Atopic dermatitis (AD) is a multifactorial, chronic inflammatory skin disorder accompanied by pruritus, dry skin, abnormal immune responses, and impaired epidermal barrier, which has no influence on normal, non-atopic individuals [1
]. Various studies indicated that the complex interaction between surrounding environmental triggers and genetic mechanisms acts on triggering AD, and this is highly correlated with immune system dysregulation [2
]. Worldwide, AD is one of the most common skin diseases, and its prevalence is estimated to be about 3% to 10% of adults and up to 20% of children, which has about tripled in industrialized countries over the past three decades [3
]. Also, AD is an initial step in “atopic march” that leads to asthma, food allergy, and allergic rhinitis, which results in cutaneous diseases of systemic disorders and reduces the quality of life in afflicted patients [5
The pathogenesis of AD is characterized by hyperactivation of a Th2-type immune response, skin barrier abnormalities, and pruritus, and the interaction of above factors brings about chronic skin inflammation [6
]. Skin barrier dysfunction is one of the typical symptoms of AD and indicates defects in skin barrier proteins [7
]. Compromised skin barrier integrity allows entry of external antigens and allergens, which activate immune responses across the skin surface. Under such conditions, pro-inflammatory cytokines including IL-6, IL-33, TNF-α, and thymic stromal lymphopoietin (TSLP) are produced by keratinocytes and Langerhans cells (LCs) [8
]. These cytokines lead to differentiate naïve CD4+
T cells into Th2 cells, and then Th2 cells secrete cytokines. These cytokines secreted by Th2 cells drive the Th2 inflammatory response by immune and inflammatory cells [9
]. The hyperactivated Th2-type immune responses induce the isotype class switch from IgM to IgE, contribute to eosinophilia, and cause pruritus [11
]. Furthermore, increased Th2 polarization not only causes an imbalance between Th1 and Th2 cells by suppressing the function of Th1 cells in AD, but it also leads to impairing skin barrier functions by reducing epithelial barrier molecules [13
]. Scratching behavior in response to pruritus caused by AD damages the skin barrier and provokes high skin sensitivity to relatively weak stimuli; as a result, subsequent pruritus exacerbates the disease cycle [6
Corticosteroids are one of the commonly prescribed anti-inflammatory drugs for AD patients, and they primarily target T cells, which are major perpetrators of AD pathogenesis [14
]. However, several studies reported that usage of corticosteroids over the long term or short term was associated with undesirable side effects such as higher rates of sepsis, dermal atrophy, rebound dermatitis, dyspigmentation, osteoporosis, venous thromboembolism, blurred vision, hypertension, and Cushing’s syndrome [15
]. Calcineurin inhibitor, as nonsteroidal therapy, is approved for the treatment of AD as an alternative to corticosteroids. However, it was reported that calcineurin inhibitors may cause lymphomas, leukemias, and malignancies [16
]. Thus, there is a need to develop and research safer and more effective anti-AD therapeutic products and therapies.
The in vivo model for AD using NC/Nga mice was established in 1997, and this inbred strain of mouse is widely used to investigate the pathogenesis of AD [17
]. 2,4-dinitrochlorobenzene (DNCB) is extensively known as an allergenic chemical, which easily stimulates the skin surface, causing hypersensitivity of the skin and contributing to inducing dermatitis in NC/Nga mice [18
]. Repeated application of DNCB induces AD-like skin lesions in mice, which indicate clinical features of human AD such as increased level of serum IgE levels and Th2 cytokines as well as a defected skin barrier [19
Noni is the fruit of Morinda citrifolia
, originating in Southeast Asia, and is distributed around Australia, the Pacific Basin, and the Caribbean [21
]. Noni has been used as a beverage, fermented tonic, and folk medicine in China, Australia, Polynesia, and Hawaii as a remedy for diabetes, heart trouble, high blood pressure, skin infections, mouth sores, and toothache. It also has a variety of biological effects including anti-hypertensive, anti-bacterial, anti-inflammatory, anti-asthma, stimulation of immune system, and anti-cancer effects; therefore, it shows substantial therapeutic potentials [22
]. We investigated the protective effects of fermented Morinda citrifolia
(F.NONI) in a DNCB-induced atopic dermatitis model in vivo. This study focused not only on the AD-like skin lesion symptoms but also the immunological balance of Th1 and Th2, and skin barrier function involved in tight junction (TJ) proteins.
2. Materials and Methods
2.1. Preparation of F.NONI
The F.NONI was provided from NST Bio (Gimpo, Korea). Morinda citrifolia (noni) fruit was collected from the NST Bio Noni Farm Co. Ltd in French Polynesia (Indonesia islands), and F.NONI was produced in the NST bio. Briefly, harvested noni fruit was washed and frozen at −27 °C to remove bacteria. Thawed noni were sliced, incubated with 2% NST 1805 (Lactobacillus plantarum) probiotics and water at 30 °C for 2–3 weeks, heated at 90 °C for 30 min, and evaporated to be concentrated. After the fermentation period, the stock ferment was filtered using Whatman filter paper and vacuum filtration to eliminate debris and fruit particles from the stock solution. The obtained solution was stored at −20 °C for further use.
2.2. HPLC-UV Analysis of F.NONI
One gram of the F.NONI extract was diluted with 5 mL of H2O-MeOH (1:1) and mixed thoroughly; the solution was collected into a 5 mL volumetric flask for HPLC analysis. The extracts were combined, filtered, and then dried in a rotary evaporator under vacuum at 50 °C. The dried extracts were re-dissolved with MeOH for HPLC analysis. Chromatographic separation was performed on a Shimadzu 20A separations module coupled with 20A UV detectors, equipped with a C18 column (4.6 mm × 250 mm; 5 μm, Waters Corporation, Milford, MA, USA). The pump was connected to two mobile phases—A, MeCN; and B, 0.1% formic acid in H2O (v/v)—and the elute data flow rate was 0.8 mL/min. The mobile phase was consecutively programmed in linear gradients as follows: 0–5 min, 0% A; and 40 min, 30% A. The UV detector was monitored in the range of 235 nm. The injection volume was 10 µL for each of the sample solutions. The column temperature was maintained at 25 °C.
NC/Nga mice (n = 48) aged four weeks were provided by SLC (Shizuoka, Japan). The mice were kept in 55% ± 5% humidity at 23 ± 3 °C in individually ventilated cages (IVCs) under specific pathogen-free (SPF) conditions with a 12 h light–dark cycle. The mice were fed a standard laboratory diet (Central Lab Animal, Seoul, Korea) and water ad libitum. All experimental procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee guidelines of Kyung Hee University (approval no. KHUASP(SE)-18-079), and the drop-out mice were zero until the day of the final experiment.
2.4. Induction of AD-Like Skin Lesions and F.NONI Treatment
AD-like skin lesions were induced by DNCB (Sigma-Aldrich, St Louis, MO, USA) topical application in NC/Nga mice described in the methods of our previous study [20
]. Briefly, after 1 week of acclimation, dorsal hair of NC/Nga mice was removed by using an electric shaver. After shaving hair, the mice were randomly divided into the following 6 groups, and 8 mice were allocated in each group (sample size was n
= 8 per group): nontreated control group (Normal, naïve control group), DNCB-treated group (Control, negative control group), DNCB-treated + prednisolone 3 mg/kg (Sigma-Aldrich, St Louis, MO, USA) group (PD, positive control group), and DNCB-treated + F.NONI 250, 500, 1000 mg/kg group (F.NONI 250, F.NONI 500, F.NONI 1000). To induce AD-like skin lesions, 1% DNCB was dissolved in an acetone and ethanol mixture (2:3 v/v) and then was topically applied on the shaved dorsal area (200 µL) and right ear (100 µL) twice a week for sensitization. Following the sensitization, 0.4% DNCB dissolved in an acetone and olive oil mixture (3:1 v/v) was challenged on the dorsal skin (150 µL) and right ear (50 µL) repeatedly three times a week for 9 weeks. The mice in the normal and control groups were orally administered 0.5% carboxymethyl cellulose (0.5% CMC). Administration of PD (3 mg/kg prednisolone) and F.NONI (250, 500, 1000 mg/kg) was performed daily for 4 weeks. AD-like skin lesions were decided by dermatitis score, scratching behavior, and histological and immunological parameters.
2.5. Dermatitis Score and Ear Thickness
The dermatitis score was recorded three times a week as described previously (Tuesday, Thursday, and Saturday at 14:00) [23
]. The scores graded as 0 (none), 1 (mild), 2 (moderate), or 3 (severe) were measured for each of the five symptoms (erythema/edema, dryness, erosion, excoriation, and lichenification). The total dermatitis score was quantified as the sum of all individual scores for five symptoms (maximum score: 15). The ear thickness was gauged on the right ear of each mice three times a week using a thickness gauge (Mitutoyo Corporation, Tokyo, Japan).
2.6. Scratching Behavior
The measurement of scratching behavior in experimental mice was recorded three times a week, as described in the previous study (Monday, Wednesday, and Friday at 14:00) [24
]. Briefly, after vehicle administration, mice were placed in acryl cages for at least 1 h. Then, we measured and recorded the scratching movements of the neck, ears, and dorsal skin with hind paw for 30 min, which was scored from 0 to 4 (0, none; score 2, scratching shorter than 1.5 s; score 4, scratching longer than 1.5 s). The total score of scratching behavior was determined as the sum of individual measured records.
2.7. Histological Analysis
The dorsal skin tissue of mice was cut for histological analysis and fixed in 10% neutral formalin. Then, fixed tissues were embedded in paraffin and sliced into 4 µm thick sections. The tissue sections were stained with hematoxylin and eosin (H&E) and toluidine blue (TB). The stained tissues were supplied from the Korea pathology technical center (Cheongju, Korea). After staining, dorsal images were photographed with an optical microscope (400×, DP Controller Software; Olympus Optical, Tokyo, Japan). The thickness of skin epidermis and the number of infiltrated inflammatory cells were measured from each six locations using Image J software (National Institute of Health, Starkville, MD, USA).
2.8. Serum Immunoglobulin and Cytokine Analysis
Blood was immediately collected after mice were sacrificed. The method of blood collection was performed according to the protocol approved by the institute’s animal ethics committee [25
]. Briefly, on the last day of the experiment, mice were anesthetized using inhalation of isoflurane (2–2.5%), and blood samples were withdrawn from venous vessels. The posterior vena cava technique was used as a terminal procedure of sacrifice. The serum samples were obtained from blood using a centrifuge (3000× g
, 4 °C, 15 min) and stored at −80 °C until use. The levels of Ig, thymus and activation regulated chemokine (TARC), TSLP, and histamine concentrations in serum were measured using mouse enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. (IgE, Shibayagi, Gunma, Japan; IgG1
, Enzo Life Sciences, Farmingdale, NY, USA; TARC, TSLP, and histamine, Elabscience, Huston, ID, USA).
2.9. Splenocyte Isolation and Splenic Cytokine Analysis
Spleens were isolated from each NC/Nga mouse after the last treatment, and then examinations were performed as described [26
]. Briefly, spleens were collected from NC/Nga mice aseptically, which were smashed with a sterile syringe plunger and dispersed into a single-celled suspension using a cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). After treatment with red blood cell lysing buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO, USA), the splenocytes were washed three times using RPMI-1640 (Gibco, Carlsbad, NY, USA) supplemented with 10% fetal bovine serum (Gemcell FBS; Gemini Bio-products, West Sacramento, CA, USA), 100 U/mL penicillin (CALSSON, Smithfield, UT, USA), and 50 mg/mL streptomycin (CALSSON, Smithfield, UT, USA). The isolated splenocytes were cultured in 24-well plates for 72 h at the concentration of 1 × 106
cells/well treated with 5 μg/mL concanavalin A (Con-A) (Sigma-Aldrich, St. Louis, MO, USA) in 5% CO2
incubator at 37 °C. After incubation, the supernatant was collected, and splenocytes were homogenized in lysis buffer containing cOmplete™ protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA). The lysates were centrifuged at 10,000× g
for 15 min at 4 °C and then collected. The collected supernatants of splenocyte and the lysates were frozen at −70 °C for subsequent cytokine analysis. The concentrations of cytokines (IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, IL-22, IL-31, IL-33, and IFN-γ) in the supernatants were measured using an ELISA kit according to the manufacturer’s instructions (Elabscience, Houston, ID, USA). The concentrations of protein in the lysates were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). The levels of cytokines in the supernatants were normalized to the protein concentrations of lysates.
2.10. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis
Total RNA was obtained from dorsal skin tissues using the Easy-RED total RNA extraction kit (Intron Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. After the RNA extraction, we quantified the RNA using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Complementary DNA (cDNA) synthesis was performed using a cDNA synthesis kit (TaKaRa Bio, Shiga, Japan) with the extracted RNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was executed with synthesized cDNA and SYBR Premix EX Taq (TaKaRa Bio, Shiga, Japan) into the ABI StepOnePlus™ real-time PCR system (Applied Biosystems, Waltham, MA, USA). The primer sequences are listed in Table 1
. The mRNA expression level of each gene was calculated from the cycle threshold (Ct
) value using the ΔΔCt
method and normalized to GAPDH.
2.11. Western Blot Analysis
The dorsal skin tissues were smashed using a pestle after being frozen with liquid nitrogen. Subsequently, the skin tissues were homogenized with lysis buffer containing cOmplete™ protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, ID, USA). The lysates of dorsal skin tissues were sonicated and centrifuged at 10,000× g for 15 min at 4 °C. The concentrations of protein from the supernatant were quantitated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). After quantitation, equal amounts of protein were loaded on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE; Bio-Rad, CA, USA) at 7.5% or 12% for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline with 0.5% Tween-20 (TBST) and incubated at 1:1000 primary antibody overnight at 4 °C. The following day, the membranes were treated with a horseradish peroxidase-conjugated (HRP) secondary antibody (GeneTex, Inc., Irvine, CA, USA) for 2 h at a diluted concentration of 1:5000 and were visualized using a ChemiDoc™XRS + System (Bio-Rad, Richmond, CA, USA). The expression level of each protein was analyzed by Image Lab statistical software (Bio-Rad, CA, USA) and normalized to β-actin. The primary antibodies used for Western immunoblotting were as follows: filaggrin (FLG) (GeneTex, Inc., Irvine, CA, USA), loricrin (LOR) (GeneTex, Inc., Irvine, CA, USA), involucrin (IVL) (Santa Cruz, CA, USA), occluding (OCC) (Abcam, Cambridge, MA, USA), zonula occludens-1 (ZO-1) (Abcam, Cambridge, MA, USA), and β-actin (Santa Cruz, CA, USA).
2.12. Statistical Analysis
The data are expressed as the mean ± standard error of the mean (SEM). We performed one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. All analyses were performed using the Statistical Package for Social Science Software Program 23 software (SPSS; Chicago, IL, USA). Statistical significance (p-value) was defined as follows: # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the normal; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.
Several studies have reported that AD is closely related to environmental, genetic, and immunological factors [28
]. Immune dysregulation and skin barrier abnormalities are major features in AD, which are involved in complex pathophysiology [30
]. Recent studies tried to apply the therapeutic effect of natural preparation and traditional medicine to prevent and cure AD [20
]. Morinda citrifolia
fruit, also known as noni, is reported to have antioxidant activity, which suggests that antioxidant effects may contribute to immune-modulating and anti-inflammatory activity [33
]. In a previous study, noni was reported to have beneficial immunomodulatory effects in inadequate immune responses through regulating expressions of Th1 and Th2 cytokines in vivo [34
]. In addition, a recent study reported that in inflammatory bowel disease, noni juice reduced expressions of inflammatory cytokines and preserved intestinal architecture [35
]. Iridoids including DAA and AA are the major constituents of noni, which are known to have antioxidant and free radical scavenging activities and quench lipid peroxides [36
]. Gaertneroside, a similar iridoid extracted from plants, has been shown to have regulatory effects on immune cells in vitro [37
]. DAA and AA were reported to inhibit the release of TNF-α from peritoneal macrophage cells in mice [38
]. We found that DAA and AA were considerably increased in the fermented noni with probiotics compared to the components of the noni in HPLC analysis. Thus, in this study, we investigated the effects of F.NONI in the AD model in vivo. The in vivo AD model using NC/Nga mice is suitable to screen potential anti-AD agents.
In our investigation, we found that the administration of F.NONI attenuated AD lesions and symptoms by DNCB. Administration of F.NONI considerably improved skin lesion severity and dose-dependently recovered clinical factors including dermatitis score, ear thickness, and scratching behavior (Figure 2
). In addition, the histological analysis indicated that F.NONI treatment reduced epidermal thickness and infiltration of mast cells and eosinophils (Figure 3
). Following these results, to investigate the restorative effects of F.NONI against AD, we focused on immune balance and skin barrier function through modulating expressions of AD-related cytokines.
The pathophysiology of AD has immunological abnormalities that are involved in the systemic increased Th2 response, and a combination of other T cell subset responses includes Th1, Th22, and Th17 [39
]. Penetration of antigens and allergens through a defective epidermal barrier activates keratinocytes and secretion of TARC, TSLP, and IL-33, which acts on skin resident dendritic cells and mast cells [41
]. Activated mast cells degranulate and secrete pro-inflammatory cytokines, including IL-4, IL-6, TNF-α, and histamine, which cause exacerbating AD-related symptoms including pruritus. Activated dendritic cells trigger polarization of Th2 cells from naïve T helper cells [43
]. Differentiated Th2 cells express Th2-derived cytokines such as IL-4, IL-5, IL-13, and IL-31[44
]. IL-4 and IL-13 promote isotype switching in B cells, resulting in the synthesis of IgE and generation of IgG1, and they also reduce expressions of skin barrier proteins [45
]. IL-5 plays a crucial role in eosinophil development, proliferation, and survival [46
]. IL-31 is an important pruritogenic inflammatory cytokine, which inhibits epidermal terminal differentiation and causes scratching behavior as well as exacerbation of skin barrier dysfunction [47
]. The cytokines derived from Th2 re-stimulate keratinocyte and can cause increased cytokine release, including IL-6, IL-33, TNF-α, and TSLP, from keratinocyte or epidermal dendritic cells and accelerate the progression of AD [49
]. In this study, the levels of histamine, TARC, and TSLP in serum were increased in the control group, while the F.NONI groups indicated the reduction of those cytokines (Figure 4
B–D). Splenic productions of Th2-derived cytokines, including IL-4, IL-5, IL-13, IL-31, and IL-33, were significantly increased in the control group, whereas the administration of F.NONI and PD reduced splenic production of Th2-derived cytokines (Figure 5
A). In addition, cytokine gene expressions, including IL-4, IL-5, IL-13, IL-31, IL-33, and TSLP, were increased in the dorsal skin of the control group. However, the administration of F.NONI and PD decreased the gene expressions of cytokines compared to those in the control group (Figure 6
A). These results suggest that F.NONI administration attenuated upregulation of AD-related Th2 cytokines in serum, splenocytes, and dorsal skin. In addition, the decreases of Th2 cytokines such as histamine, IL-5, and IL-31 were correlated with the decreased number of infiltrated inflammatory cells and reduced scratching behavior [43
The upregulation of Th2 cytokines including IL-4 is known to inhibit the generation of Th1 cells [50
]. The development of Th1 cells requires the presence of IL-12 and IFN-γ [51
]. IL-12 is a key factor of Th1 cell differentiation, and secreted IFN-γ by Th1 cells is known to suppress Th2 cell function and IgE synthesis as well as promote the generation of IgG2a
in B cells [2
]. The serum IgG1
ratio is a representative marker of the Th2/Th1 balance. Increased Th2-derived cytokines and deficiency of Th1-derived cytokines are hallmarks of AD, which indicates Th2/Th1 imbalance [53
]. In our study, Th1-derived cytokines including IL-12 and IFN-γ in splenocytes and dorsal skin tended to be decreased in the control group, whereas the expression of Th1-derived cytokines was normalized by the administration of F.NONI (Figure 5
B and Figure 6
B). Consequently, our data showed that F.NONI treatment decreased the levels of serum IgE compared to the control group (Figure 4
A) and also restored the Th2/Th1 balance through regulating the serum IgG1
ratio (Table 3
). In contrast, it was observed that the administration of PD not only inhibited expressions of Th1 and Th2 cytokines in serum, splenocytes, and skin, but it also downregulated the level of serum immunoglobulin compared to the control group. Results were already reported that prednisolone had inhibitory effects on allergic dermatitis in the mouse model [20
]. These data indicate that the administration of F.NONI improved AD-related immune imbalance through modulating the Th1/Th2 balance in serum, splenocytes, and dorsal skin. Taken together, these results suggest that PD acted as an immune suppressor in mice, whereas the administration of F.NONI had an effect on restoring the Th1/Th2 balance through inhibiting Th2 cells and improving Th1 cells simultaneously, which regulate the level of Th1 or Th2 differentiation.
Th17 cells are correlated with the development of AD severity and produce IL-17 and IL-22 [54
]. IL-17 induces the expression of various pro-inflammatory cytokines and is reported to have a crucial role in Th2 cell differentiation [55
]. IL-17 also causes neutrophil recruitment leading to neutrophil- and eosinophil-mediated inflammation [56
]. IL-22 is secreted by Th17 as well as Th22 and is synergistically increased with IL-17 in keratinocytes, leading to the downregulation of FLG expression [54
]. In this study, the splenic production of IL-6, IL-17, and IL-22 was significantly increased in the control group compared with those in the normal group, while the F.NONI or PD induced a decrease in the expression of IL-6, IL-17, and IL-22 compared to those in the control group (Figure 5
C). Similarly, gene expressions of IL-6, IL-17A, IL-22, and TNF-α were increased in the dorsal skin of the control group. Administration of F.NONI and PD decreased the expressions of IL-6, IL-17A, IL-22, and TNF-α compared to those in the control group (Figure 6
C). These results indicate that the administration of F.NONI modulated the AD-related immune imbalance by regulating the responses of the AD-related T cell subset, including Th17 and Th22, as well as the Th1/Th2 balance.
Impaired epidermal skin is one of the hallmarks of AD. FLG is an important structural protein in the skin barrier and promotes aggregation of the keratin cytoskeleton to build up the outermost epidermal barrier [54
]. In addition, FLG contributes to cell differentiation and is degraded into hydrophilic amino acids. The combination of FLG and hydrophilic amino acids is important for hydration and pH of the skin [40
]. LOR and IVL, natural moisturizing factors, are also essential components of the epidermal envelope, and they are cross-linked to pro-FLG and act as reinforcement proteins in the cornified envelope [58
]. Several studies reported that various cytokines were increased in AD patients and led to skin barrier dysfunction, which includes TSLP and IL-33 secreted by keratinocytes as well as Th2-derived cytokines such as IL-4, IL-13, and IL-31 [48
]. In addition, IL-22 and IL-17 were also reported to downregulate the expressions of TJ proteins and terminal differentiation genes, including FLG, LOR, and IVL [39
]. In our previous study, we reported that repeated application of DNCB on the dorsal skin of NC/Nga mice downregulated the expressions of FLG, IVL, and LOR [20
]. In the present study, protein expressions of FLG, IVL, and LOR were considerably reduced in the dorsal skin of the control group, which indicated that the epidermal barrier was impaired. The administration of F.NONI significantly recovered decreased expressions of FLG, IVL, and LOR, while PD administration slightly increased the expressions of the proteins compared with the control group (Figure 7
TJs reside below the stratum corneum and regulate selective permeability into the paracellular pathway [60
]. Flaky tail mice, AD model of FLG mutation, indicated skin barrier dysfunction and showed decreased expression of LOR and OCC [61
]. Furthermore, several studies reported that FLG mutation in humans indicated reduced expressions of TJ proteins, including Zo-1 and OCC, and disruption of TJs results in the incorporation of LC dendrites to TJs and processing of the immune response [62
]. The severity of AD is inversely correlated with expression levels of FLG and TJs [64
]. In this study, we observed that TJ protein expressions, including ZO-1 and OCC, were also reduced in the control group compared to the normal group. However, the F.NONI dose-dependently increased the protein expressions of ZO-1 and OCC compared to those in the control group (Figure 7
(Bd,e)), which indicates that F.NONI restored skin barrier impairment and dysfunction. Especially, F.NONI treatment more effectively restored TJ proteins, including Zo-1 and OCC, than PD did. These data suggest that the administration of F.NONI considerably alleviated AD skin lesions through restoring skin barrier proteins including TJs. Furthermore, these results correlated with a decreased dermatitis score and reduced expression of AD-related cytokines involved in disrupting the skin barrier in the F.NONI-treated group.