Krill Oil’s Protective Benefits against Ultraviolet B-Induced Skin Photoaging in Hairless Mice and In Vitro Experiments

Krill oil (KO) shows promise as a natural marine-derived ingredient for improving skin health. This study investigated its antioxidant, anti-inflammatory, anti-wrinkle, and moisturizing effects on skin cells and UVB-induced skin photoaging in hairless mice. In vitro assays on HDF, HaCaT, and B16/F10 cells, as well as in vivo experiments on 60 hairless mice were conducted. A cell viability assay, diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity test, elastase inhibition assay, procollagen content test, MMP-1 inhibition test, and hyaluronan production assay were used to experiment on in vitro cell models. Mice received oral KO administration (100, 200, or 400 mg/kg) once a day for 15 weeks and UVB radiation three times a week. L-Ascorbic acid (L-AA) was orally administered at 100 mg/kg once daily for 15 weeks, starting from the initial ultraviolet B (UVB) exposures. L-AA administration followed each UVB session (0.18 J/cm2) after one hour. In vitro, KO significantly countered UVB-induced oxidative stress, reduced wrinkles, and prevented skin water loss by enhancing collagen and hyaluronic synthesis. In vivo, all KO dosages showed dose-dependent inhibition of oxidative stress-induced inflammatory photoaging-related skin changes. Skin mRNA expressions for hyaluronan synthesis and collagen synthesis genes also increased dose-dependently after KO treatment. Histopathological analysis confirmed that krill oil (KO) ameliorated the damage caused by UVB-irradiated skin tissues. The results imply that KO could potentially act as a positive measure in diminishing UVB-triggered skin photoaging and address various skin issues like wrinkles and moisturization when taken as a dietary supplement.


Introduction
The human skin, consisting of three layers (epidermis, dermis, and subcutaneous) [1], serves as a vital barrier against environmental stressors like ultraviolet (UV) rays, physical trauma, and microorganisms.Direct exposure to UV radiation from sunlight can lead to acute effects such as DNA damage, suppression of DNA synthesis, cell death, and erythema.Additionally, it can also result in chronic effects like photoaging and epidermal cancer [2].Photoaging, induced by repeated UV exposure, results in histological alterations, collagen fiber damage, and uneven pigmentation, leading to wrinkled and coarse skin [3].Ultraviolet B (UVB), with a wavelength range of 280 to 315 nanometers, exhibits both beneficial and harmful effects on the skin.It can cause sunburns, premature aging, and an increased risk of skin cancer, among other characteristics [4].UVB penetrates the epidermis, leading to DNA damage and mutations over time, contributing to skin cancer development [5].

Effects of KO on Body Weight Changes
The intact control groupʹs average weight decreased from 24.93 ± 1.23 g to 21.77 ± 1.21 g on Day 0, and then increased to 32.82 ± 2.21 g on Day 104 (Figure 5).The UVBexposed hairless mice showed no significant weight difference compared to non-UVBexposed controls during the 105-day treatment.KO and L-AA groups also did not exhibit significant weight changes compared to intact controls or the UVB group.Additionally, no significant weight or mass index changes were observed in the UVB control group over

In Vivo Evidence of Anti-Photoaging 2.2.1. Effects of KO on Body Weight Changes
The intact control group's average weight decreased from 24.93 ± 1.23 g to 21.77 ± 1.21 g on Day 0, and then increased to 32.82 ± 2.21 g on Day 104 (Figure 5).The UVB-exposed hairless mice showed no significant weight difference compared to non-UVB-exposed controls during the 105-day treatment.KO and L-AA groups also did not exhibit significant weight changes compared to intact controls or the UVB group.Additionally, no significant weight or mass index changes were observed in the UVB control group over the 105 days.Similarly, experimental substance-treated groups did not show significant changes.The body mass index showed a −4.51% change in the UVB control compared to normal medium controls.For L-AA 100 mg/kg, KO 400 mg/kg, 200 mg/kg, and 100 mg/kg groups, the changes were 2.29%, 6.15%, 2.86%, and 3.15%, respectively, compared to the UVB control.control cells.

Effects of KO on Body Weight Changes
The intact control groupʹs average weight decreased from 24.93 ± 1.23 g to 21.77 ± 1.21 g on Day 0, and then increased to 32.82 ± 2.21 g on Day 104 (Figure 5).The UVBexposed hairless mice showed no significant weight difference compared to non-UVBexposed controls during the 105-day treatment.KO and L-AA groups also did not exhibit significant weight changes compared to intact controls or the UVB group.Additionally, no significant weight or mass index changes were observed in the UVB control group over the 105 days.Similarly, experimental substance-treated groups did not show significant changes.The body mass index showed a −4.51% change in the UVB control compared to normal medium controls.For L-AA 100 mg/kg, KO 400 mg/kg, 200 mg/kg, and 100 mg/kg groups, the changes were 2.29%, 6.15%, 2.86%, and 3.15%, respectively, compared to the UVB control.

Effects of KO on UVB-Induced Skin Wrinkle Formation and Skin Moisturization
The efficacy of KO in examining wrinkle formation on UVB-irradiated skin was conducted, and the length and depth of skin wrinkles were evaluated using replicas of the dorsal back skin.Exposure to UVB radiation clearly induced skin wrinkles, but their severity was alleviated by oral administration of either KO or L-AA (Figure 6a).After exposure to UVB, the mean skin length (mm) and depth (µm) significantly increased (0.69 ± 0.07 mm, 99.16 ± 17.37 µm) compared to the intact control (0.29 ± 0.05 mm, 31.16 ± 10.45 µm) (p < 0.01).However, both KO and L-AA significantly suppressed the formation of skin wrinkles in terms of length and depth (p < 0.01) (Figure 6b, c).Skin water content significantly decreased by 13.16 ± 2.23% after UVB irradiation compared to the intact control group (37.56 ± 5.47%) (p < 0.01).However, both KO (p < 0.05) and L-AA (p < 0.01) treatments significantly increased skin water content (Figure 6d).Key molecules for skin wrinkle formation and skin moisture maintenance-skin COL1 contents and hyaluronic acid contents-were significantly increased by both L-AA and KO treatments compared to UVB irradiation alone (p < 0.01) (Figure 6e,f).Moreover, the mRNA expression of hyaluronic acid and COL1-related genes COL1A1, COL1A2, Has1, Has2, and Has3 showed a significant increase compared to UVB irradiation alone (p < 0.01) (Figure 6g,h).Transforming growth factor-beta (TGF-β) stimulates collagen formation through the regulation of various cellular functions, with TGF-β1 particularly promoting the synthesis of hyaluronan via Has1 and Has2 mRNA expression.Levels of TGF-β1 mRNA were significantly decreased in UVB irradiation (0.17 ± 0.03) compared to the intact control group (1.00 ± 0.05) (p < 0.01).However, a significant dose-dependent increase in TGF-β1 mRNA expression was detected in the oral administration of KO (100, 200, and 400 mg/kg) compared to UVB irradiation (p < 0.01) (Figure 6i).The mRNA expression of UVB-induced MMP1, MMP9, and MMP13 was significantly reduced by both L-AA and KO treatments (p < 0.01) (Figure 6j).There was no significant difference in the treatment effects of UVB-induced skin wrinkle formation and skin moisturization between KO and L-AA treatments with an equal oral dosage.

Effects of KO on UVB-Induced Histopathological Changes in Skin Tissue
Histopathological analysis and Masson's trichrome staining revealed thickened epithelial tissue and abnormal collagen deposition in the dorsal back skin tissue due to UVB irradiation.However, the histopathological changes were significantly mitigated by L-AA and KO treatments (Figure 9a and Table 1).Additionally, KO treatments improved UVBinduced immunolabeled cells for oxidative stress markers (NT and 4-HNE), apoptosis markers (cleaved caspase 3 and cleaved PARP) in the epidermis, and immunoreactive cells for MMP9 in the dermis (Figure 9b and Table 2).However, there was no significant difference in the treatment effects of UVB-induced histopathological changes in skin tissue between KO and L-AA treatments with an equal oral dosage.

Discussion
Skin aging can be categorized into intrinsic aging, driven by hormonal changes and cellular aging, and extrinsic aging, caused by external factors like UV exposure, air pollution, and smoking [12].As the demand for anti-skin-aging solutions increases in the market, research and development of natural product-derived ingredients have advanced cutaneous science in skin beauty and health-related industries.Nutricosmetic products, including UV protectors, anti-wrinkle treatments, and moisturizers, are introduced to address these concerns [8].However, the long-term consumption of cost-effective and functional products raises ongoing concerns about potential risks of adverse events, harmful chemicals, and toxins [13].
Krill oil (KO) is gaining attention for its high bioavailability of n-3 polyunsaturated fatty acids (PUFAs) like EPA and DHA in phospholipid form.Despite being more expensive than fish oil, the superior bioavailability of EPA/DHA has sparked interest.Some clinical studies [9,14,15] have reported minor adverse events, such as rashes, headaches, taste changes, diarrhea, and a decreased appetite.However, KO is generally recognized as safe (GRAS) by the American Food and Drug Administration and has received Novel Food status from the European Union, confirming its safety profile [10].
In our previous studies, KO has been recognized as a marine-derived natural substance with significant activation of nuclear factor E2-related factor 2 (Nrf2) transferase and potent antioxidant properties, making it a promising raw material for health functional foods with natural antioxidant benefits [16,17].Although previous research on human immortalized keratinocyte lines and NC/Nga mice has suggested the potential benefits of KO in terms of antioxidants and anti-inflammatory effects [11,18], there is a lack of direct and detailed research on KO's role in improving skin wrinkles and moisturization.Hence, this study aims to scientifically evaluate the anti-skin-aging effects of KO.
Skin aging is linked to the activation of matrix metalloproteinases (MMPs) triggered by inflammatory cytokines in skin tissue.Frequent exposure to UV radiation accelerates skin aging by causing DNA breakdown, ROS generation, and DNA damage [19].Nutricosmetic products, known for their antioxidant and anti-inflammatory functions, are anticipated to prevent or improve skin aging.Enzymatic antioxidants have been shown to reduce UVinduced oxidative stress in skin tissue, suppressing inflammation and inhibiting apoptosis of skin cells [7,19].KO may not be an enzymatic antioxidant; our data demonstrated its inhibition of apoptosis in UVB-exposed skin tissue of mice, suggesting KO's potential in suppressing skin inflammation from UV radiation.Moreover, KO's inhibitory effects may extend to the apoptosis pathway and cell cycle arrest caused by UV-induced DNA damage [20], specifically cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidine photoproducts (6-4PP).Further research on KO's effects on CPD and 6-4PP as well as its ability to modulate CPD-photolyases and 6-4PP-photolyase repairing mechanisms would provide a clearer understanding of KO's protection against UV-induced DNA damage and apoptosis.These investigations will enhance our understanding of KO's potential as a skin-protective agent.
Previous studies have reported KO's antioxidant and anti-inflammatory effects on human immortalized keratinocyte lines [11].KO's potential to regulate ECM proteins and protect the skin through its antioxidant activity and anti-inflammatory effects was supported by its ability to suppress skin inflammation in NC/Nga mice using a phospholipidenriched alkyl phospholipid from krill [18].As a non-enzymatic antioxidant, KO builds an antioxidant defense network, protecting cells and tissues from ROS and benefiting skin health.In this study, we observed KO's concentration-dependent free radical scavenging activity.Furthermore, KO administration improved the UVB-induced decrease in GSH content by upregulating GSH reductase mRNA expression.KO also inhibited UVB-induced lipid peroxidation and superoxide anion production through the transcriptional regulation of NOX2.The observed results, confirmed through immunohistochemical analysis using NT and 4-HNE staining, suggest that KO's antioxidant activity plays a significant role.Additionally, increased ROS due to UV exposure can activate the MAPK (mitogen-activated protein kinase) signaling pathway, leading to the activation of AP-1 (activated protein-1) and subsequently promoting the expression of MMPs (matrix metalloproteinases), which can strongly contribute to the breakdown of ECM proteins like collagen and elastin [21][22][23][24].Indeed, the increased MMPs due to UV radiation can promote the degradation of ECM proteins and ultimately lead to the formation of skin wrinkles and photoaging [24].While our study did not directly investigate the UV-induced MAPK pathway and AP-1 activation, we observed that KO's antioxidant activity effectively suppressed the mRNA expression of MMP-1, MMP-9, and MMP-13.This inhibition contributed to the regulation of ECM proteins and directly prevented the formation of skin wrinkles, evident by the mean length and depth of wrinkles.
Human skin contains 28 different types of MMPs, including collagenases (MMP-1 and MMP-13) and a gelatinase (MMP-9), which increase with UV exposure [25].MMP-1 and MMP-13 not only promote ECM collagen degradation but also reduce collagen density in the dermal layer [23,26].Our study demonstrated that KO reduced UVB-induced MMP-1 activity in HDF cells, indicating its potential to inhibit ECM collagen degradation.Moreover, KO suppressed the upregulation of MMP-1, MMP-9, and MMP-13 gene expression in skin tissue induced by UVB, leading to improved skin COL1 levels and COL1A1/2 mRNA expression.This suggests that KO may inhibit MMP mRNA expression, likely through its effect on local inflammatory and neutrophil responses to UVB [27].Such MMP suppression could help maintain skin collagen levels and prevent excessive collagen degradation linked to photoaging.Additionally, our findings show that UV radiation induces an inflammatory response in the skin, with increased secretion of pro-inflammatory cytokine IL-1 and reduced expression of anti-inflammatory cytokine IL-10.KO administration appears to regulate this inflammatory state induced by UV, balancing pro-and anti-inflammatory cytokines, and contributing to both its anti-inflammatory effects and its potential skin health benefits.
In response to UVB-induced skin injury, polymorphneutrophils (PMNs) and neutrophils are recruited to the injured tissues through the action of oxygen metabolites [28].MPO, released from PMNs, is a cytotoxic enzyme that activates inflammation [29].The reduction of neutrophils infiltrating into the skin tissue can be confirmed by assessing MPO activity [30].Our study revealed that KO alleviates the UVB-induced inflammatory state in the skin and directly inhibits MPO activity, leading to reduced neutrophil recruitment to inflammatory sites [31].These findings suggest that KO administration modulates the inflammatory response induced by UVB exposure by decreasing MPO activity and subsequently limiting neutrophil infiltration into the skin.
Skin aging often leads to a reduction in hyaluronic acid, a vital component responsible for retaining water in the skin [32].Fatty acids play a crucial role in maintaining skin hydration and barrier integrity [8], while PUFA deficiency can increase water loss through the skin barrier [33].Our study found that UVB exposure and aging downregulated the genes responsible for hyaluronic acid synthesis (HAS1, HAS2, and HAS3) in the dermis [34].However, oral administration of KO reversed this downregulation, resulting in increased hyaluronic acid content in the skin.These results suggest that KO enhances skin moisturization by promoting hyaluronic acid synthesis through the regulation of HAS genes in response to UVB-induced water loss.
This study was conducted with the objective of investigating the protective effects of KO against UVB-induced skin photoaging.The existing literature has only offered limited insights into the potential skin health advantages of KO, with two or fewer studies referencing it.Our investigation demonstrated that oral administration of KO notably mitigated UVB-induced wrinkles, skin water loss, collagen degradation, and skin edema, comparable to L-AA (100 mg/kg) at the same dosage.These findings indicate the potential of KO as a functional product for preventing UVB-induced skin photoaging and enhancing skin moisturization.However, further clinical studies are necessary to comprehensively elucidate the diverse range of benefits provided by KO for skin health.wells at 450 nm was recorded using a microplate reader (Sunrise, TECAN, Männedorf, Switzerland).The relative cell viability (%) was calculated as [(OD s /OD c ) × 100], where OD s represents the absorbance of the sample at 450 nm, and OD c is the absorbance of the vehicle control at 450 nm.The results were expressed in terms of inhibitory concentration (IC) 50 , which indicates the concentration at which cell viability reaches 50% of the control.

DPPH Radical Scavenging Assay
The KO's free radical scavenging ability was assessed through the DPPH radical scavenging assay, following the method outlined by Blois [35].A 0.2 mM solution of 2,2-diphenyl-1-picryhydrazyl (DPPH; Sigma-Aldrich, St. Louis, MO, USA) in methanol was promptly prepared.Samples were diluted using distilled water to reach final KO concentrations of 0.25, 0.5, 1, 1.5, 2, 4, and 8 mg/mL, or a final L-AA concentration of 1 mg/mL.The DPPH radical scavenging activity was measured at 517 nm with a UV/Vis spectrophotometer (Optizen Pop, Mecasys, Daejeon, Republic of Korea) after a 10-min incubation.The free radical scavenging activity was calculated using the formula: DPPH radical scavenging activity (%) = 100 − [(OD s /OD c ) × 100], where OD s represents the sample's absorbance at 517 nm and ODc is the absorbance of the control treated with the vehicle at 517 nm.The results were expressed as IC 50 values, indicating the concentration needed to decrease DPPH by 50%.A positive control of L-AA at 1 mg/mL was employed.

Elastase Inhibition Assay
The elastase inhibition assay measured the release of p-nitroaniline due to proteolysis of N-succinyl-(Ala)3-p-nitroanilide by the human leucocyte elastase (Sigma-Aldrich, St. Louis, MO, USA).KO was tested at concentrations ranging from 0.25 to 8 mg/mL, while PP at 10 µM served as the standard.Elastase inhibitory activity was measured at 410 nm using a 96-well microplate reader, and the elastase inhibitory activity of each sample was calculated using equation as follows: Elastase inhibitory activity (%) = 100 − [(OD s /OD c ) × 100], where OD s is the absorbance of the experimental sample at 410 nm and OD c is the absorbance of the vehicle-treated control at 410 nm.The results were reported as IC 50 , representing the concentration at which the percentage inhibition of elastase activity was 50%.

Procollagen Synthesis Assay
HDF cells were cultured in 24-well plates (2 × 10 4 cells/well) for 24 h.Subsequently, the medium was replaced with varying concentrations of KO (0.25, 0.5, 1, 1.5, 2, 4, and 8 mg/mL) or 10 ng/mL TGF-β1 mixed with a serum-free kit, and cells were cultured for another 24 h.Procollagen levels in the culture supernatant were measured using a Procollagen type I-c-peptide (PIP) ELISA kit (MK101, Takara Bio, Tokyo, Japan), normalized by total protein content.Relative procollagen synthesis (%) was calculated as [(procollagen contents in experimental sample/procollagen contents in control) × 100].The results were presented as the 50% effective concentrations (EC 50 ), representing the concentration at which HDF cell procollagen synthesis doubled.

Hyaluronan Production Assay
HaCaT cells (4 × 10 4 cells/well) were exposed to KO (0.25, 0.5, 1, 1.5, 2, 4, and 8 mg/mL) or 1 µM of RA for 24 h.Following this, the cells were trypsinized and counted for normalization purposes.The quantification of hyaluronan synthesis was conducted using the Hyaluronan ELISA kit (DY3614, R&D Systems, Minneapolis, MN, USA).The results were normalized based on the total protein content of the supernatant.The relative hyaluronan synthesis (%) was calculated as [(hyaluronan contents in the experimental sample/hyaluronan contents in the unexposed control) × 100].The outcomes were presented as EC 50 values, indicating the concentration at which the percentage increase in hyaluronan synthesis reached a two-fold level.

In Vivo
A total of 60 SPF/VAF Outbred SKH1-hr hairless female mice (OrientBio, Seungnam, Korea) were procured.The mice were housed in groups of five per polycarbonate cage within a controlled environment at a temperature of 20-25 • C and humidity of 50-55%.Following a 7-day acclimation period, mice with normal skin and stable body weight (average 24.91 ± 1.14 g, range: 22.60-27.00g) were sorted into six groups, each consisting of 10 mice.The animal experiment was carried out with prior approval from the Institutional Animal Care and Use Committee of Daegu Haany University [Approval No. DHU2021-070, 18 August 2021].

Skin Photoaging
Following the methods used in previous studies [37,38], a UV Crosslinker system (CL-1000M, Analytik Jena, Upland, CA, USA) emitting wavelengths of 254 nm, 312 nm, and 365 nm (with 312 nm as the main wavelength) was used to induce skin photoaging in the hairless mice.The mice were exposed to UVB at a dose of 0.18 J/cm 2 , three times per week, for 15 weeks.For the intact control group, the same environment stress was applied by leaving the UV Crosslinker system powered off under the same conditions for the same duration.

Experimental Substances and Oral Administration
The experiment substances were prepared by dissolving KO at concentrations of 10, 20, and 40 mg/mL in sterile distilled water.They were administered orally using a metal gavage needle attached to a 1 mL syringe at doses of 10 mL/kg (equivalence to 100, 200, and 400 mg/kg) daily for 105 days, 1 h after UVB exposure.Taking into account the oral administration method of L-AA as investigated by Park et al. [13], L-AA was also dissolved in sterile distilled water at a concentration of 10 mg/mL and administered orally at a dose of 10 mL/kg (equivalence to 100 mg/kg) daily for 105 days, 1 h after UVB exposure.In the intact control and UVB control groups, only sterile distilled water was administered orally at the same volume and duration as the experimental substances to apply the same handling stress.The experimental substances were prepared at least once a week and stored in a refrigerator at 4 • C until use.
St. Louis, MO, USA) followed by centrifugation at 3000 rpm at 4 • C for 20 min.The resulting supernatants were neutralized with 10 N NaOH.Hyaluronan levels were measured using the mouse hyaluronic acid enzyme-linked immunosorbent assay (ELISA) kit (MBS161603, Mybiosource, San Diego, CA, USA), with readings at 450 nm on a microplate reader in ng/mL terms.4.2.6.Antioxidant and Anti-Inflammatory Glutathione (GSH) Assay Cutaneous GSH levels were assessed using a fluorescence assay as previously outlined [31].Initially, the skin (1:3, w/w dilution) was homogenized in 100 mM NaH 2 PO 4 (pH 8.0; Sigma-Aldrich, St. Louis, MO, USA) containing 5 mM EDTA.Subsequently, the homogenates underwent treatment with 30% trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) and were subjected to two rounds of centrifugation (at 1940× g for 6 min and at 485× g for 10 min).The fluorescence of the resultant supernatant was measured using a fluorescence spectrophotometer (RF-5301PC; Shimadzu Corp., Tokyo, Japan).In total, 100 µL of the supernatant was mixed with 1 mL of buffer 1 and 100 µL of o-phthalaldehyde (1 mg/mL in methanol; Sigma-Aldrich, St. Louis, MO, USA).The fluorescence intensity was measured after 15 min (k exc = 350 nm; k em = 420 nm).A standard curve was established using different concentrations of GSH (ranging from 0.0 to 75.0 µM).Protein levels in the skin homogenates were quantified following the method of Lowry et al. [43].The outcomes were expressed as µM of GSH per milligram of protein.

Lipid Peroxidation Assay
To begin, the protein content of the homogenate (10 mg/mL in 1.15% KCl) was assessed using the Lowry method [43].To evaluate lipid peroxidation, the measurement of thiobarbituric acid reactive substances (TBARS) was employed as previously elucidated [44].In this procedure, trichloroacetic acid (10%; Sigma-Aldrich, St. Louis, MO, USA) was added to the homogenate to precipitate proteins.Following this step, the mixture underwent centrifugation (3 min, 1000× g).The protein-free sample was then extracted, and thiobarbituric acid (0.67%) was introduced.This mixture was subjected to a water bath at 100 • C for 15 min.The intermediate product of lipoperoxidation, MDA, was quantified by measuring the difference between absorbances at 535 and 572 nm on a microplate spectrophotometer reader.The findings were reported as nM/mg of protein [45].

Superoxide Anion Production
The measurement of superoxide anion production in skin tissue homogenates (10 mg/mL in 1.15% KCl) was conducted through the nitroblue tetrazolium (NBT) assay [46].In brief, 50 µL of the homogenate was incubated with 100 µL of NBT (1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) in 96-well plates at 37 • C for 1 h.Subsequently, the supernatant was cautiously removed, and the reduced formazan was dissolved by adding 120 µL of 2M KOH and 140 µL of DMSO.The reduction of NBT was measured at 600 nm using a microplate reader.The protein content was employed for the normalization of data.

Determination of IL-1β and IL-10 in Skin Tissues
On the 105th day of UVB irradiation, dorsal back skin tissue samples were obtained from the region around the gluteal area.Skin tissue homogenates were subjected to the measurement of IL-1β and IL-10 contents using mouse IL-1β (ab100705; Abcam, Cambridge, UK) and IL-10 (ab108870; Abcam, Cambridge, UK) enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer's instructions.Optical density readings were taken at 450 nm using a microplate reader.

Edema Evaluation
The impact of the test substances on UVB-induced skin edema was evaluated by observing an increase in the weight of the dorsal skin.Following the continuous oral administration of the test articles for 15 weeks, the dorsal skin was removed.A consistent area (6 mm diameter) was then demarcated using a punch, and the weight of this standardized area was measured following established methodologies [13,47].The outcome was determined by comparing the skin weight across different groups and was expressed in grams of skin (g/6-mm diameter of dorsal skin).

Myeloperoxidase (MPO) Activity
UVB-induced leukocyte migration to the skin was assessed using the MPO kineticcolorimetric assay as per established protocols [31,48].Skin samples were collected in 400 µL of a 50 mM K 2 HPO 4 buffer (pH 6.0; Sigma-Aldrich, St. Louis, MO, USA) containing 0.5% hexadecyltrimethylammonium bromide (Gibco, Carlsbad, CA, USA), homogenized using a bead beater and ultrasonic disruptor, then centrifuged.The resulting supernatant was stored at −150 • C. For the assay, 30 µL of the sample was mixed with 200 µL of 0.05 M K 2 HPO 4 buffer (pH 6.0) containing 0.167 mg/mL o-dianisidine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 0.05% hydrogen peroxide.Absorbance was measured at 450 nm after 5 min using a UV/Vis spectrophotometer (OPTIZEN POP, Mecasys, Daejeon, Korea).MPO activity was compared to neutrophil standards, and skin protein levels were determined using the Lowry method [43].Results were expressed as MPO activity (number of total neutrophils/mg of protein).

Real-Time Polymerase Chain Reaction (RT-PCR)
The mRNA expressions of TGF-β1; p38 MAPK; AKT; COL1A1 and 2; Has 1, 2, and 3; MMP-1, 9, and 13; Nox2, and GSH reductase in the prepared dorsal back skin tissues were evaluated using real-time RT-PCR in accordance with prior studies [13,47].To elaborate, RNA extraction was carried out using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and RNA concentration and quality were determined using the CFX96 TM Real-Time System (Bio-Rad, Hercules, CA, USA).To eliminate any DNA contamination, samples underwent treatment with recombinant DNase I (DNA-free; DNA-free DNA removal kit; Cat No. AM1906, Thermo Fisher Scientific Inc., Rockford, IL, USA).RNA was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Cat No. 4368813, Thermo Fisher Scientific Inc., Rockford, IL, USA) in accordance with the manufacturer's instructions.The analysis was performed utilizing the ABI Step One Plus Sequence Detection System (Applied Biosystems, Foster City, CA, USA), and their expression levels were normalized relative to the vehicle control.The following thermal conditions were applied: 10 min at 94 • C, followed by 39 cycles of 15 s at 94 • C, 20 s at 57 • C, and 30 s at 72 • C. The data was normalized using β-actin mRNA expression through the comparative threshold cycle method [49].The oligonucleotide primer sequences for PCR are provided in Table S1.

Histopathology
Dorsal back skin samples from the gluteal regions were crossly trimmed and fixed in 10% neutral buffered formalin for 24 h for histopathological observation.Paraffin blocks were created using an automated tissue processor (Shandon Citadel 2000, Thermo Scientific, Waltham, MA, USA) and embedding center (Shandon Histostar, Thermo Scientific, Waltham, MA, USA), and 3~4 µm sections were prepared using automated microtome (RM2255, Leica Biosystems, Nussloch, Germany), followed by preparation of 3 to 4 µm sections through automated microtome.These sections were stained with hematoxylin and eosin (HE) for general histopathology and Masson's trichrome (MT) for collagen fibers.A histological examination was performed using light microscopy (Model Eclipse 80i, Nikon, Tokyo, Japan) with a camera system (ProgRes TM C5, Jenoptik Optical Systems GmbH, Jena, Germany) and image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Bernaby, BC, Canada).Various parameters such as epithelial microfolds (folds/mm of epithelium), epithelial thicknesses (µm/epithelium), inflammatory cell count in the dermis (cells/mm 2 of dermis), and collagen fiber distribution were quantified using computer-assisted image analysis (%/mm 2 of dermis) according to our previously established methods [13,47].The histopathologist was unaware of group distribution during analysis.Central regions of samples were selected for observation, resulting in a minimum of one field per dorsal back skin tissue and a total of 10 histological fields per group.4.2.9.Immunohistochemistry Immunoreactivities against nitrotyrosine (NT), 4-hydroxynonenal (4-HNE), cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and MMP-9 on the dorsal back skin were visualized with specific antibodies (refer to Table 2) using an avidin-biotin-peroxidase complex (ABC) and peroxidase substrate kit (Vector Labs, Burlingame, CA, USA) [38,47].Endogenous peroxidase activity was blocked by incubating in methanol and 0.3% H 2 O 2 for 30 min.Non-specific binding of immunoglobulin was prevented by using a normal horse serum blocking solution for 1 h in a humid chamber, following epitope retrieval in 10 mM citrate buffers (pH 6.0) by heating (95-100 • C).Primary antibodies were incubated overnight at 4 • C in a humidity chamber, followed by biotinylated secondary antibodies and ABC reagents.Sections were exposed to the peroxidase substrate kit for 3 min at room temperature.Between each step, all sections were rinsed three times in 0.01 M phosphate buffered saline.Positive immunoreactivity was considered for epithelial cells with over 40% immunoreactivity density for each antiserum in comparison to the background-NT, 4-HNE, cleaved caspase-3, PARP, and MMP-9.The mean numbers of cleaved caspase-3 and PARP, as well as NT and 4-HNE-immunolabeled epithelial cells (% of cells/100 epithelial cells) were quantified using an automated image analysis process and histological camera system.MMP-9 immunoreactive fiber percentages were calculated in the dermis (%/mm 2 ).The histopathologist performed the analysis in a blinded manner with respect to group distribution adapted from prior methodologies [38,47].

Statistical Analyses
All collected data were presented as mean ± SD.In vitro and in vivo data were subjected to multiple comparison tests, including the Levene test to assess variance homogeneity.One-way ANOVA followed by Tukey's HSD test was applied to data with no significant deviations from variance homogeneity, while Dunnett's T3 test was used for data with significant deviations.Nonparametric comparisons were performed using the Kruskal-Wallis H test and Mann-Whitney u test.Statistical significance was considered for p-values < 0.05.Statistical analysis was conducted using SPSS for Windows (Release 27.0).

Conclusions
In this investigation, we assessed KO's potential in mitigating UVB-induced skin photoaging.Our experimental outcomes demonstrated that oral administration of KO significantly attenuated UVB-induced wrinkle formation, skin water loss, and collagen degradation.These advantageous effects were attributed to the anti-inflammatory, antiapoptotic, and antioxidant properties inherent in KO, which exhibited similarity to L-AA (100 mg/kg) at an equivalent oral dose level.With these convincing outcomes, KO stands out as a promising candidate for the development of functional products aimed at preventing skin photoaging when used as a dietary supplement.

Figure 4 .
Figure 4.The effects of KO on hyaluronan synthesis: Data are presented as the mean ± SD.KO, krill oil (Superba TM Boost); RA, retinoic acid; HaCaT, human keratinocytes; ** p < 0.01 as compared with control cells.

Figure 4 .
Figure 4.The effects of KO on hyaluronan synthesis: Data are presented as the mean ± SD.KO, krill oil (Superba TM Boost); RA, retinoic acid; HaCaT, human keratinocytes; ** p < 0.01 as compared with control cells.

Figure 5 .
Figure 5. Body weight changes on the days after UVB irradiation and oral administration: KO (100, 200, and 400 mg/kg) or L-AA (100 mg/kg) was orally administrated once a day for 105 days after 1 h of UVB irradiation.The body weights were measured every week.Data are presented as the mean ± SD (n = 10, significance compared with intact control mice).

Figure 5 .
Figure 5. Body weight changes on the days after UVB irradiation and oral administration: KO (100, 200, and 400 mg/kg) or L-AA (100 mg/kg) was orally administrated once a day for 105 days after 1 h of UVB irradiation.The body weights were measured every week.Data are presented as the mean ± SD (n = 10, significance compared with intact control mice).

Figure 8 .
Figure 8. Effects of KO on UVB-induced oxidative stress: (a) GSH contents in the skin tissue; (b) MDA level in the skin tissue; (c) Superoxide anion production in the skin tissue; (d) GSH reductase mRNA expression level in the dorsal back skin tissue; (e) NOX2 mRNA expression level in the dorsal back skin tissue.Data are presented as the mean ± SD (n = 10, significance difference vs. intact control; * p < 0.05, ** p < 0.01, vs. UVB-irradiated control mice; # p < 0.05, ## p < 0.01).

Table 1 .
General histomorphometrical analysis of dorsal back skin, taken from unexposed intact or UVB-exposed hairless mice.

Table 2 .
Immunohistomorphometrical analysis of dorsal back skin, taken from unexposed intact or UVB-exposed hairless mice.