1. Introduction
Oats (
Avena sativa L.) have been used for therapeutic purposes since the 12th century, including as dietary supplements and natural remedies for various health conditions [
1]. The potent antioxidant effect of oats is derived from avenanthramides and tocopherols, which act as naturally occurring free radical scavengers [
2]. Oats, characterized by their high beta-glucan content, contribute to the gradual absorption of nutrients and the reduction in blood cholesterol levels [
3]. Additionally, oats serve as a representative plant agent for the treatment of skin diseases. The secretion of anti-inflammatory transforming growth factor β is promoted and dry skin is moisturized through the colloidal extracts of oats, signifying their effectiveness in alleviating conditions of irritated and inflamed skin [
4]. Moreover, research has demonstrated that oats can enhance skin moisture levels by modulating the expression of junctional proteins [
5,
6]. These findings have initiated the development of various cosmetics using oats to improve skin conditions [
7]. Researchers have identified various phytochemical ingredients, such as unique polyphenols, avenanthramides, saponins, flavonoids, and high beta-glucans, which are associated with pharmaceutical effects on the human body [
1]. However, despite these ongoing studies and the commercialization of oat-based products, the exact molecular mechanism and role of the detailed compounds of oats in treating skin diseases remain only partially understood.
Plant sprouts have garnered attention due to their high levels of health-beneficial phytochemicals and their environmentally friendly characteristics [
8]. In a previous study, valuable compounds were extracted and utilized from oat sprouts, with various metabolites isolated from different oat cultivars and identified as glycosylated flavonoids and steroidal saponins [
9]. Building upon previous research on oats, we hypothesized that oat sprouts might possess anti-inflammatory properties and could alleviate skin lesions, particularly those requiring moisturization. Therefore, the aim of this study was to investigate the therapeutic effects of oat sprouts and their mechanisms of action in in vivo and in vitro skin irritation models.
Contact dermatitis represents a severe skin condition resulting from exposure to external irritants. Repeated contact with sensitized allergens leads to the development of allergic contact dermatitis (ACD), characterized by intense itching and discomfort. Inflammatory responses and the integrity of the skin barrier are recognized as the principal contributing factors in contact dermatitis [
10,
11]. ACD primarily occurs owing to the activation of allergen-specific cytotoxic T cells, which are primed by the cutaneous dendritic cell–hapten complex during the sensitization phase. These T cells proliferate, migrate from the lymph nodes into the blood, and recirculate between lymphoid organs [
12]. Mast cells play a crucial role as initiators of the initial immune response during the sensitization phase [
13]. They are known to facilitate rapid vascular changes that promote the migration of dendritic cells during the initial sensitization phase. Furthermore, the secretion of tumor necrosis factor α (TNFα) by mast cells has a similar effect on cutaneous dendritic cells [
14].
Skin barrier function is crucial for maintaining skin homeostasis and health. As keratinocytes in the epidermis undergo differentiation and stratification, they synthesize proteins and lipid molecules to create mechanical and chemical barriers shielding the skin from various irritants. Proteins such as filaggrin, involucrin, and loricrin assemble with outer lipids on the skin surface to form a tough mechanical barrier known as the cornified envelope [
15,
16]. In addition, these barrier proteins affect the assembly of collagen fibers that provide structural integrity to the skin. Moreover, they break down into moisturizing factors, enhancing skin hydration [
17]. Genetic defects in the formation of this barrier have been linked to various skin diseases, such as atopic dermatitis, contact dermatitis, and psoriasis [
11,
18]. Similarly, several studies demonstrated a correlation between genetic mutations in the filaggrin-coding gene (
FLG) and increased onset of atopic and ACD [
10,
19]. Defective skin barriers can exacerbate the progression and prognosis of these diseases by increasing the risk of exposure to allergens and pathogenic microorganisms. Moreover, this inflammatory state triggers the secretion of various chemokines, cytokines, and immune mediators, damaging skin integrity and barrier function and aggravating the situation. Given the significance of the skin barrier function in the onset and progression of skin diseases, enhancing the barrier function may be a crucial therapeutic approach for improving overall skin health.
To validate the therapeutic efficacy of oat sprouts against chronic ACD, an ACD model was induced in SKH-1 mice by subjecting them to repeated oxazolone challenges. This model was chosen to mimic human ACD, characterized by mixed type 1 and type 2 immunity [
20]. Subsequently, clinical parameters associated with inflammation and skin barrier function were evaluated at clinical levels following treatment with oat sprout extract (OSE). Additionally, the potential of OSE to regulate the immune response and modulating epidermal barrier function was examined. After confirming the therapeutic efficacy, the focus shifted to elucidating the molecular mechanisms underpinning its effects. Furthermore, experiments were conducted to verify the function and mechanism of the active compounds isolated from oat sprouts using an in vitro model of lipopolysaccharide (LPS)-induced skin irritation with immortalized human keratinocytes (HaCaT).
3. Discussion
In this study, ACD was induced using oxazolone, a hapten, through initial sensitization, followed by repeated challenges. The pathological features of oxazolone-induced lesions exhibit similar characteristics to the disease observed in daily observations [
12]. Using this model, a significant modulation of the immune status in oxazolone-induced ACD lesions was observed upon OSE treatment. Specifically, in the in vivo ACD model, the topical application of OSE led to several notable changes. These changes included a decrease in IgE levels, reduced mast cell infiltration and IL-5 secretion, as well as a decrease in the T cell population within splenocytes. Furthermore, when OSE was administered in combination with both topical and oral routes, an upregulation of the MAPK signaling pathway and increased protein expression levels of filaggrin, loricrin, and involucrin were observed. Additionally, when the three main constituents of OSE, namely AB, dAB, and IV2A, were applied to keratinocytes, reduced expression levels of IL-6, TNF-α, and IFNG were observed. Simultaneously, the protein expression of filaggrin was found to be increased.
Both human and mouse ACD often exhibit a combined type 1 and type 2 immune response [
20]. Notably, our results unveiled a decrease in the composition of CD4
+ and CD8
+ T-cells following OSE treatment in vivo. Furthermore, the expression of T cell-related cytokines within the skin tissue was modulated. Cytokines associated with the type 1 response, namely IL-8, TNF α, and IFNG, exhibited reductions in the in vitro irritation model after treatment with OSE components. Conversely, cytokines linked to type 2 immunity, such as IL-6 in the in vitro model and IL-5 in the in vivo model, showed decreases. These findings indicate that OSE treatment is effective for ACD, which represents a mixed immune response involving both type 1 and type 2 immunity. Consequently, despite the absence of visible differences, there was a significant reduction in plasma IgE concentration and local mast cell infiltration density. It is plausible that OSE inhibits the priming of systemic hapten-specific T cells in regional lymph nodes by reducing mast cell recruitment during the sensitization phase. However, it is important to note that this study exclusively analyzed the immune status of the mice on day 21 after repeated exposure to oxazolone. Future investigations should delve into the sensitization and elicitation phases over time to establish clear causal relationships.
Skin barrier loss is another significant pathological factor in developing contact dermatitis [
11], as it leads to a high chance of exposure to allergens and pathogens, permitting them to penetrate deeper layers of the skin. Various proteins involved in skin barrier formation are encoded by the epidermal differentiation complex (EDC) genes at the 1q21 locus of the human chromosome [
21]. Among these, involucrin initially forms bonds with ceramides, and loricrin acts as a crosslinking component, reinforcing this barrier [
22]. Filaggrin is an essential component of the epidermal barrier, as it forms a cytofilament and keratin matrix. Moreover, during the final process of filaggrin cleavage, it is cleaved into natural moisturizing factors that comprise up to 10% of the stratum corneum and are reported to be significant skin humectants [
21,
23]. Therefore, appropriate keratinocyte proliferation and differentiation with the balanced formation and assembly of these proteins constitute the principal part of a healthy skin barrier.
In this study, OSE significantly increased the expression level of EDC proteins in the oxazolone-induced ACD model, particularly when administered orally. In addition, the expression levels of the structural proteins, loricrin and involucrin as well as the functional filaggrin, were also increased. Similar to previous reports regarding the clinical effect of oats on the skin [
6], TEWL in early lesions decreased in our study. This shows that OSE could exhibit skin-protective effects by strengthening skin barrier function in ACD through modulating EDC protein expression.
These proteins are formed during keratinocyte differentiation in the skin. MAPK signaling pathways are involved in the proliferation, differentiation, and inflammatory responses of mammalian cells and comprise three large families, ERK, JNK/SAPK, and p38 [
24]. MAPK signaling pathways have been shown to play a significant role in keratinocyte differentiation and EDC expression. Filaggrin decreases with MAPK inhibitors [
25], and ERK promotes epidermal proliferation [
26]. In this study, the expression of phosphorylated ERK1/2, SAPK/JNK, and p38 consistently increased following OSE treatment, particularly after additional oral administration. These results indicate that the increased expression of EDC proteins by OSE is strongly related to the activation of the MAPK pathway, followed by the promotion of keratinocyte differentiation. Similarly, the STAT3 pathway is involved in skin proliferation, and it reportedly downregulates filaggrin expression [
26]. In our model, STAT3 activation decreased after oral administration of OSE, with upregulated filaggrin expression. Therefore, these results suggest that OSE can stimulate the MAPK signaling pathway while simultaneously inhibiting the STAT3 pathway within the skin, modulating EDC protein synthesis, and reinforcing skin barrier formation in ACD. Notably, these events primarily occurred when OSE was orally administered.
Finally, the study aimed to elucidate the roles of various phytochemicals in oat sprouts in mediating their anti-inflammatory and barrier-reinforcing activities. In the in vitro model, the three compounds extracted from oat sprouts showed significant efficacy in inhibiting pro-inflammatory cytokines such as IL-6, IL-8, TNFα, and IFNg, which surged along with LPS-induced damage. Among the three compounds, dAB showed the most pronounced anti-inflammatory effect, decreasing all cytokines oversecreted with LPS. Previous studies have demonstrated that avenanthramides and polyphenols from oats mediate antioxidative and anti-inflammatory functions [
27]. Based on these results, it can be inferred that saponins (AB, dAB) and flavonoids (IV2A) are responsible for the anti-inflammatory effects that oat sprouts have on the skin. In addition, considering that some inflammatory cytokines such as TNFα can downregulate EDC [
28], the suppression of inflammation by the saponins and flavonoids may be associated with the increased expression of EDC. Together with the in vivo results, these results show that oat sprouts are highly effective in recovering healthy skin after being damaged by irritants.
These three compounds showed remarkable effects regarding reconstructing damaged skin barrier function in the HaCaT model. The expression levels of filaggrin were significantly diminished after LPS treatment, indicating that LPS greatly disrupted skin barrier function and homeostasis. However, many of these damages were repaired after treatment with the compounds. For example, filaggrin expression significantly recovered with AB and dAB to the level of the non-irritating controls. With filaggrin being the most powerful humectant among barrier proteins [
23], our study proposes that the moisturizing effect of oat sprouts originates from the increased filaggrin expressed by its unique steroidal saponin, avenacoside B. Moreover, oat sprouts can be used as a therapeutic reagent for ACD and other diseases such as atopic dermatitis or asthma, which are characterized by a disrupted skin barrier. Verifying the additional function of these chemicals in the purified state to elicit more potent novel therapeutic efficacy in sprout oats is essential.
4. Materials and Methods
4.1. Preparation of Oat Sprout Extracts
The plants utilized in this study were prepared following the method described in our previous journal publication [
9]. Korean oat cultivars were planted in a growth chamber at the National Institute of Crop Science (NICS), Rural Development Administration (RDA), Jeonbuk, Republic of Korea. The growth conditions were as follows: temperature, 18–20 °C; humidity, 60–70%; illumination intensity, 3300–5500 lux; alternating 9 h of light and 15 h of dark. Harvested seedlings were dried for 2 days at 25 °C and freeze-dried at −78 °C. They were then dissolved at 1 mg/mL in 80% methanol and filtered through a 0.2 um polytetrafluoroethylene syringe filter.
4.2. Mouse Model of ACD
Six-week-old female SKH-1 hairless mice were purchased from Orient Bio (Gyeonggi-do, Republic of Korea). Prior to the experiment, all mice were acclimated for 1 week at room temperature under a light–dark cycle. The experimental protocols adhered to in this study received approval from Konkuk University IACUC (KU20005). Skin lesions were induced on the back skins of the mice by topically exposing them to 1% oxazolone (Merck & Co., Rahway, NJ, USA) diluted in acetone on day 0. Starting from 7 days after the initial exposure, the same region of their backs was exposed every other day to 200 μL of 0.1% oxazolone diluted in ethanol until day 21. The first group (ACD) received no treatment except oxazolone sensitization, while the remaining groups (ACD-S and ACD-SPO) were sprayed daily with a 3% total OSE solution from day 7 to day 21. The last group (ACD-SPO) received a daily oral administration of 100 mg/kg OSE for the same duration.
4.3. Clinical Evaluation and Sample Collection
TEWL and SCH for each mouse were assessed using the GP Skin Barrier Pro instrument (GP Bio, Gyeonggi-do, Republic of Korea) on day 14 before sensitization and treatment and again on day 21 before euthanasia. Macroscopic changes in the skin were documented using a stereoscopic microscope for clinical evaluation. Clinical scores were determined by three blinded investigators, who considered the severity and distribution of the lesions. The scoring criteria can be found in
Supplementary Table S1 [
29]. The animals were euthanized on day 21, and both blood and dorsal skin tissues from the exposed areas were collected for the analysis of cytokine levels, mRNA, and protein levels. Additionally, spleen specimens were collected for the analysis of lymphoid subpopulations.
4.4. Histopathological Assessment
Half of the collected skin tissues were fixed in 10% neutral-buffered formalin. Thin slide specimens (4 µm) were prepared through paraffinization and tissue sectioning using a microtome. These sections were then deparaffinized in xylene, rehydrated through an alcohol gradient, and stained with hematoxylin and eosin. Comprehensive microscopic examinations, including measurements of epidermal and dermal thicknesses, were conducted. Mast cells were stained with toluidine blue (pH 2.0). For immunohistochemistry, the hydrated tissue sections were boiled using a TintoRetriever pressure cooker (Bio SB, Goleta, CA, USA) in sodium citrate buffer (pH 6.0) to detect loricrin. For the primary antibody, anti-loricrin (1:200, ab85679, Abcam, Cambridge, UK) was used. The antibody-labeled sections were incubated with secondary antibodies conjugated with Alexa fluor 488 (Abcam). Then, the sections were mounted in 4′-6-diamidino-2-phenylindole (DAPI) containing media (H-1800, Vector Laboratories, Newark, CA, USA) and analyzed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The other half of the skin samples were promptly frozen after sacrifice using liquid nitrogen and stored in a −80 °C deep freezer.
4.5. Flow-Cytometry
Splenocytes were obtained from splenic tissues collected during in vivo experiments. The tissues were minced in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco™, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (WELGENE Inc., Gyoungsangbuk do, Republic of Korea), and 1% penicillin–streptomycin (Gibco™, MA, USA), and 1X mycoeraser (RDTECH, Busan, Republic of Korea). Subsequently, they were filtered through a 70 µm cell strainer to isolate single lymphoid cells. Furthermore, the samples were gently layered onto Histopaque-1083 (Sigma Aldrich, St. Louis, MO, USA) in a 1:2 ratio and centrifuged for 45 min at room temperature at 400×
g. The opaque interface was collected and washed twice with Dulbecco’s Phosphate Buffered Saline. Then, 1 mL of Live/Dead working solution from the Fixable Aqua Dead Cell Stain kit (Thermo Fisher, Waltham, MA, USA) was added, and the samples were incubated for 30 min at 4 °C. Next, they were washed, and TruStain FcX Plus (BioLegend, San Diego, CA, USA) was added. After incubating for 10 min at 4 °C, a mixture of surface-staining antibodies was added directly. Samples were incubated for 60 min at 4 °C. Finally, the samples were washed twice with buffer before the flow cytometric analysis. The gating strategy is illustrated in
Figure 2. Details about the antibodies used for staining are provided in
Supplementary Table S2.
4.6. Cell Culture and Treatments
HaCaT cells were cultivated in DMEM, supplemented with 10% FBS, 2% penicillin–streptomycin (Gibco™, MA, USA), and 1% Mycoeraser™ (RDTECH, Busan, Republic of Korea) in a humidified chamber at 37 °C with 5% CO2. Cells were seeded in 24-well plates at a density of 2.5 × 104 cells/well for real-time PCR and in 6-well plates at a density of 1.25 × 105 cells/well for immunoblotting. Subsequently, the cells were subjected to a 4 h period of serum starvation by incubating with media containing 1% FBS, followed by treatment with 10 µg/mL of LPS (Sigma Aldrich, MO, USA) for 24 h to induce an irritated state. Next, the medium was replaced with DMEM containing 1 µM of dissolved oat sprout compounds, and the cells were incubated for an additional 24 h. The cell supernatants were collected, and further RNA and protein extraction processes were performed.
4.7. Real-Time Quantitative Reverse Transcription–Polymerase Chain Reaction (qPCR)
RNA was extracted from the cells and homogenized skin tissues using the RNeasy Mini Kit (QIAGEN, Germantown, MD, USA). cDNA was synthesized using a reverse transcription kit from the same company, excluding genomic DNA. Real-time PCR was conducted using the SYBR Green probe to investigate the effects and mechanisms of oat sprouts on the skin lesions. Each PCR procedure followed these conditions: 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Detailed information about the primers employed in this research is provided in
Supplementary Table S3.
4.8. Immunoblotting
Proteins were obtained from dorsal skin tissue using the Tissue Protein Extraction Reagent (Thermofisher, Waltham, MA, USA). A protease and phosphatase inhibitor cocktail from the same company was added according to the manufacturer’s instructions. Proteins from scraped cells were obtained using the Mammalian Protein Extraction Reagent and inhibitor cocktail from the same company. The concentrations of the extracted protein samples were quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were prepared in Laemmli Sample Buffer (Bio-Rad), separated by SDS–polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Merck & Co., CA, USA). They were blocked with a commercial blocking buffer (Bio-Rad) for 5 m at RT, followed by incubation with primary antibodies at 4 °C overnight. Subsequently, the membranes were incubated with secondary antibodies for 2 h at RT. Immunoreactivity was evaluated through densitometric analysis of protein band images using the ImageJ software version 1.51j8 (NIH, Bethesda, MD, USA). Information about the antibodies used for immunoblotting is provided in the
Supplementary Table S4.
4.9. Enzyme-Linked Immunosorbent Assay (ELISA)
Plasma samples from the in vivo experiments and cell supernatants were used to compare the cytokine secretion levels in each group. Commercial ELISA kits for plasma IgE (K3231082, Komabiotech, Seoul, Republic of Korea) and histamine (ab213975, Abcam, Cambridge, UK) concentrations were used for the analysis. In addition, ELISA kits for TNFα (K0331131), IL-8 (K0331216), MCP1(K0331218), and IL-6 (K0331194) were obtained from Komabiotech (Seoul, Republic of Korea) to compare cytokine secretion levels between in vitro groups. The assays were conducted according to the manufacturer’s instructions.
4.10. Statistical Analysis
Statistical analyses and graphing were conducted using GraphPad Software Prism V9 (Graphpad Software Inc., La Jolla, CA, USA). Significant differences between groups were assessed using a one-way analysis of variance, followed by Bonferroni’s multiple comparison post hoc test for parametric measures. A significance threshold of p < 0.05 was applied.