Skin aging is a complex process caused by both extrinsic and intrinsic factors [1
]. Intrinsic aging is a natural process associated with physiological, hormones, genetic, and cellular metabolic changes [2
]. By contrast, extrinsic aging is the result of various environmental causes, including air pollution and exposure to sunlight. Ultraviolet (UV) irradiation, a major cause of external changes to the skin, increases production of intracellular reactive oxygen species (ROS) and pro-inflammatory cytokines [3
]. ROS production caused by exposure to high UV radiation is closely related to oxidative stress in dermal cells [4
]. In addition, excessive ROS can indirectly generate DNA changes and membrane imbalances, resulting in a decrease in collagen synthesis. This oxidative stress is responsible for skin aging and disease development [5
]. Because skin is the most important and largest surface barrier in the body, it is essential to prevent UV-induced injury in the reconstituted human epidermis.
Metalloproteinases (MMPs), one the most important protease families, can modulate expression of collagen genes in the extracellular matrix (ECM) and is a major component of the cellular microenvironment [6
]. ECM, a highly dynamic structure in the skin, is influenced by the external environment and continuously undergoes remodeling processes, such as regeneration and degradation [7
]. For instance, recent work showed that UVB radiation causes a molecular chain reaction through upregulation of MMPs expression in the dermis and epidermis [8
]. Furthermore, UVB irradiation also promotes cutaneous inflammation [9
]. In particular, UVB-induced secretion of metalloproteinases-1 (MMP1) promotes inflammation in oxidatively stressed environments. In addition, transient expression of MMP1, MMP3, and MMP13 correlates with ECM degradation, cell adhesion, tissue remodeling, and proliferation. [10
]. UVB exposure is also associated with secretion of cytokines, including Interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNFα), in skin fibroblasts [11
]. TNFα plays a critical role in photoaging by activating the expression of elastase and collagenase, which damage the skin [11
]. Treatment with bioactive natural compounds has the potential to control these pathways and prevent photo-damage. As awareness of the harmful effects of chronic UVB exposure has grown, an increasing amount of research attention has been devoted molecules with anti-aging effects on the skin [12
Skin aging is also related to loss of skin moisture. The major glycosaminoglycan hyaluronic acid (HA), an ECM component in skin dermal tissue, is involved in aspects of skin health such as hydration, cell regeneration, development, and wound healing [13
]. Due to its net anionic charge and nonsulfated glycosaminoglycan structure, HA promotes water capture and regulates tissue hydration. HA is synthesized directly in the ECM by hyaluronan synthase (HAS1, HAS2, and HAS3) [14
]. Reductions in the levels of hyaluronan acid synthase (HAS) enzymes are associated with downregulation of HA, implying that these proteins are primarily responsible for HA synthesis. Therefore, to prevent loss of skin moisture, it is important to increase HAS expression in skin fibroblasts.
Exopolysaccharide (EPS) consists of long-chain polysaccharides with various branches and repeating units of sugars or sugar derivatives [15
]. Recent studies showed that EPS from marine microorganisms has potential applications as a therapeutic food [12
]. In particular, natural polysaccharides produced by lactic acid bacteria (LAB) possess many biological activities, including immunomodulatory and antioxidant activities, and could be used as functional antioxidants [17
]. Moreover, the functionality and composition of EPS differs even among LABs from the same species. EPS biodiversity can result from different combinations of sugar biosynthesis pathways and genetic variability among strains [18
Skin health is associated with homeostasis of tight junctions in the intestine through skin–gut axis communication [19
]. Probiotics, including Lactobacillus
sp. and Bifidobacterium
sp., play important roles in cosmetics and aging [20
]. These microbes exist in the human gastrointestinal tract and have beneficial anti-aging effects on the skin, but the interaction has not yet been completely elucidated. In a previous study, we examined the effects of Lactobacillus plantarum
HY7714 (HY7714), a functional probiotic approved by the Korea Food and Drug Administration (KFDA), regarding skin hydration [21
]. However, it remains unclear which molecule of HY7714 affects skin health. Hence, in this study, we sought to characterize the effects of HY7714 EPS on human intestinal adenocarcinoma cells (Caco-2) and human dermal fibroblasts (HS68). Briefly, HY7714 EPS was treated on TNFα- damaged Caco-2 cell, in order to prove the protective effect on skin aging of HY7714 EPS through intestinal adhesion regulation. Caco-2 cell lines are most extensively used as a model of the intestinal barrier studies [22
]. Caco-2 tight junctions between cells serve as models of human intestinal absorption and natural compound transport across monolayers [23
]. In addition, the HS68 cell line is the most representative dermal cell used in skin research, which are related to skin elasticity and integrity through MMP related to collagen synthesis. To establish aged skin cell model, HS68 cells were irradiated with UVB and then treated with HY7714 EPS.
Gut and skin, densely vascularized organs with important immune roles, are intimately related in purpose and function [27
]. Recent work showed that gut and skin share a number of crucial characteristics, with the diet and gut microbiota affecting the skin [28
]. In particular, healthy aging is closely connected to the gut-skin communication: because the skin and intestine are the primary interfaces to the external environment, the maintenance of physiological homeostasis in both organs is essential [27
]. The mechanisms underlying this positive microbial communication have not yet been elucidated, but some are immune-based. In addition, probiotics act as positive modulators in gut health and oxidative immune regulation [29
Probiotics that affect skin health have been identified, especially among lactobacilli [30
]. For instance, a clinical study reported that L. lactis
strain H61 improved the skin elasticity of middle aged-women [31
]. Another study showed that L. rhamnosus
has a potential to improve skin hydration [32
]. Previous work by our group showed that L. plantarum
HY7714 increases skin moisture and elasticity and decreases the wrinkle depth in human subjects aged 41–59 [21
]. However, the mechanisms underlying these effects have not been fully elucidated. One attractive hypothesis is that polysaccharides produced by LAB are important factors in skin health. In this study, we investigated whether certain HY7714 polysaccharides could serve as functional substances that act on the gut–skin axis to change the properties of dermal cells.
Most lactobacillus EPS molecules are heteropolymers consisting of repeated copies of oligosaccharide units [15
]. EPS, which are produced either intracellularly or extracellularly, contribute to biological activity via specific composition, size, and branching structure, which can differ even among members of the same LAB strain [33
]. Given that microbial EPS has various effects of improving biological and cosmetic functions, it is very important to explore the biodiversity of the naturally derived LAB strains that produce high levels of EPS [34
]. The yield of EPS synthesized by Lactobacill can be affected by composition of the medium and growth conditions [35
]. In addition, EPS properties in slime form can negatively affect the gradual loss of probiotics, so efforts should be made to standardize culture methods to maintain quality and purity of EPS. When HY7714 probiotics are cultured, slippery substances containing EPS are secreted into the surroundings, making centrifugation difficult. On the other hand, another strain of L. plantarum
, HY7711, can be centrifuged because it produces a different kind of EPS. We noted these differences in microbial properties, and then compared and analyzed the specific structure of HY7714 and HY7711 EPS. HPLC analyses performed under two different column conditions revealed that HY7714 and HY7711 EPS have different proportions of the same three monosaccharides, ribose:mannose:glucose, in a ratio of 4.0:1.5:1.0 (HY7714) or 1.5:2.0:1.0 (HY7711), respectively. The molecular species in HY7714 EPS and HY7711 EPS, which account for 30% of the total mass, was about 80 kDa and 57 kDa, respectively. Thus, we can infer that HY7714 EPS has a specific phenotype due to the inherent structural characteristics of complex polysaccharide composition. These findings were corroborated by the fact that HY7714 also produced sugar units with a higher molecular weight, as revealed by GPC analysis, indicating that its EPS is larger.
The most valuable application of lactobacillus EPS to date has been improvement of the texture and mouthfeel of fermented milk products [36
]. According to a recent hypothesis, EPS can remain for long periods of time in the gastrointestinal tract, improving colonization by probiotics [37
]. Consistent with this, in HY7714 EPS-treated Caco-2 cells, mRNA levels of ZO-1
were higher than in cells treated with TNFα alone, which increases intestinal tight junction permeability. This elevated permeability is accompanied by a reduction in ZO-1
levels, resulting in leakage of pro-inflammatory cytokines into blood vessels and other tissues [38
]. According to our data, HY7714 EPS decreased the secretion of IL-1β
in TNFα-treated cells and restored them to their usual levels. According to recent studies, a few types of matrix metalloproteinase (MMP) play vital roles in the development of inflammation in intestinal epithelial cells. Multiple studies have reported that IL-1β and TNFα increase expression of MMP [39
]. At the same time, MMPs play a role in promoting ECM degradation following UVB damage in skin dermis and epidermal cells. Notably in this regard, the gut environment induces redistribution of skin homeostasis after UV irradiation [41
UV exposure damages the structure and function of skin, and has therefore been implicated in sunburn, immunity, cancer, and photoaging [43
]. UV light is composed of UVC (200–280 nm), UVB (280–315 nm), and UVA (315–400 nm) [26
]. In particular, UVB irradiation promotes the production of ROS and induces the overexpression of MMP1, MMP3, and MMP9 in human fibroblasts, resulting in the destruction of collagen and ultimately to wrinkle formation [44
]. In this study, we investigated whether the protective effects of HY7714 EPS against UVB irradiation are governed by its ability to protect against oxidative stress and MMP expression. We found that UV-induced oxidative damage and induction of MMPs in HS68 cells were significantly decreased by HY7714 EPS treatment. Therefore, HY7714 EPS has the potential to alleviate UVB damage of dermal connective tissue, a collagenous ECM. MMPs, which degrade cutaneous protein, are upregulated by UVB irradiation, resulting in a loss of elasticity and promotion of wrinkle formation. The MMP can be classified into several subgroups: collagenases (MMP-1, MMP-8, MMP-13), gelatinases (MMP-2, MMP-9), and stromelysins (MMP-3, MMP-10, MMP-11). As our data show, HY7714 EPS decreased the mRNA levels of MMP1
, which are upregulated by UVB exposure, and significantly decreased the protein level of MMP13. By contrast, HY7711 EPS did not inhibit MMP activation. In summary, HY7714 EPS protects the skin by inhibiting ECM degradation.
HA present in skin cells is synthesized by hyaluronic acid synthase, a membrane-bound enzyme expressed by keratinocytes and dermal fibroblasts. HAS produces HA of varying lengths, with HAS1 and HAS2 mainly synthesizing large units of HA polymers [46
]. Meanwhile, decomposition of HA in skin dermal cells following UVB exposure causes wrinkles and loss of elasticity and moisture. According to our data, HY7714 EPS significantly increased the mRNA expression levels of control HAS1
in UVB-exposed HS68 cells. It is already known that upregulation of HAS2
mRNA expression plays a pivotal role in HA synthesis. Moreover, we found that HY7714 EPS upregulated the mRNA level of serine palmitoyl transferase (SPT1), a major enzyme involved in the biosynthesis of ceramide in skin cells. Thus, HY7714 EPS may promote synthesis of HA and ceramide.
UVB exposure also contributes to the inflammatory response by driving generation of ROS and secretion of mediators such as inflammatory cytokines. Subsequently, ROS accumulation mediates the UVB-induced expression of MMP1. We observed that UVB irradiation rapidly increased ROS levels, whereas HY7714 EPS inhibited ROS in UVB-exposed cells in a dose-dependent manner. The increase in pro-inflammatory cytokines caused by UVB exposure drives photoaging in skin dermal cells. Indeed, UVB irradiation in HS68 cells promoted acute inflammation, resulting in stimulation of IL-1β, IL-6, IL-13, and tumor necrosis factor (TNFα). However, HY7714 EPS significantly decreased the levels of interleukin in a dose-dependent manner. Taken together, these findings suggest that HY7714 EPS regulates photoaging by attenuating ROS regulation and secretion of pro-inflammatory cytokines.
In summary, our findings reveal a novel effect of HY7714 EPS, namely, improving the function of intestinal tight junctions in human-derived Caco-2 cells. In addition, HY7714 EPS increased the mRNA levels of MMPs, which can affect damaged skin cells, thereby decreasing the expression of pro-inflammatory cytokines. Moreover, HY7714 EPS attenuated ECM degradation by promoting HA synthesis and inhibiting MMP expression in dermal HS68 cells. Therefore, HY7714 has the potential to benefit skin health through the microbiome–skin–gut axis. These findings reveal that EPS from HY7714 is a biologically effective substance with potential value as a cosmetic or nutraceutical.
4. Materials and Methods
4.1. EPS Isolation and Purification
EPS was obtained from supernatant of L. plantarum by ethanol precipitation. Briefly, culture media of L. plantarum HY7714 was separated by centrifugation (8000× g for 20 min at 4 °C). Chilled ethanol was slowly added to the supernatant and incubated for 24 h at 4°C; the precipitate was recovered by centrifugation (8000× g for 20 min at 4 °C). For purification, 4% (v/v) trichloroacetic acid was added, and the sample was incubated 4°C for 2 h to remove proteins. The supernatant was filtered through a 0.45 μm cellulose nitrate filter (Millipore, Bangalore, India) to remove remaining protein, and the remaining ethanol was evaporated off, yielding purified EPS. EPS was at 4 °C prior to chemical and physical analyses.
4.2. Molecular Weight Analysis of EPS
Molecular weight of the purified EPS was checked on a gel permeation chromatograph (GPC) equipped with TSK gel guard PWXL, TSK gel GMPWXL, and TSK gel G-2500 PWXL (7.8 × 300 mm) columns (TOSOH, Tokyo, Japan) in conjunction with a refractive index detector. Two hundred microliters of 3 mg/mL EPS was injected and eluted with 0.1 M NaNO3 solution at 40 °C at a flow rate of 1.0 mL/min. Data were detected and processed using the EcoSEC software (Tosoh Bioscience, Tokyo, Japan). Molecular weights were calculated using pullulan standards (Sigma-Aldrich, St. Louis, MO, USA).
4.3. Determination of Monosaccharide Composition of EPS
Five milligrams of purified EPS and standards were dissolved in 1.6 mL of 2 N H2SO4 at 100 °C for 6 h. The EPS solution was neutralized with 3.3 mL of 1 N NaOH solution. The remaining protein and fat in the resultant lysates was removed by addition of 12% (v/v) Biggs-Szijarto solution (Sigma-Aldrich), centrifugation (13,000× g, 10 min, 4°C), and filtration through a 0.45 μm regenerated cellulose (RC) filter (Whatman, Kent, England). To analyze the monosaccharide composition, 20 µL samples were analyzed by high-performance liquid chromatography (HPLC) (1260 Infinity, Agilent, Santa Clara, CA, USA) equipped with a Shodex Asahipak NH2P-50 4E column (4.6 mm × 250 mm, 5 μm) and eluted with 72% acetonitrile at a flow rate of 0.8 mL/min. The separated components were monitored using a refractive index (RI) detector. To crosscheck, samples were analyzed on an Agilent 1260 Infinity instrument equipped with an Imtakt Unison UK-Amino (UKA66, 4.6 mm × 250 mm, 3 μm, Imtakt, Tokyo, Japan) column and eluted with methanol at a flow rate of 1.2 mL/min. The separated components were also monitored using a RI detector. The column was calibrated with a molecular mass standard, and a standard curve was established for each column condition.
4.4. Cell Culture
HS68 (CRL-1635) human dermal fibroblasts obtained from American Type Culture Collection (Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 1% penicillin–streptomycin (P/S) and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in humidified air containing 5% CO2. The cells were seeded in 12-well plates (5 × 104 cells/well) for 24 h and used between passage numbers 6 and 15.
Caco-2 human intestinal adenocarcinoma cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM essential medium supplemented with 1% P/S and 10% FBS at 37 °C in humidified air containing 5% CO2. Cells were fully differentiated for 21 days, and growth medium was refreshed every 2 days.
4.5. EPS Treatment and UVB Irradiation
To investigate the protective effect of EPS, HS68 cells were cultured in growth medium for 24 h to reach 80% confluence. The cells were pretreated with various concentrations (0.1, 1, or 5 μg/mL) of EPS in serum-free medium for 24 h. After the medium was replaced with PBS, the cells were exposed to UVB irradiation at 30 mJ/cm2 using an Ultraviolet Cross-linker (UVP, Upland, CA, USA). After UVB exposure, the cells were immediately treated with EPS in serum-free medium for an additional 24 h.
4.6. Cell Viability
To determine the appropriate concentrations of EPS for use in subsequent investigations, we performed cell viability assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). HS68 fibroblasts were seeded in 96-well plates at 1 × 104 cells/well and incubated overnight. Stock solutions of EPS were prepared in distilled water. The cells were treated with EPS (0, 0.01, 0.1, 1, 5, or 10 μg/mL) for 24 h; non-treated cells were used as negative controls. Twenty microliters of a 5 mg/mL MTT solution was added to each well, and the cells were incubated for a further 4 h, leading to the formation of purple formazan crystals. After the MTT-containing medium was removed, 100 μL DMSO was added to elute the formazan crystals. The optical density of the formazan solution, which is associated with the enzyme activity and the number of viable cells, was quantified at 570 nm on a BioTek ELISA reader (Winooski, VT, USA).
4.7. Measurement of MMP1, HA, TNFα, IL-1β and IL-6
Cell culture medium was collected, and MMP1 was quantified using commercial ELISA/calorimetric assay kits (CUSABIO, Houston, TX, USA; CSB-E04672h). HA in medium was determined using ELISA/calorimetric assay kits (CSB-E04805h). To induce MMP1 overexpression, HS68 cells were treated with 100 ng/mL TNFα with or without EPS. Cytokines including TNFα, IL-1β and IL-6 in medium were detected using commercial ELISA/calorimetric assay kits (BD OptEIA™, BD-555212, BD-557953, BD-555220).
4.8. Measurement of Intracellular ROS
Cells were cultured and pretreated with EPS (0.1, 1, or 5 μg/mL) for 24 h, washed twice in PBS, and then exposed to 30 mJ/cm2 UVB. After that, the cells were stained with 10 μM DCFH-DA and analyzed using a Axiovert 200M fluorescence spectrophotometer (Zeiss, Oberkochen, Germany). Intercellular ROS levels, visualized by DCFH-DA fluorescence, were measured on a BioTek Synergy H1 hybrid microplate reader (BioTek, Winooski, VT, USA) with excitation and emission wavelengths of 485 nm and 530 nm, respectively.
4.9. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis
RNA was isolated from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from 2 μg RNA on a thermal cycler (Bio-Rad, Hercules, CA, USA) using Maxime RT PreMix (iNtRON Biotechnology, Seongnam, Korea); the reaction ran for 60 min. The cDNA was analyzed by qPCR (Applied Biosystems, Carlsbad, CA, USA) using the TaqMan Probe-Based Gene Expression analysis system in combination with TaqMan Gene Expression Master Mix containing ROX (Applied Biosystems). Quantification of MMP1 (Hs00899658_m1), MMP2 (Hs01548727_m1), MMP3 (Hs00968306_g1), HAS1 (Hs00987418_m1), HAS2 (Hs00193435_m1), SPT1 (Hs00370543_m1), TNFα (Hs99999043_m1), IL-1β (Hs01555410_m1), IL-6 (Hs00174131_m1), IL-13 (Hs00174379_m1), ZO-1 (Hs01551861_m1), OCL1 (Hs00170162_m1), and GAPDH (Hs03929097_g1) transcripts was performed using gene-specific primers. Expression data were normalized against the corresponding level of GAPDH. To compare mRNA levels between groups, relative mRNA levels were calculated using the 2(−ΔΔCT) method.
4.10. Western Blot Analysis
Cells were lysed in lysis buffer (iNtRON Biotechnology, Seoul, Korea), and lysate protein concentrations were quantified using a protein assay kit (Bio-Rad). Equal amounts of protein were subjected to SDS-PAGE and electro-transferred to membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (TBS-T) for 1 h, washed with TBS-T, incubated with primary antibodies overnight at 4°C, and then exposed to horseradish peroxidase-conjugated secondary antibodies. Antibodies targeting MMP1, MMP13, collagen type I alpha 1, (COLa1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA).
4.11. Statistical Analysis
Data are expressed as means and standard deviations (SDs). Data were analyzed by one-way ANOVA, followed by Duncan’s test (IBM SPSS Statistics Version 20.0, Chicago, IL, USA). Statistical significance was defined as p < 0.05 (a > b > c > d).