are rich in various bioactive substances such as vitamin C, vitamin E, catechin, kaempferol, quercetin, and β-carotene. Studies have been performed to analyze the nutritional value and various physiological activities of barley, but no study has systematically evaluated the different beneficial properties of barley [48
]. According to the flavonoid database 1.0 [52
], barley sprout contains a relatively higher level of apigenin (4′,5,7-trihydroxyflavone, flavonoid), a type of phenolic compound, than other crops. Apigenin exerts health-promoting effects and is known to reduce the risk of chronic disease owing to its low toxicity [54
]. Further, apigenin has been reported to exhibit remarkable effects against cancerous cells [55
To confirm the applicability of natural resources, it is imperative to prove the effectiveness of the main ingredients contained in the resources. Therefore, this study aimed to evaluate apigenin, the main ingredient of barley sprout, for its anti-allergic effects on basophils (RBL-2H3) and anti-inflammatory effects on macrophages (RAW264.7). In addition, we investigated the effects of apigenin on human epidermal keratinocytes (HaCaT) to determine its potential as a natural substance for the prevention of AD.
3. Materials and Methods
Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Biowest (Kansas City, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS, 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG), and monoclonal anti-DNP-IgE were supplied by Sigma–Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Gaithersburg, MD, USA). Primary antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Lyn, Lyn, p-Syk, Syk, p-PLCγ, PLCγ, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), and FcεRIγ, from LSBio (Seattle, WA, USA). SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea), and human filaggrin, AQP3, and HA ELISA kits were obtained from CUSABIO (Seoul, Korea). Rat basophilic leukemia (RBL-2H3, ATCC® CRL-2256) and murine macrophage (RAW264.7, ATCC® TIB-71) cells were procured from the American Type Culture Collection (ATCC), while human immortalized keratinocyte (HaCaT) cells were obtained from Prof. Lee of Chosun University in Korea.
3.2. Cell Culture
RBL-2H3, RAW264.7, and HaCaT cells were maintained in DMEM containing 10% FBS and 1% antibiotics (penicillin and streptomycin). The cells were incubated at 37 °C in a 5% CO2 humidified incubator.
3.3. Cell Viability Assay
RAW264.7, RBL-2H3, and HaCaT cell viability were analyzed using the MTT assay [108
]. RAW264.7 cells were treated with quercetin (15 μM) and apigenin (20, 40, 60, 80, and 100 μM) in the absence or presence of LPS (1 μg/mL). RBL-2H3 and HaCaT cells were treated with various concentrations of apigenin (RBL-2H3: 5, 10, 20, 30, and 40 μM; HaCaT: 10, 20, 30, 40, and 50 μM). After incubation for 24 h, 0.5 mg/mL of MTT solution was added to each well. The supernatants were discarded, and the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and transferred to a 96-well plate. Absorbance (570 nm) was measured using a microplate reader (TECAN, Männedorf, Switzerland).
3.4. NO and β-Hexosaminidase Release Assay
RAW264.7 cells were seeded in 24-well plates in 5 × 104 cells/well and incubated for 24 h. The cells were treated with LPS (1 μg/mL) and apigenin (20, 40, 60, 80, and 100 μM) for 24 h. The supernatant was mixed with Griess reagent in a 96-well plate for 10 min, and the absorbance was measured at 530 nm using microplate reader. The amount of NO produced was calculated using a sodium nitrite (NaNO2) standard curve.
For β-hexosaminidase release assay, RBL-2H3 cells were seeded in 24-well plates at 2 × 105 cells/well and treated with 0.5 μg/mL DNP-IgE for 24 h. The medium was removed and washed with Tyrode buffer (119 mM sodium chloride (NaCl), 5 mM potassium chloride (KCl), 2.5 mM calcium chloride (CaCl2), 1.19 mM magnesium sulfate (MgSO4), 10 mM HEPES, 5 mM glucose, and 1 mg/mL BSA, pH 7.3). The cells were incubated with apigenin (5, 10, 20, and 30 μM) for 20 min, and then with 100 ng/mL DNP-BSA for 1 h. The supernatants (50 μL) were incubated with a substrate buffer (3.3 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide, pH 4.5) in 96-well plates at 37 °C for 1 h. The reaction was terminated using 100 μL a stop solution (0.1 M sodium carbonate (Na2CO3)/sodium bicarbonate (NaHCO3), pH 10.2), and the absorbance was measured at 407 nm using a microplate reader. β-Hexosaminidase release activity was measured as per the following equation: β-hexosaminidase release activity (%) = (OD407 of sample/OD407 of control) × 100.
3.5. Real-Time Quantitative PCR
Total RNA was prepared from cells using Trizol Reagent (Thermo Scientific, Seoul, Korea) according to the manufacturer’s instructions. Total RNA (1 μg/mL) and 50 μM oligo-dT primer were mixed in 15 μL of DEPC-water and reacted at 70 °C for 5 min. After the reaction, 2 μL of 100 mM dithiothreitol, 2 μL of 10 mM dNTP, 5 μL of 5× RT buffer, and 1 μL of 200 unit/μL M-MLV RTase (Bioneer, Daejeon, Korea) were added to synthesize cDNA in a reaction at 25 °C for 5 min, followed by 42 °C for 60 min and 70 °C for 15 min. Real-time quantitative PCR was performed on Rotor-Gene 6000 (Qiagen, Seoul, Korea) using SensiFast SYBR No-ROX kit, 10 pM of each primer (Supplementary Table S1
), and 100 ng of cDNA. After amplification, the melting curve analysis was performed to confirm the specificity of the reaction. The end-point cycle threshold (Ct) used for real-time PCR quantification was defined as the number of PCR threshold cycles. The relative quantification of the target gene expression level was assessed using the ΔΔ
3.6. Western Blot Analysis
Cell pellets were resuspended in 50 μL radioimmunoprecipitation (RIPA) lysis buffer (Thermo Scientific, Seoul, Korea) containing 1× protease inhibitor cocktail and phosphatase inhibitor cocktail (GenDEPOT, Seoul, Korea). Total protein concentration was determined using the Bradford 1× dye reagent (Bio-Rad, Seoul, Korea) at 595 nm. Equal amounts of protein (8 μg) were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated protein bands were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Seoul, Korea). The membrane was blocked with 5% BSA (GenDEPOT, Seoul, Korea) and incubated with 1:1000 diluted primary antibodies (p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Lyn, Lyn, p-Syk, Syk, p-PLCγ, PLCγ, FcεRIγ, and β-actin) at 4 °C for 16 h. Western blot signals were visualized using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotech, Dallas, TX, USA) and developed with EZ-western Lumi FemtoTM Kit (Dogen, Seoul, Korea). The samples were scanned using the C-Digit blot scanner (LI-COR Biosciences, Lincoln, NE, USA), and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
FLG, AQP3, and HA production was measured using an ELISA kit as per the manufacturer’s instructions (CUSABIO, Seoul, Korea). Briefly, HaCaT cells (6 × 104 cells/well) were seeded and treated with apigenin (20 μM) for 24 h. FLG, AQP3, and HA levels were measured at 450 nm wavelength using a microplate reader (TECAN, Männedorf, Switzerland).
3.8. Statistical Analysis
All data are expressed as mean ± standard deviation (SD) of three independent experiments. Statistical significance from the control group was evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s test and t-test using Prism software (GraphPad Software Inc., La Jolla, CA, USA).
In this study, we evaluated the effect of apigenin on major mediators of inflammatory and allergic responses in RAW264.7 and RBL-2H3 cells. Apigenin (100 μM) effectively inhibited NO production and cytokine expression (IL-1β, IL6, COX-2, and iNOS) as well as the phosphorylation of ERK and JNK associated with MAPK signaling pathway in RAW264.7 cells. Apigenin (30 μM) also inhibited the expression of cytokines (TNF-α, IL-4, IL-5, IL-6, IL-13, and COX-2) and FcεRIα/γ as well as the phosphorylation of signaling molecules (Lyn, Syk, PLCγ1, ERK, and JNK) corresponding to allergic responses pathway in RBL-2H3 cells. Moreover, apigenin (20 μM) significantly induced gene or protein expression (filaggrin, loricrin, AQP3, HA, HAS-1, HAS-2, and HAS-3) in HaCaT cells of molecules that play an important role in the physical barrier and water retention properties of the skin. Further, it increased the expression of antimicrobial peptides (HBD-1, HBD-2, HBD-3, and LL-37) that play an important role in acting as chemical barriers of HaCaT cells. However, apigenin has a very low solubility in water. Therefore, it is necessary to develop delivery systems such as liposomes, polymeric micelles, nanosuspension in order to improve absorption and bioavailability in consideration of absorption, distribution, metabolism, and excretion (ADME). Although additional research is warranted, the results of the present study highlight apigenin as a potential candidate for alleviating immune-related diseases and AD.