Protective effects of an aqueous extract from Pinctada fucata martensii mucus on ultraviolet A-induced skin photoaging: in vitro and in vivo evidence

Ultraviolet A (UVA) radiation is a primary extrinsic driver of skin photoaging, yet effective marine-derived photoprotective countermeasures remain underexplored. The Akoya pearl oyster (Pinctada fucata martensii, Pinctada martensii) mucus represents a largely underexplored source of water-soluble marine natural products. In the present study, a low-molecular-weight aqueous fraction (designated AKN) was prepared via direct aqueous extraction and 3 kDa ultrafiltration without enzymatic hydrolysis, preserving the native constituent profile. AKN was systematically profiled using orthogonal untargeted LC–MS and targeted compositional analyses, with taurine and betaine accounting for 8.0% and 4.1% of the dry weight, respectively. Its biological effects were evaluated in UVA-exposed L929 murine fibroblasts, LPS-stimulated BV2 murine microglial cells, and a UVA-induced BALB/c mouse skin photoaging model, using ergothioneine (EGT) as a positive control. In cellular assays, AKN markedly suppressed the expression of key senescence regulators (p16, p21, p53), attenuated pro-inflammatory mediators (Tnf, Il6, Il1b), and reduced intracellular reactive oxygen species. In mouse skin, topical AKN restored Col1a1 transcription and improved histological architecture by suppressing epidermal hyperplasia and collagen depletion, with 4 mg/mL AKN providing pronounced protection alongside 4 mg/mL ergothioneine. Overall, these findings establish P. martensii mucus as a promising source of natural, hydrolysis-free photoprotective agents and support further bioassay-guided identification of its primary active constituents.

Skin aging is an inevitable physiological process driven by intrinsic genetic programming and extrinsic environmental stressors, among which ultraviolet A (UVA, 320–400 nm) radiation is the primary contributor due to its deep dermal penetration (Tang et al., 2024; Huang and Chien, 2020). Skin photoaging is characterized by epidermal structural alterations, extracellular matrix (ECM) degradation, wrinkle formation, and chronic low-grade inflammation (Salminen et al., 2022; Liu H. et al., 2024; Song et al., 2025). At the cellular and molecular levels, chronic UVA exposure generates excess reactive oxygen species (ROS), overwhelming endogenous antioxidant defenses and initiating sustained oxidative stress. This redox imbalance subsequently activates pro-inflammatory cascades—principally the nuclear factor-κB (NF-κB) axis—and upregulates matrix metalloproteinases (e.g., MMP-1, MMP-3, and MMP-9), leading to accelerated degradation of type I/III collagen fibers and loss of extracellular matrix (ECM) integrity. Concurrently, persistent photo-oxidative stress provokes DNA damage and arrests the cell cycle in dermal fibroblasts, activating the p16/p21/p53 signaling axis to induce premature cellular senescence and a senescence-associated secretory phenotype (SASP), which further perpetuates chronic low-grade inflammation and tissue breakdown (Huang and Chien, 2020; Casale et al., 2018; Feng et al., 2024). Although conventional preventive agents—including topical retinoids, antioxidants, and peptide-based cosmeceuticals—exhibit photoprotective potential, their practical applications are often hampered by retinoid-induced skin irritation, chemical instability of antioxidants, or limited transdermal delivery and enzymatic instability of bioactive peptides (Griffiths, 1995; Lin et al., 2005; Kraeling et al., 2015), underscoring the necessity of exploring safe, multi-target natural alternatives.

Marine organisms have long been recognized as a rich reservoir of structurally diverse bioactive compounds with anti-inflammatory, antioxidant, and anti-aging properties, and a growing body of evidence has highlighted marine peptides, polysaccharides, and small molecules as promising candidates for skin protection (Liu et al., 2019; Lai et al., 2026; Wei et al., 2022). Among marine bivalves, Pinctada martensii is a commercially vital aquaculture species extensively farmed in the northern South China Sea (principally along the coasts of Guangdong, Guangxi, and Hainan) and the coastal waters of southwestern Japan (such as Mie, Ehime, and Nagasaki prefectures) (Zhang et al., 2021; Hao et al., 2019). With annual marine pearl harvests in East Asia reaching tens of metric tons, this industry concurrently generates thousands of metric tons of oyster soft-tissue biomass and mucosal secretions as underutilized processing by-products each year (Ministry of Agriculture and Rural Affairs of the People’s Republic of China, 2024). To date, most research on P. martensii bioactive has focused on processing discarded soft tissues via enzymatic hydrolysis (e.g., using trypsin, alcalase, or flavourzyme) to yield functional hydrolysates with antioxidant, anti-inflammatory, and tissue-repair activities (Liu et al., 2019; Wei et al., 2022; Zhang et al., 2022; Shen et al., 2023).

In contrast, P. martensii mucus has received limited attention as a source of biologically active natural constituents. Unlike tissue-derived bioactives that require harsh enzymatic digestion, raw bivalve mucus naturally secretes a rich cocktail of endogenous functional constituents, including low-molecular-weight peptides, free amino acids, polysaccharides, and trace minerals. Utilizing direct aqueous extraction without traditional enzymatic hydrolysis offers clear advantages, as it preserves the native bioactivity and molecular integrity of these constituents while eliminating potential enzyme residues and processing complexity. However, whether a hydrolysis-free aqueous fraction derived exclusively from P. martensii mucus can effectively combat UVA-induced skin photoaging, and through which cellular pathways, remains largely uninvestigated.

To address this knowledge gap, the present study prepared a low-molecular-weight aqueous fraction (AKN) directly from P. martensii mucus via direct aqueous extraction and ultrafiltration. We systematically characterized the chemical composition of AKN using targeted compositional assays and untargeted LC–MS profiling. Furthermore, we evaluated its anti-senescence, anti-inflammatory, and ECM-protective properties in L929 fibroblasts and BV2 microglia in vitro, and verified its protective efficacy in a UVA-irradiated BALB/c mouse model in vivo, using ergothioneine (EGT) as a positive control. Overall, this study aims to establish a scientific foundation for valorizing P. martensii mucus as a natural, hydrolysis-free anti-photoaging agent for functional cosmetics and skin healthcare.

Preparation of AKN

Fresh mucus was collected from Pinctada fucata martensii during routine oyster shucking by local pearl farmers. During shucking, the soft tissues and mucus were separated, and the mucus fraction was collected separately. Approximately 1 L of mucus was obtained from 100 oysters. The collected mucus was mixed with ultrapure water at a mucus-to-water volume ratio of 3:1 and homogenized at 4 °C for 2 h. The homogenate was centrifuged at 10,000 × g for 30 min at 4 °C. The resulting supernatant was sequentially filtered through 0.45 μm and 0.22 μm membrane filters and subsequently subjected to ultrafiltration using a 3 kDa molecular-weight-cutoff membrane (Millipore, Burlington, MA, USA). The permeate was collected, lyophilized, and stored at −20 °C until use. The resulting low-molecular-weight aqueous fraction was designated AKN. No enzymatic hydrolysis was performed during the preparation process. For in vitro experiments, AKN was dissolved in ultrapure water. For topical in vivo administration, AKN was dissolved in a PEG400/ultrapure water mixture.

Chemical composition analysis of AKN

Most compositional analyses of AKN—including targeted quantification of taurine and betaine, as well as total amino acid profiling—were performed by the Institute of Analysis, Guangdong Academy of Sciences (China National Analytical Center, Guangzhou, China). Taurine was quantified according to the Chinese National Food Safety Standard (GB 5009.169–2016), and betaine content was measured using the Reinecke salt precipitation method. The total amino acid composition was analyzed via pre-column phenyl isothiocyanate (PITC) derivatization coupled with HPLC. Briefly, lyophilized AKN (∼10 mg) was hydrolyzed with 6 M HCl at 110 °C for 24 h under vacuum. After drying the hydrolysate, the residue was derivatized with PITC in acetonitrile/triethylamine/water at room temperature for 1 h and extracted with n-hexane. Separation was performed on an HPLC system (Agilent 1,260 Infinity II, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (4.6 × 250 mm, 5 μm) at 40 °C. Peak identification and quantification were accomplished using external reference amino acid standards. Monosaccharide profiling and inorganic cation/anion analyses were conducted by Shanghai Fuda Testing Technology Group Co., Ltd (Guangzhou, China), with monosaccharide composition analyzed via ion chromatography. All analyses were performed using freeze-dried AKN powder. Unless otherwise specified, data were corrected for sample mass and dilution factors and expressed on a dry-weight basis. Detailed profiles for total amino acids, inorganic ions, and monosaccharides are provided in Supplementary Tables S1–S3, respectively.

Untargeted LC–MS profiling of AKN

AKN powder was dissolved in pure water at a concentration of 1 mg/mL. The solution was vortexed and filtered through a 0.22 μm membrane before LC–MS analysis. Orthogonal chromatographic profiling was performed using reversed-phase C18 and HILIC separations. For reversed-phase analysis, a Hypersil GOLD VANQUISH C18 column (100 × 2.1 mm, 1.9 μm; Thermo Fisher Scientific) was used with water containing 0.1% trifluoroacetic acid as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B. The gradient was programmed from 5% to 95% B over 15 min, maintained at 95% B until 20 min, returned to 5% B at 20.01 min, and maintained at 5% B until 25 min. For HILIC analysis, an ACQUITY UPLC BEH HILIC column (100 × 2.1 mm, 1.7 μm; Waters) was used with 10 mM ammonium acetate in water as mobile phase A and acetonitrile as mobile phase B. The HILIC gradient was maintained at 95% B for 1 min, decreased to 50% B from 1 to 8 min, maintained at 50% B until 10 min, returned to 95% B at 10.01 min, and maintained at 95% B until 15 min. The flow rate was 0.280 mL/min for both separations. Raw LC–MS data were processed using Compound Discoverer software (version 3.3; Thermo Fisher Scientific, Waltham, MA, USA) for peak detection, retention-time alignment, feature extraction, and molecular annotation. MS/MS spectra were searched against the online mzCloud spectral library, and candidate molecular structures were further queried against the ChemSpider database. Because authentic standards were not analyzed under identical chromatographic conditions, the reported compounds were considered putatively annotated. Representative total ion chromatograms are shown in Supplementary Figure S1.

Cell culture and treatments

L929 mouse fibroblasts and BV2 mouse microglial cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were used between passages 15 and 25 and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, in a humidified incubator at 37 °C with 5% CO 2. For UVA-induced senescence, L929 cells were irradiated using a UVA lamp system with an emission spectrum of 320–400 nm and a peak emission at λ max = 365 nm (Philips, Amsterdam, Netherlands) at an irradiance of 4.17 mW/cm 2, delivering a single dose of 10 J/cm 2 over 40 min. To eliminate optical interference and photochemical artifacts from colored media components (e.g., phenol red and riboflavin), culture medium was aspirated and L929 cells were washed twice with PBS. Following irradiation, cells were allowed to recover in complete medium for 24 h. For inflammatory stimulation, BV2 cells were treated with 1 μg/mL lipopolysaccharide (LPS, Escherichia coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA) for 10 h. For the UVA damage model, AKN (400 μg/mL) was pre-administered 12 h prior to UVA irradiation. Meanwhile, AKN was co-treated simultaneously with LPS in BV2 cells.

Senescence-associated β-galactosidase (SA-β-gal) staining

SA-β-Gal staining was performed 72 h after UVA exposure using a commercial SA-β-Gal staining kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. In brief, L929 cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and incubated with fresh SA-β-Gal staining solution (pH 6.0) at 37 °C for 12–16 h in a humidified incubator without CO 2. Bright-field images were captured with an inverted microscope (Nikon, Tokyo, Japan), and the percentage of SA-β-Gal-positive (blue-stained) cells was quantified from at least five random fields per group using ImageJ software (v1.53, NIH, Bethesda, MD, USA).

RNA extraction and RT-qPCR

Total RNA was extracted from L929 cells, BV2 cells, or mouse dorsal skin tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using a PrimeScript RT Reagent Kit (TaKaRa, Kusatsu, Japan). Quantitative real-time PCR was performed on a QuantStudio five system (Applied Biosystems, Waltham, MA, USA) using SYBR Green PCR Master Mix. The thermal cycling conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and a final melt-curve analysis. β-Actin was used as the internal reference, and the relative expression levels were calculated by the 2 − ΔΔCt method. All reactions were performed in triplicate. Primer sequences are listed in Supplementary Table S4.

UVA-induced mouse skin photoaging model

Six-to seven-week-old male BALB/c mice (specific-pathogen-free (SPF), body weight 18–22 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed under SPF conditions (22 °C ± 2 °C, 50% ± 10% relative humidity, 12 h light/dark cycle) with free access to standard chow and water. After a 5-day acclimation period, the dorsal hair was shaved with an electric clipper 3 days before the first UVA exposure. Mice were randomly divided into five groups (n = 6 per group) using a computer-generated randomization list: (i) CTRL (untreated), (ii) UVA (model), (iii) UVA +1 mg/mL AKN, (iv) UVA +4 mg/mL AKN, and (v) UVA +4 mg/mL EGT (positive control). Thirty minutes before each UVA irradiation, the corresponding solution (200 µL) was applied topically to the dorsal skin; CTRL and UVA groups received an equal volume of solvent. Mice were exposed to UVA (365 nm, 400 mJ/cm 2 per session) for 16 days using a UVA lamp placed 30 cm above the dorsal skin. The cumulative UVA dose and the animal welfare endpoints (body weight, skin appearance, behavior) were monitored throughout the experiment. Group allocation and outcome assessment were performed by investigators blinded to the treatment assignment. At the end of the experiment, mice were euthanized by cervical dislocation under isoflurane anesthesia, and dorsal skin samples were collected, photographed, and processed for histological and molecular analyses.

Histological analysis

Dorsal skin samples were fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at 5 µm thickness. For hematoxylin–eosin (H&E) staining, sections were stained with hematoxylin for 5 min and eosin for 2 min. For Masson’s trichrome staining, a commercial kit (Solarbio, Beijing, China) was used according to the manufacturer’s instructions. Slides were observed and photographed under a light microscope (Nikon, Tokyo, Japan). Epidermal thickness was measured in at least five randomly selected fields per sample using ImageJ, and collagen fiber density was quantified as the average optical density (AOD) of the blue-stained area.

Western blot analysis

Dorsal skin tissues were homogenized in RIPA lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors. The protein concentration was determined using a BCA protein assay kit (Beyotime). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk in TBST for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against iNOS (1:1,000, Abcam, Cambridge, United Kingdom), p-P65 (1:1,000, Cell Signaling Technology, Danvers, MA, USA), p16 (1:1,000, Abcam), and GAPDH (1:5,000, Proteintech, Wuhan, China). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, Proteintech) for 1 h at room temperature, and the signals were visualized using an enhanced chemiluminescence (ECL) substrate (Beyotime) and captured with a chemiluminescence imaging system (Tanon, Shanghai, China). Band intensities were quantified by densitometry using ImageJ and normalized to GAPDH.

Statistical analysis

All quantitative data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Differences among multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p -value <0.05 was considered statistically significant. Asterisks (*) in the figure panels denote statistical significance compared with the model group.

Preparation and compositional characterization of AKN

AKN was prepared from P. martensii mucus through aqueous extraction, centrifugation, 3 kDa molecular-weight-cutoff ultrafiltration, and freeze-drying, without enzymatic hydrolysis (Figure 1A). Quantitative compositional analysis showed that freeze-dried AKN contained taurine (8.0%, w/w), betaine (4.1%, w/w), total amino acids after hydrolysis (6.0%, w/w), inorganic components (72.0%, w/w) and total sugars (0.15%, w/w) (Figure 1B). Amino acid profiling after acid hydrolysis showed that glycine was the predominant amino acid, followed by glutamic acid, proline, alanine, and valine, whereas the remaining measured amino acids were present at levels below 0.1% (Supplementary Table S1). Detailed ion analysis showed that chloride and sodium were the predominant inorganic ions, followed by sulfate, bicarbonate, potassium, and magnesium (Supplementary Table S2). Monosaccharide analysis further revealed that the carbohydrate fraction was composed primarily of galactose and glucose together with lower amounts of other monosaccharides (Supplementary Table S3). Complementary untargeted LC–MS analyses using reversed-phase C18 and HILIC separations generated distinct chemical profiles of AKN (Supplementary Figure S1). Prominent features were putatively annotated as taurine and betaine under both chromatographic conditions, whereas a feature putatively assigned to hypoxanthine was observed only under the HILIC conditions. Taurine and betaine were further validated by independent targeted quantification. Collectively, these analyses provide a quantitative overview of the major constituents and a comprehensive qualitative chemical fingerprint of AKN.

AKN exhibits anti-senescence, anti-inflammatory, and antioxidant activities in vitro

To assess the biological potential of AKN, we first evaluated its anti-senescence, anti-inflammatory, and antioxidant activities in well-established in vitro models, because these three activities are tightly interlinked in the photoaging process and jointly maintain cutaneous homeostasis (Dimri et al., 1995; Li et al., 2024; Kandan et al., 2020). Cellular senescence is commonly characterized by cell-cycle arrest and the upregulated expression of cyclin-dependent kinase inhibitors (p16, p21) and the tumor suppressor p53 (Li et al., 2024; Chen et al., 2006; Beausejour et al., 2003). UVA-irradiated L929 mouse fibroblasts were employed as the senescence model due to their well-documented oxidative-stress response (Jiang et al., 2019; Xia et al., 2024), while lipopolysaccharide (LPS)-stimulated BV2 microglia were utilized to evaluate general anti-inflammatory capacity (Dai et al., 2015).

Prior to investigating the photoprotective effects against UVA-induced damage, the basal biocompatibility of AKN was evaluated in L929 fibroblasts using the MTT assay. AKN treatment alone (without UVA irradiation) across the tested concentrations (7.81–1,000 μg/mL) exerted no detectable cytotoxicity, maintaining cell viability above 95% compared with the untreated control (Supplementary Figure S3). Having established its non-toxic profile, we next evaluated whether AKN pretreatment could mitigate UVA-induced cellular senescence. As shown in Figures 2A–C, UVA irradiation markedly increased the mRNA levels of p16, p21, and p53 in L929 cells compared with the untreated control. AKN pretreatment significantly attenuated these three senescence markers compared with the UVA-irradiated model group (P < 0.05), although the expression levels remained higher than those in the untreated baseline control. In LPS-stimulated BV2 cells, AKN significantly reduced the mRNA expression of the canonical pro-inflammatory cytokines TNF- α, IL-6, and IL-1 β (Figures 2D–F), suggesting that AKN interferes with the pro-inflammatory transcriptional program activated by microglial stimulation. Consistent with this anti-inflammatory profile, AKN also decreased the intracellular ROS level in BV2 cells, as measured by DCFH-DA fluorescence (Figure 2G). Together, these in vitro data indicate that AKN exerts protective effects against oxidative and inflammatory challenges.

AKN attenuates UVA-Induced senescence in L929 cells

To provide morphological evidence corroborating the molecular findings, we performed senescence-associated β-galactosidase (SA-β-Gal) staining, the gold-standard histochemical marker of cellular senescence (Jiang et al., 2019), in UVA-irradiated L929 cells with or without AKN treatment. Compared with the non-irradiated control, UVA-irradiated L929 cells displayed a markedly enlarged and flattened morphology, accompanied by a pronounced increase in the proportion of SA-β-Gal-positive cells (blue-stained cytoplasm), confirming the successful establishment of the senescent phenotype. Notably, treatment with AKN (400 μg/mL) markedly decreased the proportion of SA-β-Gal -positive cells and largely restored normal cell morphology (Figure 3). These morphological data reinforce the conclusion from Section 3.2 that AKN effectively alleviates UVA-induced fibroblast senescence.

AKN ameliorates UVA-Induced skin photoaging in mice

To evaluate whether the in vitro protective effects translate to an in vivo setting, we established a UVA-induced skin photoaging model in BALB/c mice, utilizing topical ergothioneine (EGT, 4 mg/mL) as a positive control due to its well-documented photoprotective activity via the PI3K/AKT/Nrf2 signaling pathway (Li et al., 2024). Dorsal skin appearance was monitored throughout the experimental period. As shown in Figure 4, mice in the UVA model group developed typical photoaging phenotypes, including pronounced wrinkles, rough and dull skin texture, and a loss of elasticity, consistent with established macroscopic evaluation criteria (Kligman, 1989; Fisher et al., 2002; Liu S. et al., 2024).

Topical application of AKN at 1 mg/mL resulted in visibly smoother skin and shallower wrinkles compared with the UVA model group. This protective effect was even more pronounced at 4 mg/mL, where the dorsal skin appearance closely resembled that of the normal (non-irradiated) control group. Notably, topical application of 4 mg/mL AKN resulted in marked improvements in skin texture and wrinkle appearance alongside the positive control EGT. These results demonstrate that topical AKN effectively ameliorates UVA-induced macroscopic skin damage in this mouse model, with 4 mg/mL AKN demonstrating robust photoprotective efficacy alongside the reference control EGT.

AKN restores gene expression in UVA-Irradiated mouse skin

To elucidate the molecular basis of the in vivo protective effect, we quantified the mRNA levels of key genes associated with cellular senescence, ECM remodeling, and inflammation in mouse dorsal skin by RT-qPCR (Figure 5). UVA irradiation significantly upregulated the senescence-associated genes p16, p21, and p53 and the matrix metalloproteinase gene Mmp9, and significantly downregulated the collagen-encoding gene Col1a1, reproducing the transcriptional signature of photoaged skin (Casale et al., 2018; Feng et al., 2024; Fisher et al., 2002; Devos et al., 2023; Lee et al., 2021). The inflammation-related gene iNOS was also induced, consistent with the chronic low-grade inflammatory state that characterizes photoaging.

Treatment with AKN attenuated these transcriptional alterations. Specifically, 1 mg/mL AKN significantly decreased the mRNA levels of p16, p21, p53, and iNOS compared with the UVA model group. At 4 mg/mL, AKN further suppressed Mmp9 expression and increased Col1a1 levels. Together, these results indicate that AKN protects skin tissue against UVA-induced senescence, inflammation, and ECM degradation.

AKN improves skin histology and collagen deposition

To evaluate the structural consequences of AKN treatment at the tissue level, hematoxylin–eosin (H&E) and Masson’s trichrome staining were performed on dorsal skin sections (Figure 6). Compared with the control group, the UVA model group displayed a markedly thickened epidermis, a disorganized dermal architecture, and a striking reduction in collagen fiber density, as visualized by Masson’s trichrome staining. These histological alterations recapitulate the well-documented histopathological features of UVA-induced photoaging, in which chronic UV exposure drives epidermal hyperproliferation and dermal ECM depletion (Kligman, 1989; Fisher et al., 2002; Liu S. et al., 2024).

AKN treatment mitigated UVA-induced alterations, with 4 mg/mL AKN exhibiting greater efficacy across evaluated endpoints. Topical application of 1 mg/mL AKN exhibited a partial reduction in epidermal thickness and a modest recovery of dermal collagen. Treatment with 4 mg/mL AKN resulted in a more pronounced reduction in epidermal thickness, a more organized dermal structure, and a substantial restoration of collagen deposition compared with the UVA model group. Quantitative analysis of epidermal thickness and collagen fiber density confirmed these histological observations (Figure 6B). These histological findings are fully consistent with the macroscopic skin appearance and the gene expression data, supporting the conclusion that AKN mitigates UVA-induced skin aging in mice through coordinated actions on epidermis, dermis, and ECM.

AKN modulates protein expression in mouse skin

To verify that the gene-level changes translate to the protein level, we performed Western blot analysis of iNOS, phosphorylated p65 (p-P65), and p16 in dorsal skin lysates (Figure 7). The UVA model group showed significantly elevated protein levels of iNOS and p-P65, in line with the activation of the NF-κB inflammatory axis (Zhang et al., 2025), together with an increased level of p16, consistent with the accumulation of senescent cells in the skin.

Treatment with 1 mg/mL AKN mildly reduced the protein levels of iNOS, p-P65, and p16, while 4 mg/mL AKN produced a significant reduction in all three protein markers relative to the UVA model group. These protein-level data corroborate the RT-qPCR results, further confirming that AKN attenuates UVA-induced skin photoaging through anti-inflammatory and anti-senescent signaling pathways.

In the present study, a <3 kDa fraction (AKN) was isolated from the aqueous extract of P. martensii mucus. Through a combination of cellular assays, a UVA-induced mouse photoaging model, and multi-level molecular analyses, we demonstrated that AKN attenuates cellular senescence, inflammatory responses, and oxidative stress, thereby protecting skin against UVA-induced photoaging. Notably, topical application of 4 mg/mL AKN exhibited photoprotective activity alongside the reference antioxidant ergothioneine (EGT).

The P. martensii mucus utilized here is an underutilized natural secretion from pearl cultivation by-products. Previous research on P. martensii bioactives has overwhelmingly focused on flesh/soft tissue hydrolysates produced via enzymatic digestion. For instance, hydrolysate-derived peptides (typically 2–15 amino acid residues rich in hydrophobic residues) have demonstrated antioxidant, anti-inflammatory, and wound-healing properties (Huang et al., 2023; Yang et al., 2019a; Yang et al., 2019b). Recently, Wei et al. reported that an enzymatic hydrolysate of P. martensii meat (PME) alleviated UVB-induced skin damage in mice through MMP suppression and gut–skin axis modulation (Wei et al., 2026). The present work extends this line of investigation in two significant directions. First, AKN, prepared exclusively via direct aqueous extraction and ultrafiltration without enzymatic hydrolysis, represents a compositionally distinct fraction rich in native free amino acids, small peptides, nucleosides, and polar metabolites. This distinction is paramount, as enzymatic digestion substantially alters native peptide profiles and risks destroying sensitive bioactive sequences. Second, we specifically targeted UVA radiation, which penetrates into the deeper dermis and drives long-term dermal photoaging, yet has received substantially less attention than UVB in P. martensii research (Tang et al., 2024; Huang and Chien, 2020).

At the cellular and molecular level, the protective effects of AKN were associated with coordinated interventions across interconnected pathological axes: central senescence regulators (p16, p21, p53), key inflammatory mediators (iNOS, TNF- α, Il-6, Il-1β), and ECM-remodeling genes (Mmp9, Col1a1) in both L929 cells and mouse skin, alongside suppression of intracellular ROS. These coordinated actions align with the concept that photoaging is driven by a self-reinforcing feed-forward cascade (Salminen et al., 2022; Liu H. et al., 2024; Lee et al., 2021; Freund et al., 2010): UVA-induced oxidative stress promotes inflammatory signaling (e.g., NF-κB activation), which subsequently provokes the SASP-like secretory state and triggers irreversible p53/p21 and p16 cell-cycle arrest, while simultaneously tilting matrix metalloproteinase activity over structural collagen synthesis. AKN effectively uncouples this destructive cascade at multiple regulatory nodes.

Notably, while the positive control EGT significantly attenuated senescence and inflammatory markers, its regulation of Mmp9 and Col1a1 did not reach statistical significance. The distinct transcriptional response observed with 4 mg/mL AKN highlights the unique advantage of a holistic natural matrix over a single-target antioxidant. Crucially, the biological potency of AKN should not be interpreted as a simple additive summation of its individually identified markers (taurine, betaine, and free amino acids). Rather, AKN functions as an integrated, multi-component bioactive cluster. While targeted quantification confirmed the presence of these known osmolytes and nutritional metabolites, untargeted LC–MS profiling revealed a diverse pool of uncharacterized low-molecular-weight constituents and native oligopeptide sequences. In this natural matrix, these co-extracted uncharacterized trace constituents likely act in concert with primary osmolytes to generate an ‘entourage’ or synergistic matrix effect, simultaneously engaging complementary defense pathways and reinforcing ECM structural integrity. Preserving this unhydrolyzed, native multi-component network avoids the disruption of delicate bioactive synergies that frequently occurs during harsh fractionation, conferring comprehensive protection that transcends the capabilities of isolated single compounds. This feature is reminiscent of other marine-derived multi-target protective complexes, such as the Kappaphycus alvarezii polysaccharide KP-90 (Lai et al., 2026) and the sterol ergosterol (Zhang et al., 2025).

From an application perspective, AKN is prepared using a straightforward aqueous extraction and ultrafiltration workflow that completely avoids proteolytic enzymes, organic solvents, and laborious multi-step purification. This streamlined preparation retains the native biochemical composition while facilitating future green processing and industrial standardization. The high abundance of low-molecular-weight, water-soluble constituents provides distinct formulation advantages for cosmetic and dermatological preparations. Taken together, these findings position AKN as a promising marine-derived multifunctional ingredient for topical anti-photoaging interventions.

Limitations

Several limitations of the present study should be acknowledged. First, while AKN was comprehensively profiled via targeted metabolite quantification and untargeted LC–MS fingerprinting, its precise single active constituent(s) and potential synergistic interactions remain to be fully resolved. Systematic bioassay-guided fractionation will be required in future work to pinpoint the key active principles. Second, direct cellular uptake and intracellular disposition of AKN constituents were not experimentally tracked; future investigations using labeled constituents or targeted intracellular metabolomics are warranted to elucidate their specific transport mechanisms. Third, while our findings demonstrate multi-target photoprotection against acute UVA damage, further omics-based analyses (e.g., transcriptomics or proteomics) and evaluations under chronic, low-dose UVA or combined UVA/UVB challenge are warranted to completely delineate the responsive molecular networks and real-world applicability. Fourth, although no overt skin irritation was observed during animal trials, rigorous safety evaluations—including formal sensitization, repeated-dose toxicity, long-term formulation stability, and human clinical trials—are necessary prior to translational cosmeceutical applications.

Conclusion

AKN, a low-molecular-weight aqueous fraction obtained from P. martensii mucus by direct aqueous extraction and 3 kDa ultrafiltration without enzymatic hydrolysis, attenuated UVA-associated cellular senescence, inflammatory responses, oxidative stress, epidermal hyperplasia, and collagen loss in complementary cellular and murine models. These effects were accompanied by changes in senescence-, inflammation-, and extracellular matrix-related markers, restoration of collagen deposition, and improvement of skin histology. At 4 mg/mL, AKN demonstrated protective efficacy comparable with that of the positive control EGT in attenuating senescence and inflammatory markers. Overall, this study highlights the valorization of P. martensii mucus as a natural, hydrolysis-free anti-photoaging constituent and lays a solid foundation for future bioassay-guided fractionation to pinpoint its primary active principles.