Protective Effect of Sulfated Polysaccharides from Celluclast-Assisted Extract of Hizikia fusiforme Against Ultraviolet B-Induced Skin Damage by Regulating NF-κB, AP-1, and MAPKs Signaling Pathways In Vitro in Human Dermal Fibroblasts

Our previous study evaluated the antioxidant activities of sulfated polysaccharides from Celluclast-assisted extract of Hizikia fusiforme (HFPS) in vitro in Vero cells and in vivo in zebrafish. The results showed that HFPS possesses strong antioxidant activity and suggested the potential photo-protective activities of HFPS. Hence, in the present study, we investigated the protective effects of HFPS against ultraviolet (UV) B-induced skin damage in vitro in human dermal fibroblasts (HDF cells). The results indicate that HFPS significantly reduced intracellular reactive oxygen species (ROS) level and improved the viability of UVB-irradiated HDF cells in a dose-dependent manner. Furthermore, HFPS significantly inhibited intracellular collagenase and elastase activities, remarkably protected collagen synthesis, and reduced matrix metalloproteinases (MMPs) expression by regulating nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs) signaling pathways in UVB-irradiated HDF cells. These results suggest that HFPS possesses strong UV protective effect, and can be a potential ingredient in the pharmaceutical and cosmetic industries.


Introduction
In humans, skin is the largest organ of the integumentary system. It undergoes chronological aging similar to other organs. Skin is in direct exposure to the outside environment and therefore it undergoes aging as a consequence of environmental damage [1]. Ultraviolet (UV) irradiation from sunlight is the primary environmental factor that induces human skin aging and results in pigment accumulation and wrinkle formation. Human skin is frequently affected by oxidative stress caused by continuous exposure to UV irradiation from sunlight. Skin exposed to UV and environmental oxidizing pollutants is associated with diverse abnormal reactions including inflammatory responses, epidermal hyperplasia, the breakdown of collagen, and melanin accumulation [2,3].
UV can be classified into three subtypes of UVA, UVB, and UVC, based on the wavelength. UVB has a medium wavelength and is thought to bring more cellular stress to humans compared to the other two subtypes [4][5][6]. UVB is known to be associated with human health through stimulating reactive oxygen species (ROS) generation [7,8]. The excessive ROS subsequently activate cell signaling pathways including nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs), which stimulate matrix metalloproteinases (MMPs) expression [9]. MMPs are a class of structurally similar enzymes, and play a major role in physiological and pathological tissue remodeling. The imbalance of MMP expression could lead to cartilage, cardiac, and cancer-related diseases [10]. MMPs degrade the collagenous extra cellular matrix (ECM) in connective tissues, which is the main factor of wrinkling. Therefore, an ideal MMP inhibitor or an agent that reduces the expression of MMPs may be effective against wrinkle formation, and could be thought as a promising candidate to be used as an ingredient in the cosmetic industry.
Marine organisms are rich resources of several natural compounds such as polyphenol, polysaccharide, sterol, and peptide, which possess various bioactivities including antioxidant, anticancer, anti-inflammatory, anti-obesity, antihypertensive, anti-diabetes, and UV protective activities [11][12][13][14][15][16][17][18][19][20]. Hizikia fusiforme (H. fusiforme) is an edible brown seaweed, which is distributed in the areas of the northwest Pacific, including Korea, China, and Japan. H. fusiforme has been utilized as a traditional medicine and functional food. It contains various compounds, which possesses several of bioactivities, especially polysaccharides. Many studies have reported that polysaccharides from H. fusiforme possess various bioactivities such as antioxidant, anti-inflammatory, anti-angiogenic, anticancer, osteoprotective, and immunostimulatory activities [21][22][23][24]. Our previous research displayed that sulfated polysaccharides from Celluclast-assisted extract of H. fusiforme (HFPS) possess strong free radical scavenging activity and protective effects on H 2 O 2 -induced oxidative stress in vitro in Vero cells and in vivo in zebrafish [25]. These results suggest that HFPS may possess photo-protective activity. However, the protective effects of HFPS against UVB-induced skin damage have not yet been reported. Thus, the purpose of the present study was to investigate the protective effect of HFPS against UVB-induced skin damage in vitro in human dermal fibroblasts (HDF cells).

HFPS Inhibits Collagenase from Clostridium Histolyticum and Elastase from Porcine Pancreas
Both collagenase and elastase belong to proteases that break down proteins. Collagenase is the enzyme that breaks the peptide bonds in collagen, which is the key component of animal ECM. Elastase is the enzyme that breaks down elastin, which is an elastic fiber. Collagen and elastin together determine the mechanical properties of the connective tissue. In human skin, degradation of collagen leads to decreased skin thickness, and degradation of elastin results in losing skin elasticity. These are the major characteristics of wrinkle formation in aged skin. Thus, a collagenase or elastase inhibitor could be thought as an agent to reduce skin aging.
In this study, the inhibitory effects of HFPS against commercial collagenase and elastase were measured. As shown in Figure 1, HFPS inhibits collagenase and elastase in a dose-dependent manner. The collagenase inhibitory rates of HFPS were 13.46%, 22.58%, and 49.77% at the concentrations of 50, 100, and 200 µg/mL, respectively ( Figure 1A); and the elastase inhibitory rates of HFPS were 18.78%, 38.14%, and 56.53% at the concentrations of 50, 100, and 200 µg/mL, respectively ( Figure 1B). These results indicate that HFPS may possess the activity against skin aging through inhibition of collagenase and elastase.

HFPS Promotes HDF Cell Proliferation and UVB Irradiation Damages HDF Cells
Skin is continuously exposed to UVB from sunlight, thus it is a major target of oxidative stress [26]. UVB-induced skin cell oxidative stress leads to cell damage, resulting in skin photoaging. Over the past decades, with industrial development, the oxidizing pollutants in the air and UV irradiated to the earth have increased. This is becoming a serious issue that threatens the skin health of humans. Therefore, finding nontoxic and effective agents that can protect dermic damage would be valuable for medical and cosmetic industries.
As the first step to evaluate protective effects and mechanisms of HFPS against UVB-induced skin damage in HDF cells, we employed various levels of UVB irradiation to induce cell damage in HDF cells, and determined the cytotoxicity of HFPS at different concentrations on HDF cells. As Figure 2 shows, UVB irradiation significantly decreased the viability of HDF cells in a dose-dependent manner ( Figure 2A). Furthermore, the optimal UVB dose applied to HDF cells based on the 50% growth inhibitory dose was determined to be 50 mJ/cm 2 . We then assessed the effect of HFPS alone on HDF cells. The cytotoxicity results ( Figure 2B) suggest that HFPS is non-toxic on HDF cells and promotes HDF cells proliferation in a dose-dependent manner. This promotion of proliferation was comparable in cells treated with 100 µg/mL and 50 µg/mL samples. From these results, 50 mJ/cm 2 was determined as the optimal UVB dose applied to HDF cells and 100 µg/mL was selected as the maximum concentration of HFPS for further study.

HFPS Improves Cell Viability and Scavenges Intracellular ROS Generated in UVB-Irradiated HDF Cells
UVB irradiation induces skin damage though stimulated intracellular ROS generation [27]. Recently, identification of ROS scavengers from natural resources has been given more attention. Our previous study isolated phlorotannins from marine algal, Ecklonia cava, and evaluated their UV protective effect. The results displayed that phlorotannins reduced intracellular ROS induced by UVB irradiation and improved cell viability in a dose-dependent manner [26]. Zeng et al. investigated the protective effect of polysaccharides from Ganoderma lucidum against UVB-induced photoaging. The results suggest that polysaccharides from Ganoderma lucidum significantly reduced ROS levels and improved the viability of UVB-irradiated cells [28].
In the present study, HDF cells were pretreated with HFPS and irradiated with UVB at a dose of 50 mJ/cm 2 . Cell viability was subsequently measured by MTT assay and intracellular ROS level was analyzed by DCF-DA assay. As Figure 3 shows, cell viability was decreased while intracellular ROS level was increased after UVB irradiation. Treatment with increasing concentrations of HFPS (25, 50, and 100 µg/mL) improved cell viability by 11.78%, 14.97%, and 19.21% ( Figure 3A) and intracellular ROS scavenging by 21.95%, 36.44%, and 48.14% ( Figure 3B). These results indicate that HFPS possesses a protective effect against UVB-induced cellular damage via ROS clearance in HDF cells. Cell viability was measured by MTT assay and intracellular ROS level was measured by DCF-DA assay. The data are expressed as the means ± SE (n = 3). * p < 0.05, ** p < 0.01 as compared to UVB-exposed group and ## p < 0.01 as compared to control group.

HFPS Inhibits Intracellular Collagenase and Elastase Activities in UVB-Irradiated HDF Cells
Aged skin is thin and inelastic due to degradation of collagen and elastin in the ECM of connective tissue. Collagenase and elastase are key enzymes during collagen and elastin degradation. UVB stimulates the activities of fibroblast collagenase and elastase in the dermis consequently causing wrinkle formation [29,30]. As Figure 4 shows, the relative collagenase and elastase activities of UVB-irradiated HDF cells were significantly increased compared with non-irradiated cells. However, relative activities of both enzymes were decreased in the cells pretreated with HFPS in a dose-dependent manner. These results suggest that HFPS may act as an inhibitor of fibroblast collagenase and elastase and may prevent wrinkle formation induced by UVB irradiation. The data are expressed as the means ± SE (n = 3). * p < 0.05, ** p < 0.01 as compared to UVB-exposed group and ## p < 0.01 as compared to control group.

HFPS Protects Collagen Synthesis and Reduces MMPs Expression Levels in UVB-Irradiated HDF Cells
MMPs, particularly MMP-2 and MMP-9, have been identified as being central to degradation of ECM [31]. In addition, MMP-1 degrades two major structural proteins, type I and type III collagen [32]. Collagen is synthesized as a precursor molecule, procollagen, which contains additional peptide sequences. These sequences are cleaved off during collagen secretion, thus, a number of sequences can indirectly reflect collagen synthesis level. We determined procollagen type I carboxy-terminal peptide (PIP) to investigate collagen synthesis level.
As Figure 5 shows, UVB irradiation significantly decreased collagen synthesis in HDF cells, and HFPS dose-dependently protects collagen synthesis ( Figure 5A). Furthermore, MMPs' expression levels were significantly increased in UVB-irradiated HDF cells but decreased in HFPS pretreated cells ( Figure 5B-F). These results suggest that HFPS effectively protects collagen synthesis and reduces the expression of MMPs. Collagen synthesis level was reflected by the amounts of PIP, and the amounts of PIP and MMPs were measured by the commercially ELISA kits, based on the manufacturer's instructions. The data are expressed as the means ± SE (n = 3). * p < 0.05, ** p < 0.01 as compared to UVB-exposed group and ## p < 0.01 as compared to control group.

HFPS Inhibits Nuclear Factor Kappa B (NF-κB) Activation, Reduces Activator Protein 1 (AP-1) Phosphorylation, and Suppresses Mitogen-Activated Protein Kinases (MAPKs) Activation in UVB-Induced HDF Cells
NF-κB is a protein complex that controls cytokine production, transcription of DNA, and cell survival. NF-κB plays an important role in immune responses, and dysregulation of NF-κB is associated with various diseases such as cancer, inflammation, and aging [33]. Many studies have reported that UVB irradiation can activate NF-κB, and the activation of NF-κB can induce MMPs expression [34][35][36]. As Figure 6 shows, UVB irradiation significantly increases nuclear levels of NF-κB (p65 and p50); however, HFPS treatment remarkably reduces nuclear NF-κB levels in UVB-irradiated HDF cells in a dose-dependent manner. The relative amounts of NF-κB expressions and AP-1 phosphorylation were compared with C23. The data are expressed as the means ± SE (n = 3). * p < 0.05, ** p < 0.01 as compared to UVB-exposed group and ## p < 0.01 as compared to control group.
MAPKs are a type of protein kinases, which are involved in directing cellular responses to different stimuli, such as heat shock, mitogens, and pro-inflammatory cytokines. MAPKs regulate various cell functions including proliferation, differentiation, gene expression, cell survival and apoptosis [37,38]. The activation of MAPKs occurs through the phosphorylation of p38, Jun N-terminal kinase (JNK), and extracellular-regulated protein (ERK) signaling pathways. AP-1(c-Jun) is a nuclear transcription factor, which is phosphorylated after the activated MAPKs were translocated to the nucleus [39]. Subsequently, the expression of MMPs is up-regulated [33,40]. In the present study, the activated AP-1 and MAPKs levels were detected by Western blot analysis. The results indicate that UVB irradiation significantly phosphorylates AP-1 and HFPS remarkably reduces the phosphorylated AP-1 (p-c-Jun) levels in a dose-dependent manner ( Figure 6). In addition, HFPS treatment effectively suppresses UVB-induced p38, JNK, and ERK phosphorylation in UVB-irradiated HDF cells (Figure 7). These results indicate that HFPS regulates NF-κB activation, AP-1 phosphorylation, and MAPKs activation in UVB-induced HDF cells. The data are expressed as the means ± S.E (n = 3). * p < 0.05, ** p < 0.01 as compared to UVB-exposed group and ## p < 0.01 as compared to control group.

Measurement of Inhibitory Effect on Collagenase from Clostridium Histolyticum
To measure the collagenase inhibitory activity, a weight of 1 mg of azo dye-impregnated collagen was mixed with 800 µL of 0.1 M Tris-HCl (pH 7.0), 100 µL of 200 units/mL collagenase (stock solution), and 100 µL sample and incubated at 43 • C for 1 h under shaking condition. Subsequently, the reaction mixture was centrifuged at 3000 rpm for 10 min and the absorbance of the supernatant was detected at 550 nm in a microplate reader (BioTek Synergy HT, Woburn, MA, USA).

Measurement of Inhibitory Effect on Elastase from Porcine Pancreas
The elastase inhibitory activity was evaluated base on a method reported by Kraunsoe et al. (1996) [41]. In brief, the reaction mixture contained 650 µL of 1.015 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide (dissolved in Tris-HCl, pH 8.0) and 50 µL of sample. The reaction mixture was vortexed and incubated for 10 min at 25 • C. After incubation, a volume of 50 µL of 0.0375 units/mL elastase enzyme solution was added to the reaction mixture and, following vortexing, the reaction mixture was incubated for 10 min at 25 • C in a water bath. The amount of released p-nitroaniline was assessed by measuring absorbance at 410 nm using a microplate reader.

Cell Culture and UVB Irradiation
HDF cells (ATCC ® PCS20101™) were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). HDF cells were cultured in DMEM and F-12 mixed with a ratio of three to one supplemented with 10% heat-inactivated FBS, 100 unit/mL of penicillin and 100 µg/mL of streptomycin. Cells were sub-cultured every 5 days. Cells were incubated at 37 • C under humidified atmosphere containing 5% CO 2 in an incubator (Sanyo MCO-18AIC CO 2 Incubator, Moriguchi, Japan). UVB irradiation was carried out using a UVB meter (UV Lamp, VL-6LM, Vilber Lourmat, France), equipped with a fluorescent bulb emitting 280-320 nm wavelength with a peak at 313 nm. HDF cells were irradiated at a dose of 50 mJ/cm 2 of UVB in 1 × PBS. Cell medium was subsequently replaced with serum free medium and incubated until analysis.

Cell Viability Assay
Cytotoxicity of HFPS on HDF cells was assessed by a colorimetric MTT assay. Briefly, cells were seeded at a concentration of 5.0 × 10 4 cells per well in 24-well plates. After 24 h, cells were treated with HFPS (25, 50, and 100 µg/mL) for 48 h, and their viabilities were determined by the method described previously [42,43].

Determination of Intracellular ROS Level and Cell Viability
For intracellular ROS level analysis, HDF cells were treated with HFPS and incubated for 30 min. Subsequently, cells were treated with DCFH-DA (stock, 500 µg/mL) and incubated for 30 min. After incubation, cells were exposed to UVB (50 mJ/cm 2 ) and incubated for 1 h at 37 • C. The fluorescence intensity of cells was determined according to the method described previously [44].
To analyze the protective effect of HFPS against UVB-induced cell damage, HDF cells were treated with HFPS and incubated for 2 h at 37 • C. Cells were then exposed to 50 mJ/cm 2 of UVB and incubated for 48 h. Cell viability was assessed by MTT assay using the protocol described previously [43,45].

Determination of Relative Intracellular Elastase and Collagenase Activities
HDF cells were seeded in 100 mm culture dishes at a density of 2.0 × 10 6 cells per dish and incubated for 24 h. Cells were pretreated with HFPS and incubated for 2 h. Following incubation, cells were irradiated with UVB. After 48 h incubation, cells were harvested and lysed with 0.1 M Tris-HCl (pH 7.6) buffer containing 1 mM PMSF and 0.1% Triton-X 100, followed by sonication for 5 min on ice. The lysates were centrifuged (4000 rpm, 20 min) at 4 • C. Supernatants were quantified for their protein content and were used as the fibroblastic enzyme solution. The relative elastase and collagenase activities were measured by the method described by Suganuma et al. [46].

Determination of Collagen Synthesis Level and MMPs Expression Levels by Enzyme-Linked Immunosorbent Assay (ELISA)
HDF cells were incubated with HFPS for 2 h, and exposed to UVB (50 mJ/cm 2 ). After 48 h incubation, the culture media were collected and used for assessment of MMPs expression levels and PIP level that reflect the level of collagen synthesis. The amounts of PIP and MMPs were measured by commercial ELISA kits, based on the manufacturer's instructions.

Western Blot Analysis
The effect of HFPS on the expressions of NF-κB, p-c-Jun, and MAPKs were assessed by Western blot analysis performed as described previously [43,47]. In brief, cells were pretreated with HFPS and irradiated with UVB. After 1 h (for MAPKs assay) or 6 h (for NF-κB and p-c-Jun assay) incubation, cells were harvested. Proteins were extracted with the PRO-PREP protein extraction kit (iNtRON Biotechnology, Sungnam, Korea). The protein level of each sample was measured by a BCA™ kit. Total proteins (50 µg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to pure nitrocellulose membranes. Membranes were blocked with 5% skim milk for 3 h at room temperature and incubated with primary antibodies overnight at 4 • C. After washing with TBS-T buffer, membranes were incubated with secondary antibodies for 3 h at room temperature. Finally, the protein bands were visualized using an ECL western blotting detection kit and exposed on X-ray films.

Statistical Analysis
The experiments were performed in triplicate. All data are expressed as the mean ± SE. Significant differences between the groups were determined using the unpaired Student's t-test (using Statistical Product and Service Solutions 11.5 statistical software). Values of * p < 0.05, ** p < 0.01, and ## p < 0.01 were considered as significantly different.

Conclusions
In conclusion, in the present study, the protective effects of HFPS against UVB-induced skin damage in vitro in HDF cells were investigated. The results indicate that HFPS significantly protected collagen synthesis and reduced MMPs expression in UVB-irradiated HDF cells by regulating NF-κB, AP-1, and MAPKs signaling pathways. These results suggest that HFPS possess strong UV protective effect and has potential to be used as an ingredient in pharmaceutical and cosmetic industries.