Protective effect of sulfated polysaccharides from a Celluclast-assisted extract of Hizikia fusiforme against ultraviolet B-induced photoaging in vitro in human keratinocytes and in vivo in zebrafsh

Human skin covers the body surface and is exposed to various environmental factors, such as temperature and ultraviolet (UV) irradiation. UV irradiation can impair the ability of basal keratinocytes to maintain skin homeostasis. UV can be classifed into three subtypes: UVA, UVB, and UVC, based on its wavelength. UVB has a medium wavelength (280–320 nm) that has various adverse efects on human skin including sunburn, edema, erythema, hyperpigmentation, immune suppression, photoaging, and cancer (Park et al. 2010). These adverse efects are frequently mediated by increased levels of reactive oxygen species (ROS). Dysfunctional skin loses the ability to protect itself against oxidative stress induced by ROS, with consequent damage to the cutaneous tissues, a process commonly known as "photoaging" (Zaid et al. 2007). Therefore, an ideal ROS scavenger or an agent that reduces ROS production may be efective against photoaging and could be a promising compound for use in the pharmaceutical and cosmeceutical industries.

Marine algae are rich in bioactive compounds, such as polyphenols, sterol, peptides, and polysaccharides, which possess various bioactivities including anticancer, antiinfammatory, anti-obesity, anti-hypertensive, antioxidant, and UV-protective activities (Ariede et al. 2017; Wang et al. 2018c). Marine algae are especially rich in sulfated polysaccharides, possessing numerous health benefts (Wijesekara et al. 2011; Ruocco et al. 2016). Recently, marine algaederived sulfated polysaccharides have been reported in many studies to possess cosmeceutical efects. Ji et al. evaluated the UV-protective efects of a sulfated polysaccharide fraction from Sargassum fusiforme (SFP-P1) in human keratinocytes (HaCaT cells) and found that SFP-P1 reduced oxidative stress and suppressed matrix metalloproteinase (MMP) expression (Ji et al. 2017). Kim et al. investigated the anti-photoaging efects of low molecular-weight fucoidan (LMF) isolated from Ecklonia cava on UVB-irradiated mice and found that LMF reduced oxidative stress, suppressed infammation, and inhibited the expression of MMPs in UVB-irradiated mice (Kim et al. 2018).

Hizikia fusiforme (H. fusiforme) is one of most popular edible algae, mainly cultured in the Northwest Pacifc, including China, Korea, and Japan. It has been used as a food ingredient and herbal medicine for hundreds of years (Li et al. 2006). H. fusiforme contains a particular high amount of polysaccharides that possess various bioactivities (Wang et al. 2018a). In a previous study, we suggested that sulfated polysaccharides isolated from a Celluclast-assisted extract of H. fusiforme (HFPS) possess strong antioxidant activities in vitro and in vivo (Wang et al. 2018b). These results suggest that HFPS possesses photo-protective potential. Until now, the anti-photoaging efects of HFPS had not been investigated. Therefore, in this study, we investigated the protective efect of HFPS against UVB-induced photoaging in vitro in HaCaT cells and in vivo in zebrafsh.

Over the past three decades, the use of marine algae-derived polysaccharides for biological, biomedical, nutriceutical, and cosmeceutical applications has been reported by many investigators. Sulfated polysaccharides are one of the major constituents of marine brown algae (Li et al. 2006). The edible brown alga, H. fusiforme, contains various bioactive compounds and has a long history as a food and medical ingredient. H. fusiforme is especially rich in sulfated polysaccharides, which have various health benefts (Wang et al. 2012). Dobashi et al. isolated sulfated polysaccharides from H. fusiforme and evaluated their anticoagulant activity (Dobashi et al. 1989). Jeong et al. reported the immune-modulating activities of sulfated polysaccharides extracted from H. fusiforme (Jeong et al. 2015). In our previous study, we isolated sulfated polysaccharides from a Celluclast-assisted extract of H. fusiforme (HFPS) and evaluated its antioxidant activity. The results indicated that Celluclast-assisted extract (HF) showed a high extraction yield (44.00%) and polysaccharide content (48.05%). HFPS was isolated from HF by ethanol precipitation and the yield was 29.35%. HFPS is sulfated polysaccharides that contains 55.05% polysaccharides and 7.78% sulfate content. HFPS was comprised of fucose (53.53%), glucose (5.95%), galactose (23.15%), and xylose (17.37%). In addition, HFPS possesses strong ROS scavenging efects as well as cytoprotective efects against hydrogen peroxide-induced cell death in vitro in Vero cells and in vivo in zebrafsh (Wang et al. 2018b). These results suggest that HFPS possesses photo-protective potential. Thus, in this study, we investigated the protective efect of HFPS against UVB-induced photoaging in vitro in HaCaT cells and in vivo in zebrafsh.

As shown in Fig. 1a, there was no signifcant decrease in the viability of HaCaT cells treated with diferent concentrations of HFPS, suggesting that HFPS is not toxic to HaCaT cells at concentrations ranging from 6.25 to 100 μg/mL. Thus, 100 μg/mL was considered a safe concentration and used in the rest of the study. The protective efect of HFPS against UVB-induced HaCaT cell damage was evaluated by measuring intracellular ROS levels and the viability of UVB-irradiated HaCaT cells. As shown in Fig. 1b, UVB signifcantly increased intracellular ROS levels; however, the intracellular ROS levels of HFPS-treated HaCaT cells were dose-dependently decreased. As shown in Fig. 1c, the viability of UVB-irradiated HaCaT was 52.45%, whereas the viabilities of HFPS-treated HaCaT cells were 56.54%, 61.85%, and 86.37% at concentrations of 25, 50, and 100 μg/mL, respectively. In addition, an investigation of UVB-induced apoptosis showed that the apoptosis body formation and the number of apoptosis bodies in the cells irradiated with UVB were signifcantly decreased in a dose-dependent manner (Fig. 2). These results indicated that HFPS possesses potent UV-protective efects owing to the scavenging of intracellular ROS, improving cell viability, and reducing apoptotic body formation in UVB-irradiated HaCaT cells.

Figure 1.  Protective efect of HFPS against UVB-induced HaCaT cell damage. a Cytotoxicity of HFPS on HaCaT cells; b intracellular ROS levels in UVB-irradiated HaCaT cells; c viability of UVB-irradiated HaCaT cells. Cell viability was measured by an MTT assay and intracellular ROS levels were determined by a DCF-DA assay. The experiments were conducted in triplicate, and the data are expressed as the mean ±standard error (SE). ^*P < 0.05, ^**P < 0.01 when compared to the UVB-treated group and ^##P < 0.01 when compared to the control group

Figure 2.  Apoptotic body formation levels in UVB-irradiated HaCaT cells. a Nuclear morphology of normal cells; b UVB-irradiated cells; c, d, e HFPS-treated cells; f the relative apoptotic body formation levels of UVB-irradiated HaCaT Cells. The apoptotic body formation was observed under a fuorescence microscope after Hoechst 33342 staining. Relative apoptosis levels were measured using Image J software. ^*P < 0.05, ^**P < 0.01 as compared to the UVB-treated group and ##P < 0.01 as compared to the control group

Zebrafsh (Danio rerio) are a popular in vivo model used to study human disease. Zebrafsh irradiated with UVB were successfully used to evaluate the anti-photoaging efects of algae-derived compounds in our previously published studies (Cha et al. 2012; Ko et al. 2011). Therefore, zebrafsh were used to investigate the in vivo photo-protective efect of HFPS in this study. In this study, ROS generation, cell death, nitric ox 50, and 100 μg/mL, respide (NO) production, and lipid peroxidation levels were measured using diferent fuorescence probes. As shown in Fig. 3, the ROS levels in zebrafsh irradiated with UVB were signifcantly increased compared to non-irradiated zebrafsh. However, the ROS levels in the HFPS-treated zebrafish decreased in a dose-dependent manner. In addition, the cell death level of UVB-induced zebrafsh was 120.15% increased comparing to non-irradiated zebrafsh (Fig. 4); however, the cell death levels in HFPS-treated zebrafsh were 34.60%, 65.28%, and 89.14% decreased at the concentration of 25, 50, and 100 μg/mL, respectively. As shown in Fig. 5, NO generation in the zebrafsh irradiated with UVB was signifcantly increased compared to non-irradiated zebrafsh. Whereas the NO generation of HFPS-treated zebrafsh were dose-dependently decreased. Furthermore, the lipid peroxidation level of UVB-irradiated zebrafsh was 194.43% compared to the nonirradiated group (100%). However, HFPS markedly reduced lipid peroxidation levels to 158.99%, 125.57%, and 115.04% at concentrations of 25, 50, and 100 μg/mL, respectively (Fig. 6). These results demonstrated that HFPS is efective against UVB-induced in vivo damage, reducing ROS levels, suppressing cell death, inhibiting NO production, and attenuating lipid peroxidation in zebrafsh.

Figure 3.  Protective efect of HFPS against UVB-induced ROS production in zebrafsh. a Zebrafsh under fuorescence microscope; b levels of ROS. The relative fuorescence intensities of zebrafsh were determined using Image J software. The experiments were conducted in triplicate, and the data are expressed as the mean± standard error (SE). ^**P < 0.01 when compared to the UVB-treated group and ##P < 0.01 when compared to the control group

Figure 4.  Protective efect of HFPS against UVB-induced cell death in zebrafsh. a Zebrafsh under fuorescence microscope; b measured levels of cell death. The relative fuorescence intensities of zebrafsh were determined using Image J software. The experiments were conducted in triplicate, and the data are expressed as the mean ±standard error (SE). ^**P < 0.01 when compared to the UVB-treated group and ##P < 0.01 when compared to the control group

Figure 5.  Protective efect of HFPS against UVB-induced NO production in zebrafsh. a Zebrafsh under fuorescence microscope; b levels of NO. The relative fuorescence intensities of zebrafsh were determined using Image J software. The experiments were conducted in triplicate, and the data are expressed as the mean ± standard error (SE). ^**P < 0.01 when compared to the UVB-treated group and ##P < 0.01 when compared to the control group

Figure 6.  Protective efect of HFPS against UVB-induced lipid peroxidation in zebrafsh. a Zebrafsh under fuorescence microscope; b levels of lipid peroxidation. The relative fuorescence intensities of zebrafsh were determined using Image J software. The experiments were conducted in triplicate, and the data are expressed as the mean± standard error (SE). ^**P < 0.01 when compared to the UVBtreated group and ^##P < 0.01 when compared to the control group

The in vitro and in vivo UV-protective efects of HFPS were investigated in the present study. The results indicated that HFPS possesses strong in vitro and in vivo UV-protective efects. In conclusion, the present study results suggest that HFPS could efectively attenuate skin damage induced by UVB irradiation, so could be considered for use as an ingredient in the pharmaceutical and cosmeceutical industries. In addition, the pure fucoidans of HFPS need to be further purifed and examined to determine their physicochemical characteristics as well as their potential functions.

The fuorescent probe 2′, 7′-dichlorodihydrofurescin diacetate (DCFH-DA), acridine orange, diaminofuorophore 4-amino-5-methylamino-2′, 7′-difuorofuorescein diacetate (DAF-FM DA), 1, 3-Bis (diphenylphosphino), propane (DPPP), 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma Co. (St. Louis, MO, USA). Dulbecco's modifed Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). All other chemicals used in this study were of analytical grade.

HFPS was prepared in our previous study, the separation, and analysis procedures were described by Wang et al. (Wang et al. 2018b). In brief, H. fusiforme was hydrolyzed by Celluclast and the Celluclast-assisted extract of H. fusiforme (HF) was obtained. HF was precipitated by ethanol and the crude polysaccharides from HF were obtained and named as HFPS. The polysaccharide content, sulfate content, and the monosaccharide constituents of HFPS were then determined.

The HaCaT cell line was purchased from Korean Cell Line Bank. The HaCaT cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 units/mL) and streptomycin (100 µg/mL) at 37 ℃ under a humidifed atmosphere containing 5% CO₂. HaCaT cells were subcultured every 3 days and seeded at a density of 1.0 × 10⁵ cells/mL in a 24-well plate. UVB irradiation was imposed using a UVB meter (UV Lamp, VL-6LM, Vilber Lourmat, France) with a fuorescent bulb emitting at 280–320 nm with a peak at 313 nm. HaCaT cells were irradiated at a dose of 30 mJ/cm² of UVB in PBS (Wang et al. 2017). After UVB irradiation, cells were incubated with serum-free DMEM media until analysis.

The cytotoxicity of HFPS on HaCaT cells was evaluated with an MTT assay. HaCaT cells were seeded and incubated for 24 h. The cells were treated with HFPS at a fnal concentration of 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, 50 µg/ mL, and 100 µg/mL. After 24 h of incubation, MTT stock solution (2 mg/mL) was added to each well. After 3 h, the supernatant was aspirated, the formazan crystals were dissolved in DMSO, and the absorbance was measured by a microplate reader at 540 nm.

To determine the efect of HFPS on UVB-irradiated HaCaT cells, intracellular ROS levels, cell viability, and apoptosis body formation were measured. The intracellular ROS levels of UVB-irradiated HaCaT cells were determined using a DCF-DA assay and cell viability was measured by an MTT assay following the protocols described in a previous study (Wang et al. 2017). Apoptosis body formation in UVBirradiated HaCaT cells was determined by Hoechst 33342 nuclear staining according to the method described by Wijesinghe et al. (Wijesinghe et al. 2013). The stained cells were observed using a fuorescence microscope equipped with a Cool SNAP-Pro color digital camera (Olympus, Japan). Apoptosis prevalence was determined using Image J software.

Adult zebrafsh were purchased from a commercial market (Seoul Aquarium, Korea) and maintained per the manufacturer's protocol (Cha et al. 2011). The embryos were collected after natural spawning induced by light.

At 2 day post-fertilization (dpf), the zebrafsh larvae (15/group) were incubated with HFPS (25 µg/mL, 50 µg/ mL, and 100 µg/mL) for 1 h. They were then washed with embryo media and exposed to UVB (50 mJ/cm²) (Heo and Jeon 2009). After 6 h, the zebrafsh were stained with DCFH-DA (20 μg/mL, 1 h), acridine orange (10 μg/mL, 30 min), DAF-FM-DA (10 μM, 3 h), and DPPP (3 μM, 1 h) to measure ROS, cell death, NO production, and lipid peroxidation, respectively. The zebrafsh were anesthetized and photographed under the microscope with a Cool SNAPProcolor digital camera (Olympus, Japan). The fuorescence intensity of the individual zebrafsh was quantifed using image J software.

All experiments were conducted in triplicate. The data are expressed as the mean ± standard error (SE). One-way ANOVA was used to compare the mean values of each treatment using SPSS 12.0. Signifcant diferences between the means were identifed by Tukey's test. Signifcance was established as ^*P < 0.05 or ^**P < 0.01 as compared to the UVB-treated group, and ^##P < 0.01 as compared to the control group.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1A6A1A03033553).

Lei Wang and You-Jin Jeon conceived and designed the experiments; Lei Wang, Jae Young Oh, Hye-Won Yang, Hyun Soo Kim performed experiments and analyzed data; Lei Wang and You-Jin Jeon wrote the paper.

The authors declare that they have no confict of interest.

We declare that all applicable international, national, and/or institutional guidelines for sampling, care, and experimental use of organisms for the study have been followed and all necessary approvals have been obtained.