Introduction

Healthy skin contributes to the prevention of infection, pressure ulcer development, incontinence-associated dermatitis, and skin tears [2,8]. It also prevents skin dryness by inhibiting the evaporation and loss of water. The extracellular lipid matrix is composed of a lipid complex, including ceramide (50%), cholesterol (25%), and free fatty acids (10%) [10]. Clinically, dry skin is dehydrated and has a low elasticity, which results in a rough and damaged outermost horny layer. It is an uncomfortable condition marked by scaling, itching, and cracking. The water content in skin is affected by environmental factors, skin metabolism, lipid layers, or natural moisturizing factors [5]. Psoriasis and atopic dermatitis (AD) are chronic and relapsing inflammatory skin diseases caused by a skin barrier disorder. Although the pathophysiology of AD is not fully known, it involves a complicated interaction of environmental and genetic factors that induce abnormalities in the structure and function of the epidermal barrier and immune system. It is now well established that moisturizers play an important role in preventing skin inflammation in AD, including reducing the amount of topical corticosteroid use [13,17]. The presence of probiotic bacteria in the intestinal microbiota is also known to correlate with protection against atopy. Probiotics, such as lactobacilli, show very promising evidence to recommend their addition to foods for the prevention and treatment of allergic diseases [12].

Lactic acid bacteria (LAB) are known for their healthpromoting effects such as the nonspecific enhancement of the immune system, protection against intestinal infection, reduction of serum cholesterol level, and antioxidant properties [11]. Several strains of LAB also have effects on the production of cytokines, such as IL-12, IL-10, TNF-a, TGF-b, IL-8, and RANTES, and on cell proliferation in human intestinal epithelial cells [18]. Lactobacillus plantarum is a well-documented probiotic that has been used in clinical trials for the regulation of the immune system and treatment of gastrointestinal diseases with probiotics [6]. The beneficial and protective properties of probiotic grampositive bacteria might be due to cell wall components, including lipoteichoic acid (LTA). The biggest issue in the preparation of single components, however, may be the complicated processes, which are time- and cost-consuming work. On the other hand, the preparation of L. plantarum K8 lysates is simple and cost-saving. In this study, we tried to identify the role of L. plantarum K8 lysates on the regulation of immune activity. For this, we examined the hyaluronic acid production in HaCaT cells, and assessed the attenuation effect of L. plantarum K8 lysates on AD mice. The moisturizing effect of L. plantarum K8 lysates-containing candy on the human face and forearm was also examined.

Materials and Methods

Preparation of L. plantarum K8 Lysates

L. plantarum K8 (KCTC 10887BP) was cultured in 10 L of MRS broth (Himedia Laboratories, Mumbai, India) containing soy bean peptone at 37℃ for 18 h, and then cells were harvested by centrifugation at 8,000 rpm for 10 min. After washing in phosphate-buffered saline (PBS) three times, 50 g of cells (wet weight) was disrupted by sonication. To obtain active components from disrupted cells, additional sonication was performed (up to 9 times). Disrupted bacteria (L. plantarum K8 lysates) were freeze-dried and packed under clean room conditions. Active components such as LTA and aglycone isoflavone were examined by phosphate assay and LC-MS/MS, respectively, and are described in the Supplementary Materials.

Manufacturing of L. plantarum K8 Lysates-Containing Candies.

We manufactured candies using common ingredients. Experimental candy contained 2.1% L. plantarum K8 lysates, whereas control candy contained no lysates. The information on candy ingredients is described in Table S1.

Mice

SKH-1 hairless female mice (6 weeks old) were purchased from Central Lab, Animal Inc. (Seoul, Korea). They were kept in individual cages at 24 ± 2℃ and 50 ± 10% moisture condition, and fed nutritionally balanced rodent food (Central Lab. Animal Inc.) and sterilized water. The mice were cared for and used in accordance with the guidelines of the animal ethics committee of Gyeonggi Bio Center. To develop a mouse model of atopic dermatitis using skin sensitization, each mouse was exposed to 200 µl of immune-disturbing material containing acetone:olive oil (3:1) and 2.5% 2,4-dinitrochlorobenzene (DNCB; Sigma, MO, USA). After 3 days of exposure, mice were treated with 150 µl of 1.0% DNCB at 3-day intervals. Mice were allocated to three random groups (n = 8/group): normal control group, AD group, and atopy-treated group. The treated group was orally administered by gavage with L. plantarum K8 lysates (1 × 109 CFU/mouse, day) in 100 µl of water.

Histopathological Examination

The dorsal skins of the experimental mice were removed on the final day of the schedule and fixed in 10% neutral-buffered formalin and embedded in paraffin. Serial paraffin sections (5 µm) were stained with hematoxylin and eosin (H&E), and the skin layer was examined by NIS-Elements (Nikon, Japan).

Clinical study with Healthy Volunteers

Forty-one (41) healthy volunteers aged 25 to 60 years with dry and dark skin were recruited from the campus of Kyung Hee University (Yongin, Korea), where the trial was conducted. All volunteers signed an informed consent form before participation. The Medical Ethics Committee of Kyung Hee University approved the trial (KHUSBC 2013-011). Participants were randomly assigned to two groups (control and experimental groups), and then randomly assigned to two subgroups groups within each group to examine each side of the face. Participants took four control candies or experimental candies a day (two candies in the morning and two candies in the evening) for 8 weeks. Participants were banned from using certain functional cosmetics, external application, and dietary supplement from two weeks before the study to the end of the study.

Skin Hydration Measurement

Hydration under the dead skin (10-20 µm of the stratum corneum) was examined using the Corneometer CM825 according to the manufacturer’s instructions (Courage+Khazaka Electronic GmbH, Cologne, Germany). To determine whether epidermal permeability barrier function was altered in participants, transepidermal water loss (TEWL) was assessed using a Vapometer (Delfin Technologies Ltd., Kuopio, Finland). Additionally, the interior of the forearm and front cheek were examined with D-squame Black Tape. The image of the collected dead skin samples was magnified 70 times using Charm View (Moritex, Japan) and analyzed with Image-Pro Plus software (Media Cybernetics Inc., MD, USA).

Statistical Analysis

The average of the value estimated before and after 4 and 8 weeks of administration of candies was statistically analyzed according to the time and between groups. Data represent the mean ± SEM and th e normality test was confirmed by Sh apiroWilk test. The statistical significance at different time points was tested by the paired t-test (p < 0.05) when the data fitted the normality test (95% confidence), or by Wilcoxon test (p < 0.05) when the data did not fit the normality test. Statistical significance between groups was performed by independent t-test (p < 0.05) when the data fitted the normality test, or by Mann-Whitney test (p < 0.05) when the data did not fit the normality test. SPSS 20.0 software was used for the analysis.

Results

Preparation of L. plantarum K8 Lysates and Candy Manufacturing

To obtain L. plantarum K8 lysates, a general sonication method was used. About 30~50 g of L. plantarum K8 (w et weight) was resuspended in distilled water after 3-5 washes, and then subjected to sonication. Cell wall component, such as lipoteichoic acid (LTA), was extracted from the disrupted cells. LTA is a major pathogenassociated molecular pattern (PAMP) molecule of gram-positive bacteria. LTA mediates innate immune and inflammatory responses [14] similar to the recognition of lipopolysaccharide (LPS) in gram-negative bacterial sepsis [7]. Interestingly, the repetition of the sonication step increased the extraction of active components, such as LTA and aglycone isoflavone, within the disrupted bacteria. The amount of LTA (Fig. S1A) and aglycone isoflavone such as daidzein and genistein (Fig. S1B), for example, increased with the number of sonication steps, and peaked after the 8th sonication and decreased with the 9th sonication step. The TNF-α inhibitory effect of the L. plantarum K8 lysates against LPS was increased by repeated sonication (7 to 8 times) in THP-1 cells as compared with fewer sonication steps (fewer than 4 times), indicating that more active components might be extracted from the cell wall by increasing the sonication number (data not shown). L. plantarum K8 lysates did not induce cell death (data not shown), and the hyaluronic acid contents increased up to 78~85% with cells treated with L. plantarum K8 lysates compared with the positive control N-acetyl-D-glucosamine (NAG)-treated cells, indicating that L. plantarum K8 lysates may have a moisturizing effect (Fig. 1). The candies used in this study had the same ingredients, except that the experimental candy contained 2.1% L. plantarum K8 lysates (Table S1). After the manufacture of candies, index components, such as LTA (Fig. S2) and aglycone isoflavone (Table S2) of L. plantarum K8 lysates, were examined by liquid chromatography-mass spectrometry (LC/MS).

Fig. 1.Hyaluronic acid increased with L. plantarum K8 lysates. To examine the level of hyaluronic acid expression, HaCaT cells were treated with L. plantarum K8 lysates at the indicated dosages and 20 mM N-acetyl-D-glucosamine (NAG) was used as a positive control. The secretion of hyaluronic acid was examined by ELISA.

Assessment of Clinical and Epidermal Permeability Barrier Function in Mice

Clinical assessment of AD was performed by measuring the horny layer of the skin. The horny layer increased significantly in the DNCB-treated group compared with the negative control, whereas oral administration of L. plantarum K8 lysates effectively attenuated the horny layer formation (Fig. 2A). Oral administration of L. plantarum K8 lysates also decreased epidermal thickening in DNCB-treated skin (Fig. 2A, H&E staining). Epidermal permeability barrier function was assessed by measuring the TEWL. The TEWL increased significantly in the DNCB-treated group by 600%, 580%, and 440% after oral administration for 4, 6, and 8 weeks, respectively, compared with that in the negative controls. The damage to barrier function after the 8 weeks oral administration was reduced by L. plantarum K8 lysates (12.6%, p = 0.0115) compared with that in the AD mice that received DNCB treatment only (Fig. 2B). The thickness of mouse skin was increased in the DNCB-treated group by 450%, 320%, and 250% after oral administration for 4, 6, and 8 weeks, respectively, as compared with the control. Oral administration of L. plantarum K8 lysates decreased skin thickness by 10%, 35% (p = 0.0365), and 40% (p = 0.0425) in comparison with skin thickness in the DNCB-treated group (Fig. 2C). These results suggest that oral administration of L. plantarum K8 lysates improves the clinical signs of AD and reduces damage to epidermal barrier function in mice.

Fig. 2.Effect of oral administration of L. plantarum K8 lysates on skin histology in the mouse model. (A) AD was induced by DNCB sensitization, and L. plantarum K8 lysates were orally administered for 8 weeks. Immunohistochemical staining showed that the skin epidermal layer decreased in the experimental group as compared with the control group. The differences in TEWL value (B) and epidermal thickness (C) between control and experimental groups are shown. AD, atopic dermatitis; NG, non-treated group; CG, DNCB-treated control group; TG, DNCB and L. plantarum K8 lysates-treated experimental group.

L. plantarum K8 Lysates-Containing Candy Increased Skin Hydration

The hydration contents of the front cheek and interior of the forearm of the volunteers were examined before initiation of the study, and 4 and 8 weeks after the administration of candy. A statistically significant difference was found using an independent t-test and Mann-Whitney test between the control and experimental groups. The hydration on the face and forearm increased in the experimental group (L. plantarum K8 lysates-containing candy-administered group) after 4 weeks of administration, compared with hydration prior to testing. Both the control and experimental groups showed that the hydration increased on the face (5.87% in control and 19.01% in experimental) and forearm (5.49% in control and 16.86% in experimental) at 8 weeks after administration of the candy. A significant increase in hydration in the experimental group as compared with the control group was shown on the face after 4 and 8 weeks of administration, while hydration increased on the forearm after 4 weeks of administration (Table 1).

Table 1.†p-value : Independent t-test (*p < 0.05, **p < 0.01,***p < 0.001). †p-value^: Mann-Whitney test.

L. plantarum K8 Lysates-Containing Candy Decreased TEWL Values

A statistically significant difference in TEWL values between control and experimental groups was observed at 8 weeks (paired t-test, p < 0.01; Wilcoxon signed rank test, p < 0.05). When compared with the control group, TEWL values were significantly decreased in the experimental candy-administered group on both the face and forearm (Table 2). The decrease of TEWL in the experimental group was about 8% on the face and 14% on the forearm as compared with the control group. No statistical differences were observed between the TEWL values of the two groups at 4 weeks.

Table 2.†p-value: Paired t-test (*p < 0.05, **p < 0.01,***p < 0.001). †p-value ^: Wilcoxon signed rank test. °Intensification factor (IF): (WX – W0)/W0 × 100, calculated by mean value.

L. plantarum K8 Lysates-Containing Candy Decreased the Thickness of the Horny Layer

The horny layer was significantly decreased on the face in the experimental group after 4 and 8 weeks, whereas both the control and experimental groups showed a significant decrease in the horny layer on the forearm. The thickness of the horny layer was found to be significantly reduced on the face (18.48%) and forearm (29.61%) of experimental group volunteers after oral administration of L. plantarum K8 lysates-containing candy for 4 and 8 weeks, as compared with the control group (3.53% on face and 14.27% on forearm) (Table 3). A statistically significant difference was found by using an independent t-test (p** < 0.01 and p*** < 0.0001) and Mann-Whitney test (p < 0.05). Images of the horny layer from the control and experimental groups are shown in Fig. S3. Skin horny layer was reduced in volunteers who took L. plantarum K8 lysates-containing candy, on both the face (Fig. S3A) and forearm (Fig. S3B).

Table 3.†p-value : Independent t-test (*p < 0.05, **p < 0.01,***p < 0.001). †p-value^: Mann-Whitney test.

Discussion

This study was aimed at investigating the effects of oral administration of L. plantarum K8 lysates on the AD mouse model and the moisturizing effect of L. plantarum K8 lysates-containing candy on the human face and forearm. It is known that AD mouse is a good animal model for the study of miniaturization effects, since moisturizing affects AD alleviation [17]. AD is a common chronic inflammatory skin disease that is increasing in prevalence, and is frequently found in infants and children [4]. The pathogenesis of AD involves a complicated interaction of environmental and genetic factors that induce the abnormalities in the structure and function of the epidermal barrier and immune system [13]. Interleukin (IL)-4 switches B cell signals to synthesize IgE, and IgE-mediated mast cell activation leads to the release of various kinds of chemical mediators, which results in infiltration of inflammatory cells into the skin lesion. On the other hand, IL-12 and interferon (IFN)-γ repress B cell-mediated IgE synthesis [16]. The elevated serum IgE levels in patients with AD also suggest that T helper (Th) 2 cytokines may participate in the disease pathogenesis [3]. In our previous study using L. plantarum lysates, we observed decreased IL-4 in peritoneal macrophages and casein-specific IgE levels from blood, indicating that L. plantarum lysates repress Th2 response. Th1-response cytokines, such as IL-12 and IFN-γ, were increased by L. plantarum lysates. The regulation of Th1/Th2 balance by L. plantarum lysates may contribute to the induction of hyaluronic acid and alleviation of AD lesions.

Nowadays, it is well established that moisturizers play an important role in preventing skin inflammation in AD, including reducing the amount of topical corticosteroid use [11,13,15,17]. Thus, the use of moisturizers is considered standard therapy for the treatment of AD. The purpose is to improve dry skin with decreased barrier function and also to prevent recurrence of inflammation. Moisturizers are externally applied compounds comprising multiple components, aiming to maintain skin integrity and appearance. Previous randomized controlled studies have shown that the use of moisturizers reduces the amount of topical corticosteroid use, and affects the skin barrier function of normal skin by reducing TEWL and susceptibility to irritants [17]. In the current study, oral administration of L. plantarum K8 cell lysates has been shown to alleviate AD in a mouse model by reducing the TEWL value. The epidermal thickness was also reduced in the L. plantarum K8 lysates-administered experimental group. This alleviation may be induced by regulatory dendritic cells and the generation of CD4+FoxP3+ Treg cells within the mesenteric lymph nodes [9]. Treg cells play a critical role in immune-mediated inflammation by preventing T-cell activation. The regulation of Treg cells by probiotic cell lysates, however, has not been studied yet.

A clinical research study with healthy volunteers showed improvement in barrier repair and function when volunteers took L. plantarum K8 cell lysate-containing candy. TEWL values were significantly decreased in the experimental group after 8 weeks of administration as compared with the control group. Although the water content on the face and forearm of volunteers increased in both the control and experimental groups, a significant increase was shown in the experimental group when it was compared with the control group. Statistically significant differences were found; sample candy containing L. plantarum K8 lysates demonstrated significantly higher capacitance readings. In contrast, control candy did not emerge as different when compared with control site values. Although the reasons for this difference were not explored in this study, it can be inferred that the active component in L. plantarum may contribute to the difference in capacitance readings.

In conclusion, we observed that the hyaluronic acid was significantly increased by L. plantarum K8 lysates in in vitro experiment. Hyaluronic acid is known to restore hydration. The alleviation of AD lesion in the mouse model also indicates that L. plantarum K8 lysates have a moisturizing effect. The experiments with healthy volunteers administered L. plantarum K8 lysates-containing candies showed increased hydration, less water loss, and decreased horny layers on the face and forearm as compared with the control group administered L. plantarum K8 lysates-free candies. Thus, our results suggest that L. plantarum K8 lysates may help to improve skin moisturization.