The skin protects the body from environmental elements such as sunlight and harmful factors. Ultraviolet (UV) rays affect the skin the most; therefore, photoaging progresses when ultraviolet rays continue to be irradiated. Among the ultraviolet rays, ultraviolet B (UVB) exposure induces increased epidermal thickness because keratinocytes continue to divide on a molecular basis, and the skin tissue structure changes after exposure to UV rays, leading to dry skin, hyperpigmentation, desquamation, and hyperkeratosis (Salminen et al., 2022).

Excessive exposure to UVB rays and increased levels of reactive oxygen species (ROS) in the skin can cause the breakdown of collagen, hyaluronan, and proteoglycan in the extracellular matrix (ECM) and shut down the synthesis of new collagen in the dermis (Pillai et al., 2005). In addition, photodamage results from the accumulation of abnormal elastin in the dermis, and matrix metallopeptidases (MMP) have been implicated in this process (Naylor et al., 2011). As a result, the skin matrix in the dermis is destroyed by UVB, causing brown spots and wrinkles (Ceni et al., 2022).

Wrinkles, a characteristic of photoaging, are generated by destruction of the ECM and fibroblasts in the dermis. Collagen, a major component of the ECM, is decomposed and fragmented, resulting in decreased collagen content (Shin et al., 2019). The main reason for this is the increase in the levels of ROS, cytokines, and MMP-1 in the skin (Oh et al., 2011). Additionally, a decrease in elastin and collagen levels can cause coarse wrinkles in the skin. Moreover, fibroblasts around the ECM interact with collagen type 1, and when the ECM collapses owing to photoaging, fibroblasts are reduced in size, exhibit decayed elongation, and change in morphology (Shin et al., 2019).

Currently, there is a growing interest in the role of functional foods or dietary supplements in the care and treatment of UVR-induced skin photodamage. Indeed, it already revealed that oral supplements such as vitamins, unsaturated fatty acids, collagen peptides, and flavonoids are absorbed in the body, reach the skin through the blood, and help improve skin functions against overexposure to UVRs (Boelsma et al., 2003). Interestingly, rich sources, including fatty fish, such as salmon, sardines, trout, and herring, promote skin health. Recently, the focus of functional foods derived from fish is primarily on collagen peptides, and there remains a need to develop new materials that can prevent and alleviate the complex symptoms of UV skin.

Proteoglycans have potential applications as cosmetic materials to protect the skin barrier against damage (Shin et al., 2019). Proteoglycans are structurally abundant components of the ECM, collagen fibers, and intracellular vesicles that facilitate cell migration and maintain the tissue structure. It is primarily composed of chondroitin sulfate and keratin sulfate (Hascall and Sajdera, 1970). They are linked to the hyaluronate backbone by linked proteins, and glycosaminoglycans (GAG) are attached to the core proteins of proteoglycans. Sulfated GAGs linked to core proteins have various roles in the skin.

Especially, it is well known that proteoglycan derived from salmon nasal cartilage is aggrecan, and its functions have been widely studied (Kakizaki et al., 2011). Indeed, numerous studies conducted in in vitro studies have revealed that salmon nasal cartilage-derived proteoglycan (SPG) plays an important role as a growth factor in normal human dermal fibroblasts (NHDF) via extracellular signal-regulated kinase (ERK) activation, indicating that SPG maintains skin homeostasis (Goto et al., 2012). Furthermore, in vivo studies revealed that oral supplementation with SPG inhibited UVB-induced skin aging (e.g., erythema, transepidermal water loss (TEWL), and decreased hydration of epidermal and dermal hypertrophy) and improved skin health compared to that in control mice (Sano et al., 2017). Therefore, we aimed to confirm its skin-protective efficacy in mice with a damaged skin barrier by UVB irradiation and to explore its detailed mode of action.

In this study, the effectiveness of SPG in moisturizing the skin and ameliorating wrinkles was evaluated after oral administration in an experimental model damaged by UVB. We also investigated the molecular mechanisms of action of SPG in UVB-induced skin photoaging. Our data demonstrates the preventive capability of SPG as a functional food ingredient against UVB-induced skin photodamage.

Sample preparation

The SPG used in this study was lyophilized powder extracted from salmon nasal cartilage using 4% acetic acid (JVECOL K, Lot. 212463). The proteoglycan content by the previously reported analysis method (Takahashi et al., 2018) was 21.5%, and the crude protein and chondroitin sulfate contents were 7% and 320 mg/g, respectively, as analyzed according to the Health Functional Food Code of the Ministry of Food and Drug Safety, Korea. Collagen peptide (CP) obtained from Naturetech Co., Ltd. (Cheonan, Korea). JVECOL K was provided by Ichimaru Pharcos, Co. Ltd (Gifu, Japan).

Cell culture

Human immortalized keratinocytes (HaCaT) and NHDF were purchased from AddexBio (San Diego, CA, USA) and the American Type Culture Collection (Rockville, MD, USA), respectively. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (GIBCO, Invitrogen) at 37°C in a 5% CO₂ incubator.

UVB irradiation and sample treatment

UVB irradiation and SPG treatment were performed as described previously with minor modifications (Subedi et al., 2017). HaCaT and NHDF cells were seeded in culture dishes and washed twice with phosphate buffered saline (PBS). Each cell line was suspended in a thin layer of PBS and exposed to UVB (60 mJ/cm²) using an UVB irradiation machine (Bio-Link BLX-312; Vilber Lourmat GmbH, Marne-la-Vallée, France). The cells were then washed twice with PBS and immediately treated with various SPG concentrations in serum-free DMEM.

Cell viability

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HaCaT cells (5×10⁵ cells/mL) were seeded in 96-well plates and incubated in a 5% CO₂ incubator for 24 h. After the cells were exposed to UVB (60 mJ/cm²) as described above, they were treated with SPG (5, 10, and 25 µg/mL) and CP (250 µg/mL, PC) for 1 d. Afterward, 0.5 mg/mL of MTT solution was added to each well and incubated at 37°C for 1 h. The solution was discarded and 200 μL of dimethyl sulfoxide was added to resolve the formed formazan. Absorbance was measured at 570 nm using a microplate reader (VersaMax, Molecular Devices, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

HaCaT and NHDF cells were irradiated with UVB (60 mJ/cm²) and treated with SPG (5, 10, and 25 µg/mL) or CP (250 µg/mL) for 1 d. To measure the levels of hyaluronan and MMP-1, the conditioned medium was collected from the treated group. Activity was analyzed using an ELISA kit (R&D Systems, Minneapolis, MN, USA). Following the manufacturer’s protocol, the absorbance of the samples was measured at 450 nm using a microplate reader (Kang et al., 2018).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RNA from HaCaT cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA concentration and purity were measured by determining the absorbance ratio using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and 500 ng of cDNA was prepared using the Takara PrimeScript RT reagent (Takara, Tokyo, Japan). The mixture was prepared using Takara TB Green Premix EX Taq (Takara), cDNA and primers were mixed, and RT-qPCR was performed. mRNA expression levels were determined by normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels using the 2^∆∆Ct method. Each primer pair was used for RT-qPCR. Human glucosylceramide synthase (hGCS): forward primer, 5′-ATGTGTCATTGCCTGGCATG-3′ and reverse primer, 5′-CCAGGCGACTGCATAATCAAG-3′; GAPDH: forward primer, 5′-AAGGTGAAGGTCGGAGTCAAC-3′ and reverse primer, 5′-GGGGTCATTGATGGCAACAATA-3′.

Animal Experiment Schedule

Hairless SKH-1 mice (male, 5 weeks old) were obtained from Orient Bio Inc. (Seongnam, Korea) and acclimated for one week before the start of the experiments (23 ± 1°C; 60 ± 5% humidity) under 12/12 h light/dark cycles. For UVB irradiation, the mice were housed in specially designed cages. The SKH-1 hairless mice were irradiated (200 mJ/cm²) with a UV irradiation system (Bio-Link BLX-312; Vilber Lourmat GmbH). The UV irradiation unit used was the same as that used in our previous study (Kang, et al., 2018). Male SKH-1 hairless mice were randomly divided into six groups (n=8): control, UVB, UVB + CP (1000 mg/kg/d), and UVB + SPG (1 or 5 mg/kg/d). All samples were dissolved in saline and administered orally daily (Fig. 1A). All experiments were performed in accordance with the Care and Use of Laboratory Animals (LCDI-2022-0038) approved by the Institutional Animal Care and Use Committee of Gachon University (Inchon, Korea).

Figure 1. Effect of salmon nasal cartilage proteoglycan (SPG) on skin wrinkle formation, TEWL, hydration, and histological changes in UVB-irradiated mouse model. (A) Schematic diagram of UVB-irradiated SKH-1 hairless mouse model. (B) Changes in body weight during the in vivo study. (C) Photographs of the skin surface and replica impression of the mouse dorsal skin. Histogram of dorsal wrinkles using a video camera and analyzed using ImageJ. (D) Changes in TEWL and hydration after repeated irradiation with UVB using the Derma Combo System. (E) Histological changes in the dorsal skin exposed to UVB irradiation after eight weeks. Ctrl: saline administration without 200 mJ/cm² UVB irradiation, NC: saline administration with 200 mJ/cm² UVB irradiation, PC: 1000 mg/kg collagen peptide (CP) administration with 200 mJ/cm² UVB irradiation, SPG: 1 and 5 mg/kg salmon nasal cartilage proteoglycan. The values are presented as mean ± SEM (n=8). *p<0.05, **p<0.01, and ***p<0.001 vs. Ctrl, ^#p<0.05, ^##p<0.01, and ^###p<0.001 vs. NC.

Evaluation of skin barrier function and skin moisturization

The TEWL and hydration were evaluated using a Dermalab Combo system (Cortex Technology, Hadsund, Denmark). Measurements were recorded 30 s after the TEWL curve had stabilized. Three measurements of the same skin area were obtained and averaged (Cho et al., 2019; Kang et al., 2019).

Wrinkle measurement

For wrinkle analysis, skin replicas were collected using a replica analysis system (Repliflo, Clinical & Derm, Dallas, TX, USA) and measured using a microwrinkle camera to estimate wrinkle depth (VC 98, Visioscan, Köln, Germany).

Ceramide content in skin tissue

The epidermis was crushed and extracted using chloroform/methanol 2:1 (v/v), and the chloroform layer containing lipids was separated. The separated lipids were then dried under N2 gas and dissolved in chloroform. Sequential lipids from the epidermis were analyzed using high-performance liquid chromatography (HPLC). The assay was performed using a Waters system (Waters Corporation, Milford, MA, USA) equipped with a photodiode-array detector. Kromasil C18 column (250 mm×4.6 mm, 5 µm) was used, and mobile phase consisted of methanol (solvent A) and distilled water (solvent B). The gradient conditions of the mobile phase were 0-5 min, 100%; 5-30 min, 0-100%; 30-55 min, 100-0% as percentage of solvent B. The injection volume was 10 µL and the flow rate was set to 1 mL/min. The ceramide fraction, separated by an absorbance of 215 nm, was quantified using an external ceramide standard, and normalized by measuring the protein content in the epidermis and dermis (Smith et al., 1981; Yano et al., 1998; Groener et al., 2007; Bakar et al., 2022).

Skin histology and immunohistochemistry (IHC) assay

Dorsal skin was fixed in 10% formalin overnight at 4°C, and each tissue was dehydrated with several concentrations of ethanol and washed with xylene. All tissues were paraffin-embedded and fixed sections with a thickness of 4 μm were fabricated. To measure the skin thickness, deparaffinized sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO, USA). To analyze total collagen density, deparaffinized sections were stained with Masson’s trichrome (Sigma-Aldrich). For immunohistochemistry, the deparaffinized sections were incubated with primary antibodies, filaggrin (dilution 1:100), involucrin (dilution 1:100), Aquaporin-3 (AQP-3, dilution 1:100), and procollagen type 1 (dilution 1:100) at 4°C for 1 d. Next, each slide was incubated with secondary rabbit and mouse IgG antibodies (dilution 1:200) for 1 h, followed by incubation with an avidin-biotin horseradish peroxidase complex (Vector Laboratories, Newark, CA, USA). All samples were analyzed and photographed under a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) at 100× magnification (Kang et al., 2018; Bang et al., 2020; Mintie et al., 2020).

Western blot analysis

HaCaT and NHDF cells were plated in 60 mm dishes for 1 d. The cells were then treated with SPG and CP as described above. The skin of SKH-1 hairless mice was collected, and the epidermal layers were separated using dispase II (catalog no. 04942078001; Roche Diagnostics, Almere, The Netherlands). The lysed cells and separated epidermis were lysed with Pro-Prep^TM solution (iNtRON Biotechnology, Seoul, Korea) and centrifuged at 10,000×g for 30 min at 4°C. Protein concentration was estimated using the Bradford assay (Bio-Rad, Hercules, CA, USA). Forty micrograms of protein were subjected to 6–0% (sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membranes in the presence of 10% methanol. Membranes were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 2 h and incubated with involucrin, filaggrin, AQP3, hyaluronan synthase (HAS1), ERK, phospho-ERK (p-ERK), c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, elastin, α-tubulin, and GAPDH with 5% bovine serum albumin in TBST overnight at 4°C. After overnight incubation, the membranes were washed thrice with TBST and incubated with a secondary antibody (rabbit anti-goat IgG-HRP; mouse anti-rabbit IgG-HRP, Santa Cruz Biotechnology, Dallas, TX, USA). Protein bands were visualized using Pierce ECL western blotting substrate (Thermo Fisher Scientific) and quantified using a ChemiDoc (Bio-Rad) (Park et al., 2021; Ahn et al., 2022; Liang et al., 2022).

Statistical analysis

Significant differences between groups were determined by one-way analysis of variance (ANOVA) using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). *p value <0.05 was considered statistically significant. Results are presented as mean ± standard error of the mean (SEM).

Changes of wrinkle formation in the dorsal skin of SKH-1 mice treated with SPG

To evaluate the effects of SPG on UVB-irradiated SKH-1 mice, we measured the body weight of each group (Fig. 1B). None of the groups showed any weight loss, indicating that body weight was not affected by UVB exposure or sample administration. The assessment revealed visible wrinkle formation in the skin of the mice treated with UVB, CP, and SPG (Fig. 1C). UVB-irradiated (NC) group showed roughness surface (12.02 ± 0.68 V/mm³, *p<0.05) in the dorsal skin. However, coarse wrinkles were reduced in mice groups which oral administrated with SPG (1 mg/kg/d of SPG group, 8.62 ± 0.49 V/mm³; 5 mg/kg/d of SPG group, 7.38 ± 0.14 V/mm³) after UVB exposure. In particular, the 5 mg/kg/d of SPG group (^###p<0.001) revealed the highest inhibitory effect of wrinkle formation in comparison with 1000 mg/kg/d of CP group (9.42 ± 0.10 V/mm³, ^##p<0.01).

Changes of TEWL, hydration, and epidermal thickness in the dorsal skin of SKH-1 mice treated with SPG

The effects of SPG were investigated by measuring basal TEWL and stratum corneum water content of the epidermal barrier. TEWL and hydration differed in the NC group. TEWL increased, and hydration decreased in the NC group. When the skin is irradiated with UVB, it becomes dry, corneous, and exhibits moisture loss. In the case of the TEWL in the SPG group (Fig. 1D), transepidermal water was lost for three weeks, but a significant difference in water loss was observed in all groups treated with SPG after four weeks (^###p<0.001). In addition, SPG (1 and 5 mg/kg/d, ^###p<0.001) inhibited the UVB-induced decreased in hydration level after four weeks of UVB irradiation compared with CP (1000 mg/kg/d, ^###p<0.001) (Fig. 1D). One of the representative features of UVB photoaging is an increase in skin thickness (Fig. 1E). Based on the histological examination results, the NC group (164.63 ± 7.68 µm, ***p<0.001) showed significantly increased skin thickness compared to the normal group (24.11 ± 1.54 µm). The 1 mg/kg/d of SPG group (156.21 ± 8.58 µm) group did not differ from the UVB group, but the thickness of the epidermis treated with 5 mg/kg/d of SPG group (54.00 ± 3.86, ^###p<0.001) µm was improved epidermis thickness more than 1000 mg/kg/d of CP group (61.36 ± 5.66, ^###p<0.001).

Changes in filaggrin, involucrin, and AQP3 levels in the dorsal skin of SKH-1 mice treated with SPG

As shown in Fig. 2A, UVB irradiation of hairless mouse skin significantly decreased the production of involucrin, filaggrin, and AQP3, which are involved in skin moisturization, as quantified by histochemical analysis. In the epidermis, the NC group displayed lower staining levels of involucrin (60.32 ± 2.37%, *p<0.05), filaggrin (58.29 ± 4.51%, **p<0.01), and AQP3 (45.17 ± 7.55 µm², *p<0.05). Whereas SPG groups were significantly restored the levels of involucrin (1 mg/kg/d of SPG group, 71.76 ± 13.59%; 5 mg/kg/d of SPG group, 105.94 ± 5.09%), filaggrin (1 mg/kg/d of SPG group, 56.80 ± 4.26%; 5 mg/kg/d of SPG group, 83.23 ± 1.75%), and AQP3 (1 mg/kg/d of SPG group, 80.62 ± 18.49%; 5 mg/kg/d of SPG group, 98.25 ± 4.94%) in a dose-dependent manner (Fig. 2A). Histochemical analysis confirmed the protein levels of involucrin, filaggrin, and AQP3 in the SKH-1 mice. Filaggrin and involucrin are proteins involved in the differentiation of keratinocytes into corneocytes, and AQP3 is related to water movement in the epidermis. We explored the protein expression levels of filaggrin, AQP3, and involucrin in the epidermis of mice after seven weeks of SPG treatment (Fig. 2B). Interestingly, the group treated with 5 mg/kg/d SPG showed the highest increase in protein levels (filaggrin, ^#p<0.05; involucrin, ^##p<0.01; AQP3, ^##p<0.01) compared with the NC group. This suggests that SPG has a potential moisturizing effect on the UVB-irradiated model.

Figure 2. Effect of salmon nasal cartilage proteoglycan (SPG) on proteins related to keratinization and water movement in a UVB-irradiated mouse model. (A) Immunohistological changes in filaggrin, involucrin, and AQP3 in the skin of SKH-1 mice exposed to UVB irradiation for 8 weeks. (×40; bar size 100 μm). (B) Western blot was used to measure the expression levels of filaggrin, involucrin, and AQP3 in skin of SKH-1 mice exposed to UVB irradiation for eight weeks. All proteins were normalized to GAPDH. Ctrl: saline administration without 200 mJ/cm² UVB irradiation, NC: saline administration with 200 mJ/cm² UVB irradiation, PC: 1000 mg/kg collagen peptide (CP) administration with 200 mJ/cm² UVB irradiation, SPG: 1 and 5 mg/kg salmon nasal cartilage proteoglycan. The values are presented as mean ± SEM (n=8). *p<0.05 and **p<0.01 vs. Ctrl, ^#p<0.05 and ^##p<0.01, vs. NC.

Effect of SPG on the expression of skin barrier and moisturizing factors

In addition, the protein expression levels of factors related factors to skin moisturization and barrier function (profilaggrin, filaggrin, involucrin, HAS1, and AQP3) were evaluated using western blot to determine the role of SPG in UVB-irradiated HaCaT cells (Fig. 3A). In the UVB-irradiated group, the protein levels of profilaggrin/filaggrin (***p<0.001) and involucrin (***p<0.001) increased, but the levels in the treated SPG groups were lower than those in the CP group. It seems to be involved in increasing the transfer from profilaggrin to filaggrin, and suggests that SPG may protect the skin barrier from UVB. In addition, the expression of AQP3, a factor involved in water movement, was upregulated by SPG. In addition, the levels of HAS1 upon treatment with SPG were upregulated compared to those in UVB-irradiated HaCaT cells, indicating that SPG might be engaged the hyaluronan content via increasing HAS1 protein levels after SPG treatment.

Figure 3. Effect of salmon nasal cartilage proteoglycan (SPG) on protein expression of keratinized, moisturized factors, and MAPKs pathway in UVB-irradiated HaCaT cells. (A) Western blot analysis to measure the expression of keratinized (profilaggrin/filaggrin, involucrin, and AQP3) and moisturized (AQP3 and HAS1) factors in UVB-irradiated HaCaT cells. (B) Western blot was used to measure the expression levels of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 in UVB-irradiated HaCaT cells. All protein levels were normalized to those of GAPDH. The values are presented as mean ± SEM (n=3-4). **p<0.01 and ***p<0.001 vs. Ctrl, ^#p<0.05, ^##p<0.01, and ^###p<0.001 vs. NC.

Effect of SPG on MAPK signaling in UVB-irradiated HaCaT

To determine the mechanism on skin barrier function of SPA in keratinocytes, we investigated the levels of proteins involved in the MAPK pathway, including ERK, JNK, and p-38, in UVB-irradiated HaCaT cells. As shown in Fig. 3B, the UVB-irradiated group showed significantly increased levels of phosphorylated-ERK (***p<0.001), JNK (**p<0.01), and p-38 (***p<0.001) compared to the control group. The SPG-treated groups showed significant inhibition of the MAPK pathway in a concentration-dependent manner and exerted stronger effects than the CP-treated group. Our results suggest that SPG treatment inhibits activation of the MAPK pathway, which subsequently prevents UVB-stimulated downregulation of the skin barrier (i.e., filaggrin and involucrin) and moisturizing factors (i.e., HAS1 and AQP3) in HaCaT.

Content of the ceramide in the skin epidermis of SKH-1 mice, and mRNA levels of GCS and contents of hyaluronan in the HaCaT cell

The proportion of ceramides is the highest among intercellular lipids that form the skin barrier. The ceramide content in the epidermis of the UVB-irradiated mice was evaluated using HPLC (Fig. 4A). Compared to the normal control (2344.92 ± 179.62 µg/100 mg), the ceramide content in NC group (737.02 ± 169.31 µg/100 mg, *p<0.05) was reduced by approximately 2.5-fold, but the treated with CP group (1519.32 ± 247.89 µg/100 mg) was recovered. A dose-dependent increase was observed in the SPG group (1 mg/kg/d of SPG group, 1078.31 ± 44.77 µg/100 mg; 5 mg/kg/d of SPG group, 1657.89 ± 17.51 µg/100 mg). Thus, SPG appears to produce the ceramide that connects the skin barrier materials via collagen peptides.

Figure 4. Effect of salmon nasal cartilage proteoglycan (SPG) on ceramide content in UVB-irradiated mouse model, and the mRNA level of human glucosylceramide synthase (hGCS) and hyaluronan content in UVB-irradiated HaCaT cells. (A) HPLC analysis of the ceramide content in mouse epidermis. (B) mRNA levels of hGCS in HaCaT cells treated with PG for 1–12 h. All mRNA levels were normalized to those of GAPDH. (C) Effect of SPG on cell viability and hyaluronan secretion in UVB-irradiated HaCaT cells. ELISA was performed to quantify hyaluronan in the supernatant of the cell culture medium. The values are presented as mean ± SEM (n=4-8). *p<0.05, **p<0.01, and ***p<0.001 vs. Ctrl, ^#p<0.05, ^##p<0.01, and ^###p<0.001 vs. NC.

Besides, the ceramide synthesis pathway in the stratum corneum proceeds through glucosylceramide or sphingomyelin was assessed. Analysis of the mRNA expression of hGCS, a synthase that helps produce glucosylceramide, showed that the mRNA levels of hGCS increased after treatment with a high dose of SPG (25 µg/mL, ^###p<0.001) for 6 h compared to those in UVB-irradiated HaCaT cells (Fig. 4B). Additionally, the level of hGCS mRNA after 6 h of SPG treatment was higher (approximately 1.3-fold) compared to that of CP (250 µg/mL, ^##p<0.01). This suggests that SPG promotes the synthesis of ceramides as potential agents for restoring the skin barrier function by upregulating hGCS.

We compared the viability of HaCaT cells treated with UVB (60 mJ/cm²), UVB + CP (250 µg/ml), and UVB + SPG (5, 10, and 25 µg/mL for one day (Fig. 4C). The viability of UVB-irradiated HaCaT cells alone (***p<0.001) was reduced by 35% compared with that of the control. The viability of HaCaT cells treated with SPG significantly increased in a concentration-dependent manner (Fig. 4C). In addition, the treated SPG at 25 µg/mL (88.09 ± 1.37%, ^###p<0.001) significantly increased the cell viability, similar to that of CP (80.43 ± 5.48%, ^#p<0.05). Furthermore, we evaluated the effect of SPG on the UVB-irradiated secretion of hyaluronan in HaCaT cells (Fig. 4C). UVB irradiation (16.64 ± 1.52 pg/mL, ***p<0.001) decreased the level of hyaluronan secretion, but the SPG treated groups (5 µg/mL of SPG, 20.09 ± 0.63 pg/mL; 10 µg/mL of SPG, 23.21 ± 1.32 pg/mL; 25 µg/mL of SPG, 29.29 ± 1.01 pg/mL) were significantly increased in a concentration-dependent manners. This result supports the moisturizing and barrier function effect of SPG (Fig. 2, 3).

Effect of SPG on the collagen synthesis in dermis of SKH-1 mice

To evaluate the total collagen and associated proteins, such as procollagen type 1, in UVB-irradiated mice, Masson’s chrome and IHC staining assays were performed. As shown in Fig. 5A, the CP (103.73 ± 8.69%) and SPG groups (1 mg/kg/d of SPG, 51.02 ± 3.15%; 5 mg/kg/d, 77.79 ± 5.53%) reverted significantly from UVB-irradiated loss of collagen density (NC group, 29.98 ± 4.52%). Moreover, we analyzed procollagen type 1 in the dermis of SKH1 mice using IHC staining (Fig. 5B). The NC group (75.19 ± 1.86%, **p<0.01) showed lower density of procollagen type1 in comparison that of the normal group, whereas the treated SPG groups (1 mg/kg/d of SPG, 103.80 ± 5.53%; 5 mg/kg/d of SPG, 113.28 ± 5.57%) significantly restored the density of procollagen type. SPG has been speculated to promote collagen synthesis through the procollagen type 1 protein. Furthermore, we confirmed the association between SPG and the MMP-1 and elastin levels in UVB-irradiated NHDF, which are affected by collagen synthesis (Van Doren, 2015). As shown in Fig. 5C and Fig. 5D, the SPG-treated groups showed significantly reduced MMP-1 levels (5 µg/mL of SPG, 1442.10 ± 123.04 pg/mL; 10 µg/mL of SPG, 1627.23 ± 106.74 pg/mL; 25 µg/mL of SPG, 1079.09 ± 135.62 pg/mL) in NHDF, similar to the CP-treated group (1633.076 ± 6.08 pg/mL, ^#p<0.05). In contrast, the SPG group showed significantly increased elastin expression, which was decreased by UVB irradiation in NHDF. These data indicate that SPG facilitates ceramide synthesis by suppressing MMP-1 and promoting elastin production.

Figure 5. Effect of salmon nasal cartilage proteoglycan (SPG) on collagen degradation in UVB-irradiated in vitro and in vivo models. (A) Masson’s trichrome staining of mouse skin tissue was performed on the last day of the experiment (×40; bar size 100 μm). (B) Immunohistological changes in procollagen type 1 in the skin of SKH-1 mice exposed to UVB irradiation for eight weeks (×40; bar size 100 μm). (C, D) ELISA kit and western blot analysis for measuring the expression of MMP-1 and elastin in NHDF cells. ELISA was performed to quantify MMP-1 in the supernatant of the cell culture medium. All protein levels were normalized to those of α-tubulin. The values are presented as mean ± SEM (n=3-4). **p<0.01 and ***p<0.001 vs. Ctrl, ^#p<0.05, ^##p<0.01, and ^###p<0.001 vs. NC.

Although UV radiation is beneficial in many ways, exposure to chronic high-dose UVRs radiation harms the eye and immune system, including the skin, and is also linked with the hallmarks of extrinsic aging (Pillai et al., 2005). More than 80% of skin aging is caused by UV exposure, and dietary supplementation with functional ingredients may protect skin from UVR-induced photodamage. Especially, overexposure of UVB rays directly causes to inflammation, oxidative stress, and DNA damages than UVA rays, because UVB having high level energy has a greater impact on the skin photoaging (Egbert et al., 2014). Therefore, there are an increasing number of reports on functional materials from natural resources that ameliorate UVB photoaging. (Hascall and Sajdera, 1970; Kashiwakura et al., 2008; Goto et al., 2012; Ito et al., 2015; Sano et al., 2017). Therefore, we aimed to provide insights into the preventive potential of a new ingredient, proteoglycans, against UVB skin aging. SPG have been reported to potentially affect other diseases (Hascall and Sajdera, 1970; Goto et al., 2012; Ito et al., 2015). However, whether SPG ameliorates photoaging by improving UVB-induced skin barrier damage has not been reported. In this study, we investigated whether SPG affects skin moisturization and wrinkle improvement by maintaining the skin barrier in keratinocytes, matrix content in dermal fibroblasts, and photoaging in the SKH-1 mouse model.

Previous studies have shown that UVB irradiation of the skin causes epidermal damage, leading to dryness, desquamation, and hyperkeratosis (Peng et al., 2020). In this study, UVB-irradiated mice models showed increased levels of TEWL, a skin barrier damage factor, which was accompanied by decreased hydration. Meanwhile, treatment with SPG (1 and 5 mg/kg/d) effectively reduced dryness and desquamation in the dorsal skin of mice because UVB-irradiated photoaging restored both TEWL and hydration (Fig. 1D). In addition, the thickness of the epidermis and the number of inflammatory cells decreased in the SPG group compared to those in the H&E-stained skin (Fig. 1E). Macroscopic observations of the epidermis have revealed that photoaged skin has a critically irregular morphology and loss of cell polarity. SPG treatment may decrease epidermal thickness by regulating the skin barrier.

UVB strongly reduces the expression of skin barrier proteins, including filaggrin, involucrin, and AQP3, which are involved in the skin barrier function (Li et al., 2022). Our findings also revealed that UVB damages the accumulation of filaggrin, involucrin, and AQP3 in mouse dorsal skin and keratinocytes (Fig. 2, 3). After treatment with SPG, each skin barrier protein significantly increased in a concentration-dependent manner in the UVB-treated keratinocytes (Fig. 3). IHC and western blotting results showed that filaggrin and AQP3-forming skin barriers increased in the SPG group, especially in the 5 mg/kg/d group (Fig. 2). In addition, the level of involucrin, which is related to water movement, increased in all SPG groups (Fig. 2). Furthermore, we compared the profilaggrin and filaggrin expression levels to confirm the formation of natural moisturizing factors (Fig. 3). The profilaggrin/filaggrin ratio decreased in high-dose SPG-treated cells compared with that in UVB-treated keratinocytes. Following the administration of SPG, HAS1 and hyaluronan secretion levels were upregulated (Fig. 3A, 4C). These results suggest that SPG can cause UVB-induced skin barrier damage by restoring the expression of barrier proteins (filaggrin, involucrin, and AQP3) and promoting moisturizing factors (HAS1 and hyaluronan). Therefore, oral SPG treatment may ameliorate the barrier function of the photoaged human skin.

Moreover, various extracellular agents including UVB trigger stress-related signaling and affect MAPKs pathway cascades via activating AP-1 and NF-κB signaling in the epidermal keratinocytes (Li et al., 2019). It already reported The MAPK pathway is involved in epithelial and endothelial tight junctions within keratinocytes, and skin barrier proteins such as AQP-3 are regulated by the MAPK pathway (Li et al., 2022). Our data showed that SPG strongly downregulated UVB-induced activation of the MAPK pathway, including ERK, JNK, and p38. Therefore, SPG may increase the expression of skin barrier proteins by suppressing the MAPK pathway (Fig. 3B).

Several studies have reported that ceramides are more potent than retinol, niacinamide, and peptides. Certain skincare products with ceramides help strengthen skin barrier function and improve hydration. In addition, high levels of ceramides result in smoother and firmer skin and reduced fine lines and wrinkles in facial skin. Therefore, ceramides may be the core anti-aging components responsible for the regulation of UVB photoaging. In this study, ceramide, a skin barrier component, was quantified using HPLC. Ceramides maintain a strong barrier by linking the cells (Coderch et al., 2003). The ceramide content of the epidermis in the SPG group was higher than that in the UVB and CP groups (Fig. 4A). Ceramides in the epidermal layer can be divided into two types, glucosylceramide and sphingomyelin, where different enzymes act in pathways to form ceramides in the stratum corneum (Cha et al., 2016). The time dependence of the mRNA levels of enzymes contributing to ceramide synthesis, which accounts for the largest proportion of intercellular lipids in HaCaT cells, was tested. UVB influenced the synthesis of ceramide to inhibit its production, and GCS levels increased in SPG-treated HaCaT cells after 6 h (Fig. 4B). It can be inferred that ceramide function is maintained when the glucosylceramide production pathway is upregulated. These animal analyses suggest that the administration of SPG to photoaging skin may increase the levels of proteins involved in moisturization and maintain the skin barrier, thus preventing skin barrier disruption and protecting wrinkles from UVB.

Sun-induced skin aging is a cumulative process, and wrinkling is one of the main symptoms of UVB-induced skin aging. First, fine lines appear, and the creases deepen upon exposure to UVB radiation, which is closely related to the loss of elastic properties of the skin (Imokawa, 2009). With dry skin, fine lines and wrinkles appear to be more exaggerated. Previous studies have demonstrated that skin wrinkling by UVB increases the degradation of the collagen matrix through MMP, resulting in a loss of elasticity due to reduced extracellular matrix proteins, such as collagen fibers (Dhital et al., 2017). Consistently, our findings showed that UVB strongly increased wrinkle formation in epidermal skin and reduced the expression levels of elastin by enhancing MMP-1 secretion in dermal fibroblasts (Fig. 1C, 5C, 5D). Meanwhile, treatment with SPG increased collagen levels, such as procollagen type 1, restored the wrinkles of skin in the UVB-irradiated mouse model, and decreased the protein expression of elastin by MMP-1, which was reduced in dermal fibroblast cells. These results imply that SPG also has protective effects against UVB-induced wrinkle formation by decreasing collagen and elastin levels, which are accompanied by MMPs, including MMP-1. To date, there have been some treatments to reduce the appearance of wrinkles in the form of cosmetics. However, there are few clinically effective anti-wrinkle products available as dietary supplements. In this respect, SPG is expected to be an interesting dietary anti-wrinkle material with a high potential.

Taken together, the levels of proteins involved in the keratinization of corneocytes constituting the skin barrier increased in the SPG-treated group, despite UVB skin damage. SPG protects the skin barrier from collapse by influencing factors constituting the skin barrier, such as corneocytes and intercellular lipids damaged by UVB, and by maintaining the ceramide and hyaluronan content, which is crucial for skin moisturization. Additionally, SPG improved the expression of skin barrier proteins by suppressing the MAPK pathway. SPG is thought to improve wrinkles by maintaining the skin barrier damaged by UVB radiation without causing skin collapse. Interestingly, SPG significantly increased ceramide levels in the epidermis of the UVB-induced photodamaged mouse skin. An increase in ceramide by treatment with SPG might ameliorate epidermal water loss by holding water in the skin and reducing the free radicals in the skin that destroy collagen. Consequently, dietary SPG, a proteoglycan found in salmon nasal cartilage, may prevent photoaging processes that can lead to skin dehydration and wrinkles (See Fig. 6). Thus, the ingestion of salmon nasal cartilage-derived functional proteoglycan components may be an attractive strategy for preventing UVR-induced skin photoaging. Moreover, further research should be carried out on whether main components such as aggrecan from SPG is the active component in anti-aging in skin and to investigate the exact mechanisms that prevent photoaging.

Figure 6. Proposed mechanism for dietary anti-skin photoaging effect of salmon nasal cartilage proteoglycan (SPG).

This research was supported by grants from the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2023R1A2C2003366), Gachon University research fund of 2020 (GCU-202008420002), and Naturetech, Co. Ltd. (Grant No. 202303360001).

The authors declare no conflict of interest.

Hae Ran Lee, Seong-Min Hong: Methodology, investigation, formal analysis, visualization, and writing – review and editing. Kyohee Cho: Methodology, investigation, formal analysis, and visualization. Seon Hyeok Kim , Eunji Ko, Eunyoo Lee: Methodology, investigation, and formal analysis. Hyun Jin Kim, Se Yeong Jeon, Seon Gil Do: investigation, funding acquisition, and project administration. Sun Yeou Kim: conceptualization, methodology, research design, writing, review, and editing.