. 2026 Mar 19;20(4):571–589. doi: 10.4162/nrp.2026.20.4.571

Oyster hydrolysate attenuates osteoarthritis progression by modulating inflammatory signaling and extracellular matrix homeostasis

Seon Yeong Ji ^1,^2,^*, Hyun Hwangbo ^1,^2,^*, Min Yeong Kim ^1,², Da Hye Kim ^1,², Su Hyun Hong ^1,², Jung-Hyun Shim ^3,⁴, Sung-Kwon Moon ⁵, Gi-Young Kim ⁶, Suengmok Cho ⁷, Yung Hyun Choi ^1,^2,^✉

PMCID: PMC13458003 PMID: 42582610

Abstract

BACKGROUND/OBJECTIVES

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage destruction and persistent low-grade inflammation, in which extracellular matrix (ECM) homeostasis plays a pivotal role. Although oyster hydrolysate (OH), derived from Crassostrea gigas (Magallana gigas), is rich in bioactive peptides and has antioxidant and anti-inflammatory properties, its effects on the pathogenesis of OA have not yet been studied. This study aimed to investigate the anti-inflammatory and chondroprotective effects of OH using in vitro and in vivo OA models.

MATERIALS/METHODS

To evaluate the protective effects of OH against OA-associated inflammation and ECM degradation, an in vitro inflammatory model was established by stimulating human chondrosarcoma SW1353 cells with interleukin (IL)-1β, followed by assessment of inflammatory and ECM-related proteins. In vivo, OA was induced by intra-articular injection of monosodium iodoacetate (MIA) into the knee joints of Sprague-Dawley rats, and arthritis-related histological, molecular, and biochemical biomarkers were analyzed in joint tissues and serum.

RESULTS

In vitro, OH reduced IL-1β-induced pro-inflammatory cytokine production and downregulated ECM-degrading enzymes, including a disintegrin and metalloproteinase with thrombospondin motifs-5, matrix metalloproteinase (MMP)-3, and MMP-13, but preserved the expression levels of anabolic proteins, such as aggrecan and collagen II. These effects were associated with the suppression of phosphoinositide 3-kinase/protein kinase B, c-Jun N-terminal kinase, p38, and nuclear factor κB cells signaling pathways. Consistently, oral administration of OH for 14 days alleviated joint swelling, preserved proteoglycan content, and improved the histological Osteoarthritis Research Society International scores in MIA-induced OA rats. OH treatment also reduced the cartilage expression levels of inflammatory cytokines and MMPs and serum levels of IL-1β, IL-6, leukotriene B4, MMP-9, C-reactive protein, and cartilage oligomeric matrix protein.

CONCLUSION

Overall, our results suggest that OH attenuates OA progression by modulating inflammatory signaling and restoring ECM homeostasis, highlighting its potential as a marine-derived candidate for OA prevention and therapy.

Keywords: Cartilage, extracellular matrix, inflammation, osteoarthritis, ostreidae

INTRODUCTION

Osteoarthritis (OA) is the most common degenerative joint disorder that is a major cause of disability in elderly individuals worldwide. Clinically, it is diagnosed based on a combination of characteristic symptoms, such as chronic joint pain, stiffness, swelling, and reduced range of motion, along with radiographic features, including joint space narrowing, osteophyte formation, and subchondral bone sclerosis [1,2]. Histopathologically, OA is characterized by the progressive destruction of the articular cartilage, synovial inflammation, and remodeling of the periarticular bone, ultimately leading to impaired joint function and diminished quality of life [3,4]. A central event in this degenerative process is the disruption of extracellular matrix (ECM) homeostasis: anabolic synthesis of key cartilage components such as aggrecan and type II collagen is suppressed, whereas catabolic degradation mediated by matrix metalloproteinases (MMPs) and aggrecanases is accelerated [5,6]. This imbalance between ECM synthesis and degradation directly contributes to cartilage erosion and loss of structural integrity [7,8]. The growing prevalence of OA, driven by aging demographics, obesity, and mechanical overuse, imposes substantial healthcare and socioeconomic burdens [9]. Despite its significant impact, existing therapies are largely palliative, with disease-modifying effectively attenuating the structural progression of OA still lacking.

Inflammation, a conserved host defense mechanism, has been extensively studied under both infectious and sterile conditions [10]. Activation of pattern recognition receptors, such as Toll-like receptors (TLRs) initiates downstream signaling cascades that converge on transcription factors including nuclear factor κB (NF-κB) and activator protein-1, thereby inducing the expression of cytokines, chemokines, and effector enzymes [11,12]. For example, NF-κB activation drives the transcription of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and MMPs, which collectively shape the inflammatory microenvironment [13,14]. Mitogen-activated protein kinase (MAPK) signaling pathways, including p38 and c-Jun N-terminal kinase (JNK) pathways, similarly enhance inflammatory gene expression and regulate immune cell recruitment [15,16,17]. Although acute inflammation eliminates pathogens and initiates repair, failure of such resolution programs leads to chronic low-grade inflammation, contributing to metabolic disorders, atherosclerosis, and degenerative diseases [18,19].

Accumulating evidence highlights the critical role of inflammation in OA pathogenesis [20,21]. Xu et al. [17] demonstrated that IL-1β and TNF-α suppress anabolic processes in chondrocytes while upregulating catabolic mediator levels, thereby tipping the balance toward cartilage breakdown. MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) are the key enzymes mediating type II collagen and aggrecan degradation, respectively, in human OA cartilage and experimental models [3,22,23]. Synovitis, previously considered a secondary feature of OA, is now recognized as an early driving factor of disease progression, with the synovial tissue producing cytokines and proteases that perpetuate cartilage damage [24,25]. Moreover, cartilage degradation products act as damage-associated molecular patterns, activating TLR-mediated NF-κB signaling in chondrocytes and synoviocytes and establishing a self-perpetuating cycle of inflammation and ECM destruction [26]. Unlike autoimmune-driven inflammatory arthritis, OA inflammation remains compartmentalized within joints; however, its presence is strongly correlated with pain severity and radiographic progression [20,27].

Marine-derived bioactive compounds have attracted increasing attention as alternatives or adjuncts to conventional therapies due to their pleiotropic bioactivities and favorable safety profiles [28,29]. Oysters, particularly Crassostrea gigas (Magallana gigas), are rich in proteins, peptides, taurine, and trace minerals and traditionally used both as nutrient sources and remedies in East Asian medicine [30,31]. Oyster extracts and hydrolysates possess antioxidant and anti-inflammatory properties; they suppress iNOS, COX-2, and pro-inflammatory cytokines in macrophage models and activate the nuclear factor erythroid 2-related factor 2-mediated antioxidant pathway [32,33,34,35]. Hydrolyzed oyster peptides exhibit enhanced bioavailability and biological activity, showing great potential as nutraceuticals [36,37,38,39]. However, despite increasing evidence of their bioactivity, the potential of C. gigas hydrolysates to modulate inflammatory signaling and ECM homeostasis in musculoskeletal disorders, particularly OA, has not yet been investigated. Therefore, this study aimed to assess the therapeutic potential of oyster hydrolysates (OHs) for OA. By focusing on its ability to modulate critical inflammatory signaling cascades, including the MAPK, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), and NF-κB cascades, and ECM regulatory processes underlying OA pathogenesis, this study sought to provide a novel marine-derived candidate to the prevent and manage of OA.

MATERIALS AND METHODS

SW1353 cell culture for in vitro experiments

SW1353 human chondrosarcoma cell line (No. HTB-94) was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Leibovitz’s (1×) L-15 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (WelGENE, Daegu, Korea) and 100 U/mL penicillin and were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

OH preparation

Freshly frozen Pacific oysters (C. gigas) were purchased from a local seafood supplier in Gyeongsang-do, Republic of Korea. The oysters (approximately 10 kg) were thawed, thoroughly washed, and soaked in distilled water adjusted to pH 6.4. Then, the oysters were enzymatically hydrolyzed by with 0.5% Alcalase 2.4 L FG (Novozyme, Bagsværd, Denmark) in a shaking incubator at 60°C for 5 h. The enzyme was subsequently inactivated by heating the mixture at 85°C for 20 min. The hydrolysate was centrifuged at 9,000 rpm for 10 min at 4°C, and the resulting supernatant was collected, concentrated under reduced pressure, and lyophilized for at least 72 h. The final yield of OH was 9.6%, and the dried powder was reconstituted in distilled water before use in experiments.

Cell viability and in vitro OA experimental design

The cytotoxicity of OH in SW1353 cells was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (Thermo Fisher Scientific) assay as previously described [40]. The cells were treated with various concentrations of OH (100, 200, 400, 600, and 800 μg/mL) for 24 h, after which cell viability was analyzed. For inflammatory stimulation, SW1353 cells were exposed to IL-1β, and the optimal concentration for inducing an OA-like environment was determined (Supplementary Fig. 1). Based on the results, the cells were stimulated with 40 ng/mL IL-1β (Abcam, Cambridge, UK) and subsequently treated with OH at a non-cytotoxic maximal concentration of 800 μg/mL for 24 h to evaluate its effects under OA-mimicking conditions.

Enzyme-linked immunosorbent assay (ELISA)

ELISA kits for IL-1β (Cat. No. SMLB00C), IL-6 (Cat. No. SM6000B), MMP-9 (Cat. No. MMPT90), and C-reactive protein (CRP, Cat. No. DY1744) were purchased from R&D Systems (Minneapolis, MN, USA). Kits for leukotriene B4 (LTB4; Cat. No. MBS727908) and cartilage oligomeric matrix protein (COMP; Cat. No. MBS2020931) were obtained from MyBioSource (San Diego, CA, USA). Levels of these factors were measured in cell culture supernatants or rat serum samples according to the manufacturers’ instructions. Notably, the serum samples were diluted 2- to 4-fold with the provided assay diluent to ensure that the measured values fell within the linear range of the standard curve.

Western blot analysis

SW1353 cells were pretreated with OH for 1 h and stimulated with IL-1β for 24 h before collection. Total protein was extracted by suspending the cells in a lysis buffer (250 mM NaCl, 25 mM Tris-Cl, 5 mM EDTA, 1% NP-40) and incubating the lysates on ice for 30 min, as previously described [41]. The cytoplasmic and nuclear fractions were isolated using the Nuclear and Cytosol Extraction Kit (Thermo Fisher Scientific). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 25 min and incubated overnight with the appropriate primary antibodies against MMP-3, MMP-13, ADAMTS-5, aggrecan, collagen-II, IL-6, IL-1β, p-PI3K, PI3K, pAKT, AKT, pJNK, JNK, p-p38, p38, p65, actin, lamin B (Santa Cruz Biotechnology, Dallas, TX, USA; Cell Signaling Technology, Danvers, MA, USA; and Abcam) at 4°C. Detailed information on the antibodies used in this study, including manufacturer, catalog numbers, and dilution conditions, is provided in Table 1. After washing thrice with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min each, the membranes were incubated with the corresponding secondary antibodies (Santa Cruz Biotechnology) at room temperature (RT) for 1 h. Following additional washes, protein bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific) and images were captured using the Fusion FX imaging system (Vilber Lourmat, Colléien, France).

Table 1. List of antibodies used for Western blot analysis.

Antibody	Manufacturer	Catalog No.	Dilution
Actin	Bioworld	BS6007M	1:25,000
ADAMTS-5	Abcam	ab41037	1:1,000
Aggrecan	Invitrogen	MA3-16888	1:500
AKT	Santa Cruz Biotechnology	sc-81434	1:1,000
p-AKT	Santa Cruz Biotechnology	sc-514032	1:1,000
Collagen-II	Abcam	ab34712	1:500
IL-1β	Abcam	ab9722	1:1,000
IL-6	Abcam	ab6672	1:1,000
JNK	Cell Signaling Technology	9252s	1:1,000
p-JNK	Cell Signaling Technology	9255s	1:1,000
Lamin B	Santa Cruz Biotechnology	sc-6216	1:1,000
MMP-3	Abcam	ab52915	1:1,000
MMP-13	Abcam	ab39012	1:1,000
p38	Santa Cruz Biotechnology	sc-7972	1:1,000
p-p38	Cell Signaling Technology	9211s	1:1,000
p65	Cell Signaling Technology	6956s	1:1,000
p-p65	Cell Signaling Technology	3033s	1:1,000
PI3K	Cell Signaling Technology	3358s	1:1,000
p-PI3K	Cell Signaling Technology	4228s	1:1,000

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ADAMTS-5, a disintegrin and metalloproteinase with thrombospondin motifs-5; AKT, protein kinase B; p-, phosphorylated; IL, interleukin; JNK, c-Jun N-terminal kinase; MMP, matrix metalloproteinase; PI3K, phosphoinositide 3-kinase.

Immunofluorescence assay

Briefly, cultured SW1353 cells were fixed with 100% methanol (SK Chemicals, Seongnam, Korea) for 5 min and incubated with a blocking buffer (5% bovine serum albumin, BSA; Sigma-Aldrich) in PBS containing 0.1% Triton X-100) at RT for 1 h. After blocking, the cells were incubated overnight at 4°C with an anti-collagen II antibody (Abcam) diluted in 2.5% BSA. The next day, the cells were incubated with the Alexa Fluor 488–labeled donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific) for 1 h in the dark [42]. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (Sigma-Aldrich).

Enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (version 6.8; https://davidbioinformatics.nih.gov). These analyses were conducted to identify the biological functions and signaling pathways significantly associated with C. gigas and OA. GO terms were categorized into 3 domains: biological processes (BPs), cellular components (CCs), and molecular functions (MFs). Data were visualized using the Wei Sheng Xin platform (https://www.bioinformatics.com.cn).

In vivo OA experimental design

Seven-week-old male Sprague–Dawley rats (230–260 g of average body weight; n = 40) were purchased from Koatech Co., Ltd. (Namyangju, Korea) and housed under specific pathogen-free conditions at the Animal Care Center of Dong-eui University College of Korean Medicine. All animal procedures were approved by the Institutional Animal Care and Use Committee of Dong-eui University (Ethical approval No. R2024-002) and adhered to the relevant guidelines and regulations. After acclimation for one week, OA was induced by intra-articular injection of 50 μL monosodium iodoacetate (MIA; 6 mg/mL; Sigma-Aldrich) into the right knee joints of rats. Rats in the normal group received 0.9% saline instead. MIA was dissolved in 0.9% saline (JW Pharmaceutical Co., Ltd., Dangjin, Korea) before injection. Seven days after MIA injection, the animals were orally administered OH (50 and 100 mg/kg) or glucosamine hydrochloride (positive control, PC, 100 mg/kg; Sigma-Aldrich) once daily for 2 weeks.

Histopathological analysis

Following euthanasia, the right knee joints were harvested, and joint swelling was assessed by measuring the knee width using a digital caliper (Bluebird, Seoul, Korea). For histological evaluation, the joints were fixed with 10% neutral buffered formalin (Junsei Chemical Co., Ltd., Tokyo, Japan) at 4°C for 24 h and decalcified in a decalcifying solution (Sigma-Aldrich) for 6 days. After routine paraffin (Leica Biosystems, Nussloch, Germany) embedding, the tissues were sectioned at a thickness of 7 μm and mounted on slides (Marienfeld Superior, Lauda-Königshofen, Germany). The sections were stained with safranin O and fast green (Sigma-Aldrich) and hematoxylin and eosin (H&E; Sigma-Aldrich), and histopathological changes were evaluated using an optical microscope [43] based on the Osteoarthritis Research Society International (OARSI).

Immunohistochemistry

For immunohistochemical staining, the tissue sections were incubated overnight at 4°C with primary antibodies against aggrecan, collagen II, MMP-3, MMP-9, MMP-13, TNF-α, IL-6, IL-1β, p-AKT, p-JNK, and p-p38. Subsequently, the sections were incubated with the appropriate secondary antibodies at RT for 1 h [44]. The stained slides were examined using the EVOS FL Auto 2 imaging system (Thermo Fisher Scientific), and the stained areas were quantitatively analyzed using Celleste Image Analysis Software (Thermo Fisher Scientific).

Statistical analysis

All statistical analyses were conducted using the GraphPad Prism software version 8.4.2 (GraphPad Software Inc., San Diego, CA, USA). Differences among groups were analyzed by one-way analysis of variance, followed by Tukey’s post hoc test. Statistical significance was set as P < 0.05.

RESULTS

Effect of OH on ECM degradation in IL-1β-stimulated SW1353 cells

To determine whether OH exerts any cytotoxic effects on chondrocytes, SW1353 cells were exposed to increasing concentrations of OH (0–800 μg/mL) for 24 h, and cell viability was assessed. Notably, OH treatment did not exert any cytotoxic effects, and viability of OH-treated cells remained comparable to or slightly higher than that of untreated controls across all tested concentrations (Fig. 1A). As no adverse effects were observed even at the highest concentration, subsequent experiments were conducted using 800 μg/mL OH. Additionally, we established an in vitro osteoarthritic model by stimulating SW1353 cells with IL-1β to mimic the inflammatory microenvironment associated with cartilage degradation. Next, we investigated the effects of OH on the balance between ECM synthesis and degradation by measuring the protein expression levels of ECM anabolic (aggrecan and collagen II) and catabolic (ADAMTS-5, MMP-3, and MMP-13) markers via Western blotting analysis. Indeed, IL-1β stimulation markedly upregulated the catabolic marker levels while decreasing the anabolic marker levels. However, OH treatment strongly attenuated the IL-1β-induced upregulation of catabolic enzymes and prevented the loss of aggrecan and collagen II (Fig. 1B). Consistently, immunofluorescence staining revealed that OH preserved collagen II expression, preventing its IL-1β-induced degradation (Fig. 1C and D). Collectively, these findings suggest that OH mitigates IL-1β-induced ECM degradation in chondrocytes, highlighting its modulatory effect on matrix homeostasis under inflammatory conditions.

Fig. 1 — OH, oyster hydrolysate; IL, interleukin; ECM, extracellular matrix; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; ADAMTS-5, a disintegrin and metalloproteinase with thrombospondin motifs-5; MMP, matrix metalloproteinase; DAPI, 4′,6-diamidino-2-phenylindole.

^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. IL-1β-treated group.

Effect of OH on inflammatory cytokine expression in IL-1β-stimulated SW1353 cells

OA development and progression are closely associated with inflammatory responses, as pro-inflammatory cytokines promote chondrocyte apoptosis and suppress ECM component synthesis [45]. To evaluate the effects of OH on inflammatory responses, we analyzed the expression and secretion of pro-inflammatory cytokines in IL-1β-stimulated SW1353 cells. As shown in Fig. 2A, IL-1β treatment markedly increased the protein expression levels of IL-1β and IL-6 compared to those in the control group; however, OH treatment attenuated this effect. To confirm whether elevated protein expression translated into increased cytokine secretion, cytokine secretion was quantified in the culture supernatants under the same treatment conditions. Indeed, IL-1β stimulation significantly increased the production of IL-1β and IL-6, whereas OH treatment reduced their secretion in a concentration-dependent manner (Fig. 2B and C). These data suggest that OH alleviates IL-1β-induced inflammatory cytokine expression and secretion in chondrocytes.

Fig. 2 — OH, oyster hydrolysate; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.

^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control; ^##P < 0.01 and ^###P < 0.001 vs. IL-1β-treated group.

Modulation of PI3K/AKT, MAPK, and NF-κB signaling pathways by OH in SW1353 cells

To further elucidate the action mechanisms of OH, we performed GO and KEGG pathway analyses of OA- and oyster-related target proteins. BP terms were enriched for inflammation, apoptosis regulation, oxidative stress responses, and NF-κB signaling (Fig. 3A). CC analysis revealed that the analyzed proteins were predominantly localized in the extracellular space and endoplasmic reticulum lumen, whereas MF categories highlighted protein- and enzyme-binding properties. KEGG pathway enrichment identified TNF-α, MAPK, IL-17, and PI3K/AKT signaling pathways, along with osteoclast differentiation and proteoglycan-associated pathways, implicating them in both inflammatory responses and cartilage/bone homeostasis (Fig. 3B). Based on these results, we further examined whether OH modulates IL-1β-induced activation of the identified pathways. Western blotting analysis indicated that IL-1β stimulation enhanced the phosphorylation of PI3K, AKT, JNK, and p38; however, OH treatment substantially attenuated this effect (Fig. 3C). As NF-κB signaling plays a central role in regulating pro-inflammatory mediators and ECM-degrading enzymes, we further assessed NF-κB localization. IL-1β treatment promoted the nuclear translocation of NF-κB in SW1353 cells; however, OH attenuated this effect (Fig. 3D). Collectively, these results suggest that OH modulates PI3K/AKT and JNK/p38 MAPK signaling and reduces NF-κB activation, thereby attenuating the IL-1β-induced inflammatory responses in chondrocytes.

Fig. 3 — OH, oyster hydrolysate; IL, interleukin; GO, Gene Ontology; OA, osteoarthritis; BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; p-, phosphorylated.

^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control; ^##P < 0.01 and ^###P < 0.001 vs. IL-1β-treated group.

Protective effect of OH on cartilage damage in MIA-induced OA rats

To evaluate the effects of OH in vivo, we established OA model rats via intra-articular injection of MIA. As shown in Fig. 4A, following MIA injection, the animals were allowed to stabilize for one-week before initiating 2 weeks of oral administration. At the end of the treatment period, joint swelling was measured using a digital caliper, and the joint sections were examined. As shown in Supplementary Fig. 2, no significant differences in body weight or serum biochemical parameters were observed among the experimental groups, indicating that OH administration did not cause systemic toxicity. Compared to the normal group, MIA-induced rats showed severe joint swelling and hemorrhage, resulting in an increased knee width. In contrast, OH-treated rats exhibited a reduction in swelling and knee joint width, comparable to those observed in the PC group (Fig. 4B and C). Histological evaluation using safranin O and H&E staining revealed marked proteoglycan loss and cartilage disruption in the MIA group, whereas OH50, OH100, and PC groups retained the proteoglycan content and exhibited reduced cartilage degeneration (Fig. 4E). Consistently, OARSI scores were significantly higher in the MIA group than in the normal group; however, OH treatment markedly reduced the OARSI scores compared to those in the MIA group (Fig. 4D). Taken together, these findings suggest that OH administration attenuates cartilage destruction and structural deterioration in vivo.

Effect of OH on ECM anabolic and catabolic factors in cartilage tissues

The maintenance of cartilage physiology depends on the balance between anabolic and catabolic processes [26]. To determine whether OH exerts similar effects in vivo to those observed in vitro, we measured the expression levels of anabolic (aggrecan and collagen II) and catabolic (MMPs) markers in cartilage tissue using immunohistochemistry. Aggrecan and collagen II expression levels were markedly reduced in the MIA group, whereas OH treatment restored their levels in a dose-dependent manner (Fig. 5A-C). In contrast, MMP-3, MMP-9, and MMP-13 levels were strongly elevated in the MIA group but substantially reduced in both the OH50 and OH100 groups (Fig. 5A and D-F). Notably, inhibitory effects of OH on MMPs expression levels were comparable to those in the PC group. These findings suggest that OH modulates the balance between ECM anabolic and catabolic factors, thereby contributing to the preservation of cartilage integrity.

Fig. 5 — OH, oyster hydrolysate; ECM, extracellular matrix; MIA, monosodium iodoacetate; OA, osteoarthritis; MMP, matrix metalloproteinase; PC, positive control.

^***P < 0.001 vs. normal group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. MIA-induced group.

Effect of OH on pro-inflammatory cytokine expression in cartilage tissues

Because pro-inflammatory cytokines are known to suppress ECM synthesis and promote OA progression [46], we examined cytokine expression in cartilage tissues by immunohistochemistry. As shown in Fig. 6, expression levels of TNF-α, IL-6, and IL-1β were substantially elevated in the MIA group compared with normal group. Notably, TNF-α expression levels were markedly reduced in the OH50 and OH100 groups, and IL-6 and IL-1β levels were significantly decreased in the OH50, OH100, and PC groups. These results suggest that OH treatment attenuates the MIA-induced upregulation of pro-inflammatory cytokine levels in cartilage tissues.

Fig. 6 — OH, oyster hydrolysate; MIA, monosodium iodoacetate; OA, osteoarthritis; TNF, tumor necrosis factor; IL, interleukin; PC, positive control.

^***P < 0.001 vs. normal group; ^###P < 0.001 vs. MIA-induced group.

Effect of OH on serum biomarkers associated with OA progression

To further assess the systemic effects of OH, we measured the serum levels of OA-associated biomarkers using ELISA. Consistent with disease progression, IL-1β and IL-6 concentrations were significantly elevated in the MIA group compared with normal group. However, this effect was markedly reduced in the OH50 and OH100 groups (Fig. 7A and B). Similar patterns were observed for multiple serum biomarkers related to inflammation and cartilage degradation. LTB4, CRP, COMP, and MMP-9 levels were significantly elevated in the MIA group but markedly reduced following OH treatment. Particularly, COMP levels showed a clear dose-dependent reduction (Fig. 7C-F). Collectively, these results suggest that OH decreases the levels of circulating biomarkers associated with joint inflammation and cartilage degradation.

Fig. 7 — OH, oyster hydrolysate; MIA, monosodium iodoacetate; OA, osteoarthritis; IL, interleukin; LTB4, leukotriene B4; CRP, C-reactive protein; COMP, cartilage oligomeric matrix protein; MMP, matrix metalloproteinase; ELISA, enzyme-linked immunosorbent assay; PC, positive control.

^***P < 0.001 vs. normal group; ^##P < 0.01 and ^###P < 0.001 vs. MIA-induced group.

Modulation of AKT/JNK/p38/NF-κB signaling pathways by OH in MIA-induced OA rats

Finally, we investigated whether the signaling pathways modulated by OH in vitro are also affected in vivo. Immunohistochemical analysis revealed that phosphorylated NF-κB p65 (p-p65) levels in the cartilage were markedly elevated in MIA group compared with normal group and significantly reduced in both the OH50 and OH100 groups (Fig. 8A and B). Similarly, phosphorylation of AKT, JNK, and p38 was strongly induced in MIA-treated rats; however, OH treatment substantially attenuated this effect (Fig. 8C-F). Consistent with the in vitro results, these in vivo findings suggest that OH limits OA progression by downregulating the AKT/JNK/p38 and NF-κB signaling pathways.

Fig. 8 — OH, oyster hydrolysate; NF-κB, nuclear factor κB; MIA, monosodium iodoacetate; p-, phosphorylated; OA, osteoarthritis; DAPI, 4′,6-diamidino-2-phenylindole; AKT, protein kinase B; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

^***P < 0.001 vs. normal group; ^###P < 0.001 vs. MIA-induced group.

DISCUSSION

OA is increasingly recognized as a multifactorial degenerative joint disease in which chronic low-grade inflammation and dysregulated ECM metabolism act synergistically to drive cartilage destruction and joint dysfunction [20,47]. Marine-derived bioactive compounds have attracted attention as potential therapeutic agents due to their diverse biological activities and favorable safety profiles [48,49]. Specifically, C. gigas-derived OH is rich in proteins, peptides, and micronutrients and exerts antioxidant and anti-inflammatory effects in immune cell models [30,35]. However, its specific role in modulating inflammatory signaling and ECM homeostasis in OA remains unclear. We hypothesized that OH attenuates OA progression by regulating inflammatory responses and matrix metabolism in the joint microenvironment. To verify this hypothesis, we investigated the chondroprotective and anti-inflammatory effects of OH using IL-1β–stimulated SW1353 chondrocytes and MIA-induced OA model, integrating molecular, histological, and biochemical analyses to evaluate its effects on cytokine production, ECM regulation, and key intracellular signaling pathways.

A central feature of OA pathogenesis is the sustained overproduction of pro-inflammatory mediators such as IL-1β, IL-6, and TNF-α, which collectively suppress ECM synthesis, stimulate chondrocyte apoptosis, and induce catabolic enzymes including MMPs and aggrecanases [46,50]. IL-1β is a key upstream cytokine triggering a cascade of inflammatory responses in the cartilage by stimulating the production of prostaglandins, nitric oxide, and various catabolic enzymes, thereby exacerbating tissue damage [51,52]. To model this inflammatory milieu in vitro, we used IL-1β-stimulated SW1353 human chondrosarcoma cells, a well-established system mimicking the cartilage catabolic and inflammatory responses [51,53]. This model is widely used to study OA-related molecular mechanisms, as SW1353 cells exhibit phenotypic and transcriptional features of articular chondrocytes and respond robustly to IL-1β stimulation by inducing catabolic enzymes and pro-inflammatory cytokines [54]. Consistent with established mechanisms, IL-1β stimulation of SW1353 chondrocytes elicited the robust upregulation of the levels of catabolic factors and downregulation of the levels of anabolic markers such as aggrecan and collagen II, in this study (Fig. 1B). However, OH treatment counteracted these changes, reducing the expression and secretion of pro-inflammatory cytokines while preserving the ECM components (Figs. 1 and 2). These results are consistent with previous reports that oyster-derived peptides modulate inflammatory mediator production in macrophage models and extend these findings to chondrocytes, a key effector cell type in OA pathology [33,55]. This alignment with established inflammatory pathways underscores the biological validity of the effects of OH. At the signaling level, OH markedly suppressed IL-1β-induced phosphorylation of PI3K, AKT, JNK, and p38, and NF-κB p65 nuclear translocation in SW1353 chondrocytes. These pathways are widely recognized as central molecular hubs integrating inflammatory stimuli and driving catabolic gene expression in the osteoarthritic cartilage [56]. Dysregulated activation of PI3K/AKT, MAPK, and NF-κB signaling enhances pro-inflammatory cytokine production and accelerates cartilage matrix degradation, thereby contributing to disease progression [57]. Particularly, persistent activation of NF-κB and MAPKs stimulates the expression of matrix-degrading enzymes such as MMPs and ADAMTS, in chondrocytes, increasing the catabolic activity and promoting structural cartilage breakdown [58]. The inhibitory effects of OH on these pathways provide a plausible mechanistic basis for its ability to reduce inflammatory cytokine production and preserve the ECM integrity in IL-1β-stimulated chondrocytes. However, OH is a complex mixture of bioactive peptides, and the specific molecular components responsible for its effects remain unknown. Therefore, future studies should characterize the active constituents of OH to elucidate its precise action mechanisms and therapeutic potential.

Our in vivo findings further substantiated the chondroprotective potential of OH by demonstrating its ability to ameliorate joint pathology and modulate the key molecular processes associated with OA progression. Effective OA interventions should ultimately confer tissue-level benefits, as improvements in cartilage structure are closely linked to functional outcomes [59]. In this study, OH administration significantly reduced MIA-induced joint swelling, preserved the cartilage morphology, and improved the OARSI scores, exerting tangible protective effects in the joint microenvironment. This structural preservation was accompanied by marked changes in ECM metabolism, consistent with previous reports that OA progression is driven by both excessive catabolic activity and impaired anabolic repair [60,61]. Notably, OH enhanced the expression of anabolic markers such as aggrecan and collagen II, while suppressing that of catabolic enzymes including MMP-3, MMP-9, and MMP-13, thereby helping to restore the balance between matrix synthesis and degradation under inflammatory stress. Moreover, OH treatment reduced the expression levels of IL-1β and IL-6 in cartilage tissues, consistent with the established role of these cytokines as key drivers of inflammatory amplification and matrix breakdown in OA pathogenesis. These local anti-inflammatory effects were paralleled by systemic changes. Serum levels of LTB4, CRP, COMP, and MMP-9 elevated in MIA-induced OA model, were significantly reduced following OH administration. LTB4 is a potent upstream mediator recruiting immune cells, stimulating IL-1β and IL-6 production, and accelerating ECM degradation, whereas serum COMP is a clinical biomarker of cartilage breakdown and disease severity [62,63]. The reduction in these biomarker levels suggests that OH not only mitigates local inflammatory and catabolic processes but also attenuates systemic disease activity, showing desirable properties as a potential disease-modifying OA intervention. From a translational perspective, these findings suggest that OH may have potential as a nutraceutical candidate for the prevention or adjunctive management of OA by modulating inflammatory responses and cartilage metabolism. However, further studies, including long-term animal studies and clinical trials, are required to confirm its safety, efficacy, and clinical applicability. These results collectively support the protective role of OH. However, the MIA-induced OA model only reflects acute chemically induced joint damage and does not fully replicate the chronic and multifactorial nature of human OA. Additionally, although our data clearly demonstrated the beneficial outcomes at the tissue and systemic levels, direct modulation of specific signaling pathways or molecular mechanisms in vivo was not assessed, necessitating further evaluation of the precise mechanistic underpinnings of these effects. Future studies using chronic or surgically induced OA models, along with targeted molecular analyses, are necessary to address these limitations and clarify the therapeutic potential of OH in clinically relevant contexts. In summary, this study demonstrated that OH attenuated the key pathological processes associated with OA by reducing inflammatory cytokine production, preserving the ECM components, and suppressing pro-inflammatory signaling pathways both in vitro and in vivo (Fig. 9). Our findings are consistent with the existing literature on oyster-derived peptides and highlight marine functional ingredients as biologically plausible therapeutics for OA management. Future studies should explore the specific bioactive components of OH, assess its efficacy in chronic OA models, and confirm its potential as a disease-modifying intervention for clinical use.

Fig. 9 — OH, oyster hydrolysate; ECM, extracellular matrix; IL, interleukin; MIA, monosodium iodoacetate; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; NF-κB, nuclear factor κB; MMP, matrix metalloproteinase; ADAMTS-5, a disintegrin and metalloproteinase with thrombospondin motifs-5; OA, osteoarthritis.

ACKNOWLEDGMENTS

The authors would like to thank Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, Republic of Korea), for letting us use flow cytometer and fluorescence microscope.

Footnotes

Funding: This work was supported by Korean Institute of Marine Science & Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries, Korea (RS-2022-KS221637).

Conflict of Interest: The authors declare no potential conflicts of interests.

Author Contributions:

Conceptualization: Ji SY, Cho S, Choi YH.
Data curation: Ji SY, Hwangbo H, Kim MY, Kim DH.
Formal analysis: Ji SY, Hong SH, Shim JH, Moon SK.
Funding acquisition: Cho S, Choi YH.
Methodology: Ji SY, Hwangbo H, Kim MY, Kim DH, Kim GY.
Project administration: Cho S, Choi YH.
Resources: Ji SY, Shim JH, Moon SK.
Software: Hong SH, Kim GY.
Supervision: Shim JH, Moon SK, Kim GY.
Validation: Hong SH, Hong SH, Kim GY.
Visualization: Hwangbo H, Moon SK, Kim GY.
Writing - original draft: Ji SY, Cho S.
Writing - review & editing: Choi YH.

SUPPLEMENTARY MATERIALS

Supplementary Fig. 1

IL-1β induced OA in SW1353 cells. (A) Cell viability of SW1353 cells treated with the indicated concentrations of IL-1β. (B) The levels of IL-6 in culture supernatants by ELISA under the same treatment conditions. (C) Western blot analysis of MMP-3, MMP-13 and collagen-II protein expression levels in SW1353 cells treated with IL-1β (10 ng/mL and 40 ng/mL) for 24 h. Data are represented as the mean ± SD (n = 3).

nrp-20-571-s001.ppt^{(792KB, ppt)}

Supplementary Fig. 2

Effects of OH on body weight and blood biochemical parameters in the MIA-induced OA model. (A) Changes in body weight and (B, C) immune organ indices (thymus and spleen) are presented as the mean values. Blood samples were collected to determine (D) AST, (E) ALT, and (F) LDH levels. Data are represented as the mean ± SD (n = 6/group).

nrp-20-571-s002.ppt^{(1.2MB, ppt)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Fig. 1

nrp-20-571-s001.ppt^{(792KB, ppt)}

Supplementary Fig. 2

nrp-20-571-s002.ppt^{(1.2MB, ppt)}

PERMALINK

Oyster hydrolysate attenuates osteoarthritis progression by modulating inflammatory signaling and extracellular matrix homeostasis

Seon Yeong Ji

Hyun Hwangbo

Min Yeong Kim

Da Hye Kim

Su Hyun Hong

Jung-Hyun Shim

Sung-Kwon Moon

Gi-Young Kim

Suengmok Cho

Yung Hyun Choi

Abstract

BACKGROUND/OBJECTIVES

MATERIALS/METHODS

RESULTS

CONCLUSION

INTRODUCTION

MATERIALS AND METHODS

SW1353 cell culture for in vitro experiments

OH preparation

Cell viability and in vitro OA experimental design

Enzyme-linked immunosorbent assay (ELISA)

Western blot analysis

Table 1. List of antibodies used for Western blot analysis.

Immunofluorescence assay

Enrichment analysis

In vivo OA experimental design

Histopathological analysis

Immunohistochemistry

Statistical analysis

RESULTS

Effect of OH on ECM degradation in IL-1β-stimulated SW1353 cells

Effect of OH on inflammatory cytokine expression in IL-1β-stimulated SW1353 cells

Modulation of PI3K/AKT, MAPK, and NF-κB signaling pathways by OH in SW1353 cells

Protective effect of OH on cartilage damage in MIA-induced OA rats

Effect of OH on ECM anabolic and catabolic factors in cartilage tissues

Effect of OH on pro-inflammatory cytokine expression in cartilage tissues

Effect of OH on serum biomarkers associated with OA progression

Fig. 7. Effects of OH on serum inflammatory and cartilage degradation biomarker levels in MIA-induced OA model. Serum levels of (A) IL-1β, (B) IL-6, (C) LTB4, (D) CRP, (E) COMP, and (F) MMP-9 were quantified using ELISA. Data are represented as the mean ± SD (n = 6/group).

Modulation of AKT/JNK/p38/NF-κB signaling pathways by OH in MIA-induced OA rats

DISCUSSION

Fig. 9. A schematic illustration depicting the protective effects of OH in inhibiting inflammatory responses and ECM degradation.

ACKNOWLEDGMENTS

Footnotes

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