Characterization and screening of a novel octapeptide from protein hydrolysates of Crassostrea gigas and its immunomodulatory effects on RAW264.7 cells

TL;DR

A new oyster-derived peptide was identified that can both stimulate and regulate immune responses, making it useful for functional foods.

Contribution

A novel immunomodulatory octapeptide (SWDNFLQR) from oyster protein was discovered and shown to regulate immune pathways.

Findings

SWDNFLQR activates TLR and PI3K-Akt pathways to enhance immune cell activity.

Higher concentrations of SWDNFLQR inhibit NF-κB, reducing excessive inflammation.

The peptide promotes immune cell proliferation, phagocytosis, and cytokine secretion.

Abstract

Food-derived bioactive peptides show great potential for functional foods, given their structural adaptability and safety. Crassostrea gigas contains abundant bioactive proteins, but the immunomodulatory mechanism of their peptides remains unclear. In this research, 29 new peptides were identified from papain-hydrolyzed oyster extracts using LC-MS/MS, and target peptide SWDNFLQR with high activity potential was obtained through bioinformatics screening and chemically synthesized. The immunomodulatory properties of SWDNFLQR were assessed through the RAW264.7 cells model and iTRAQ quantitative proteomics. SWDNFLQR enhanced proliferation, phagocytosis, and secretion of NO, TNF-α, and IL-6. It activates Toll-like receptors and the PI3K-Akt signaling pathway, modulating key proteins Fn1 and Prkacb. Notably, higher concentrations of SWDNFLQR specifically inhibited NF-κB pathway activation, indicating its dual immunomodulatory role in stimulating immune responses and mitigating excessive inflammation. This positions SWDNFLQR as a valuable component for the formulation of anti-inflammatory and immune-regulating functional foods.

•A new type of oyster peptide (SWDNFLQR) with immunomodulatory activity was identified and screened out.•Bidirectional immune regulation‌ achieved by activating TLR/PI3K-Akt while inhibiting NF-κB pathways.•Integrative approach combining LC-MS/MS, bioinformatics screening, synthetic chemistry, and proteomics.

A new type of oyster peptide (SWDNFLQR) with immunomodulatory activity was identified and screened out.

Bidirectional immune regulation‌ achieved by activating TLR/PI3K-Akt while inhibiting NF-κB pathways.

Integrative approach combining LC-MS/MS, bioinformatics screening, synthetic chemistry, and proteomics.

Figures

Results of RAW264.7 cell viability and immune index detection. (A)Effect of oyster peptides prepared by different proteases on the proliferation rate of RAW264.7 cells; (B) Effect of synthetic peptides on the proliferation rate of RAW264.7 cells; (C) Effect of synthetic peptides on the phagocytic ability of macrophages towards neutral red; Effects of SWDNFLQR on RAW 264.7 cells NO secretion(D), TNF-α, and IL-6(F)production. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Fig. 1

Effects of SWDNFLQR fraction on RAW 264.7 cells morphology.Fig. 2

protein expression profile in RAW264.7 cells. (A) PCA based on quantitative data of the selected proteins in each group (*n* = 3 for each group). (B)Differential protein statistical chart. (C) Venn diagram of three groups (Different colors represent different groups, and the numbers in the figure represent the number of intersections and the unique quantity of each group.). The volcano map of CG1-vs-Control (D), CG2-vs-Control(E), LPS-vs-Control(F) (Notes: blue and red dots represent upregulated and downregulated proteins, respectively, darker colors indicate more significant differences, and

GO function annotation enrichment analysis diagram. (A) CG1-vs-Control UP. (B) CG1-vs-Control Down. (C) CG2-vs-Control UP. (D) CG2-vs-Control Down. (E) LPS-vs-Control UP. (F) LPS-vs-Control Down.Fig. 4

KEGG enrichment analysis diagram. (A) CG1-vs-Control UP. (B) CG1-vs-Control Down. (C) CG2-vs-Control UP. (D) CG2-vs-Control Down. (E) LPS-vs-Control UP. (F) LPS-vs-Control Down.Fig. 5

PPI network analysis of SWDNFLQR. (A) CG1-vs-Control group's top 23 connectivity protein interaction network diagram. (B) The highest-scoring sub-network in the CG1-vs-Control group was elected using the MCODE algorithm. (C) CG2-vs-Control group's top 22 connectivity protein interaction network diagram. (D) The highest-scoring sub-network in the CG2-vs-Control group was elected using the MCODE algorithm. (E) LPS-vs-Control group's top 23 connectivity protein interaction network diagram. (F) The highest-scoring sub-network in the LPS-vs-Control group was elected using the MCODE algorithm.Fig. 6

The effect of SWDNFLQR on the expression of signal pathway-related proteins in RAW264.7 Cells.Fig. 7

Possible immunomodulatory mechanism diagram of SWDNFLQR.Fig. 8

Full text

Introduction

1

Bioactive peptides sourced from food, including those derived from gelatins and both animal and plant proteins, serve as functional components in food products (Zaky et al., 2022). These peptides are characterized by their excellent structural adaptability and safety, along with a range of biological functions. Such attributes have positioned food-derived bioactive peptides as a prominent focus in the fields of nutritional intervention and the creation of functional foods. Short-chain peptides, consisting of 2 to 20 amino acids, exhibit activities that are highly influenced by their sequence and three-dimensional structure. Specifically, the amino acid composition, hydrophobic properties, weight and length of the peptides, type of residues at C- and N-terminals, all influence the functional properties and biological activities of a peptide (Li et al., 2011). There is significant interest in utilizing these short-chain and small bioactive peptides due to their potential to modulate immune responses (Chalamaiah et al., 2018). In contrast to conventional synthetic immunomodulators, which may lead to adverse effects like excessive immune activation or suppression, food-derived peptides offer three key benefits: they are resistant to pepsin, exhibit dose-dependent regulatory effects, and can be produced on a larger scale through food processing methods such as fermentation and enzymatic hydrolysis (Jia et al., 2021). A concrete comparison illustrates these advantages. The food-derived antimicrobial and immunomodulatory peptide Bovine Lactoferricin B exhibits a multi-target, homeostatic mechanism by competitively binding to LPS, modulating TLR4 signaling, and inducing anti-inflammatory cytokines such as IL-10. In contrast, the synthetic anti-TNF-α monoclonal antibody Infliximab operates through a potent, single-target blockade, which, while clinically effective, carries significant risks of severe infections, lymphoma, and the development of anti-drug antibodies. This fundamental difference underscores the safety-first, balanced-modulation paradigm of food-derived peptides (Chalamaiah et al., 2018).

Aquatic life, particularly bivalves like the Pacific oyster (Crassostrea gigas), serves as an excellent source of proteins and bioactive peptides. Research has shown that peptides extracted from oysters exhibit a variety of biological functions, including anti-inflammatory, antioxidant, and anti-osteoporotic properties (Chen et al., 2024; Hao et al., 2022; Xiang et al., 2021). Additionally, these peptides can stimulate the production of sex hormones, enhance lipid metabolism, modulate gut microbiota, and boost immunomodulation. However, there are two significant challenges in the current exploration of peptides derived from oysters. Firstly, most studies concentrate on hydrolysates or fermentation products, which consist of diverse peptide mixtures that vary in stability and complexity, making it difficult to clarify their specific mechanisms of action (Kang et al., 2019; Zhang et al., 2021). Secondly, while certain peptides with defined structures, such as DSQLAPFRF and HFNPRL, known for their antioxidant and anti-osteoporotic benefits (Quan et al., 2025), as well as IEELEELERER, which has protective effects on endothelial cells and anti-inflammatory properties (Cheng et al., 2022), have been identified, the target molecules and signaling pathways associated with these peptides remain largely unclear.

Recent advancements in foodomics technologies have equipped researchers with innovative tools to explore bioactive peptides. For instance, sophisticated liquid chromatography-tandem mass spectrometry (LC-MS/MS) facilitates the reliable identification of peptides (Shuli et al., 2022), while bioactivity prediction platforms such as PeptideRanker assist in both forecasting bioactivity and conducting targeted screenings. Additionally, the chemical synthesis of peptides allows for the large-scale generation of high-purity, precisely defined peptides, effectively bypassing the limitations associated with natural extraction methods (Callmann et al., 2020). The utilization of these techniques has significantly propelled research in food peptides.

To thoroughly investigate the structure-function relationship of specific peptide sequences, we employed a systematic research workflow: “LC-MS/MS sequence identification, bioinformatics screening, high-purity chemical synthesis, independent cell functional validation, and proteomics-based mechanism exploration.” This approach allowed us to explore the immunomodulatory mechanisms of oyster-derived bioactive peptides effectively. LC-MS/MS provided raw peptide sequence data, while computational biology screening tools predicted and identified target peptides with significant immunological activity from extensive datasets. Furthermore, chemical synthesis produced high-purity target peptides for subsequent functional studies. The immunomodulatory effects and regulatory mechanisms of these synthetic peptides were quantitatively assessed using the RAW264.7 cells model. This study paves the way for molecular-level exploration of oyster-derived immunomodulatory peptides and establishes a theoretical framework for future innovative strategies in developing functional food ingredients with immune-regulating capabilities.

Materials and methods

2

Materials and reagents

2.1

All Pacific oysters (Crassostrea gigas) used in this experiment were sourced from Kongtong Island (Yantai, China). RAW264.7 cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Papain (activity 10 × 10^5^ U/g) was purchased from Nanning Pangbo Biological Engineering Co., Ltd. (Nanning, China). Alkaline protease (activity 2 × 10^5^ U/g), trypsin (250 N.F·U/mg), pepsin (activity 2.5 × 10^5^ U/g), CCK-8 assay kit, DMEM high-glucose medium, fetal bovine serum, and penicillin-streptomycin mixture were all purchased from Beijing Solabio Technology Co., Ltd. (Beijing, China). Assay kits for IL-6, IL-1β, TNF-α, and nitric oxide (NO) were all obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). acetonitrile, formic acid, and trifluoroacetic acid (chromatography grade) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). all other chemicals were at least analytical grade.

Extraction and preparation of oyster peptides

2.2

The fresh oyster meat underwent a washing process with deionized water three times, followed by a mixing step with deionized water at a mass-to-volume ratio of 1:3. This resulting mixture was then homogenized at a speed of 9000 r/min for a duration of 10 min in an ice bath. After homogenization, oyster peptides were produced through the enzymatic hydrolysis utilizing papain, trypsin, alkaline protease and pepsin. Optimal conditions were established based on the methods described in references and the instructions provided for the enzymes (Quan et al., 2025; Zhang et al., 2021), ensuring a consistent enzyme loading of 2% (enzyme to substrate ratio) and a hydrolysis period of four h. The ideal temperatures for the four proteases were maintained at 50 °C, 37 °C, 50 °C, and 37 °C, and the optimal pHs were tuned to 7, 8, 10, and 2. Subsequently, after the incubation period, the enzymes were inactivated by applying heat at 100 °C for 10 min. The hydrolysates were then subjected to centrifugation at 9000 r/min for 20 min. A tangential flow ultrafiltration system (Pall Minimate, East Meadow, NY, USA) equipped with a membrane pack featuring a molecular weight cutoff of 10 kDa was employed to isolate fractions with molecular weights under 10 kDa. This process was followed by the elimination of small-molecule impurities utilizing a 100 Da dialysis bag. The resulting oyster peptides were then obtained through vacuum freeze-drying. Then the oyster peptides prepared by degradation of four different proteases were tested separately. The viability of RAW264.7 cells served as a metric to assess and pinpoint the optimal protease for the preparation of oyster immunomodulatory peptides.

Identification of oyster peptides

2.3

The structural identification of the oyster peptides was conducted utilizing an Easy-nLC 1200/Q Exactive™ Hybrid Quadrupole-Orbitrap™ system (Thermo Fisher Scientific, Waltham, MA, USA) in combination with ESI nanospray technology. For chromatographic separation, a custom C18 column (150 μm × 15 cm, 1.9 μm, 100 Å) was employed with a 5 μL injection volume. The mobile phases utilized were: (A) 0.1% formic acid in water; (B) a solution consisting of 20% 0.1% formic acid and 80% acetonitrile (flow rate of 600 nL/min). Gradient: 4–8% B (2 min), 8–28% B (43 min), 28–40% B (10 min), 40–95% B (1 min), 95% B (10 min). The mass spectrometry settings included a spray voltage of 2.2 kV, a capillary temperature of 270 °C, and a resolution of 70,000 at 400 m/z, with an m/z range spanning from 100 to 1500. For MS/MS analysis, HCD was set at 28.0 NCE with 66 ms activation, targeting the top 20 precursor ions. Data processing was carried out using Byonic alongside species-specific databases to facilitate high-confidence identifications.

Peptide activity prediction and chemical synthesis

2.4

The BIOPEP database (https://biochemia.uwm.edu.pl/biopep-uwm/) was utilized to validate the peptide sequences. Predictions regarding biological activity were made with the help of PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/), while the analysis of physicochemical properties, including molecular weight, isoelectric point, hydrophobicity, and charge, was conducted using PepDraw (https://www2.tulane.edu/∼biochem/WW/PepDraw/). The correlation between immunomodulatory activity and amino acid composition, focusing particularly on hydrophobic and basic residues, was noted. To evaluate potential toxicity, ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) was employed. Predictions of immunological properties such as IL-6 (limited to 8–25 amino acids), IL-4, IL-10, IFN-γ, and anti-cancer effects were made utilizing the IIITD platform (https://webs.iiitd.edu.in/).This study established a three-tiered screening criterion for peptides: a) Confidence score > 300, predicted activity score > 0.8, and peptide length ≤ 10 amino acids. b) Utilizing the IIITD platform, we conducted a comprehensive prediction of IL-6, IL-4, IL-10, IFN-γ, and anticancer activities for initially screened peptides. For peptides with ≥8 amino acids, all five predicted activities were required to be positive; for peptides with <8 amino acids, positive predictions were required for IL-4, IL-10, IFN-γ, and anticancer activities. c) All candidate peptides were verified by ToxinPred to ensure they were non-toxic and non-allergenic.

Peptides, exceeding 98% purity, were produced through solid-phase methods by Sangon Biotech (Shanghai, China), then dissolved in PBS, consisting of 1.47 mM KH₂PO₄, 8.10 mM Na₂HPO₄, 2.68 mM KCl, and 137 mM NaCl at pH 7.4, and subsequently stored at −80 °C.

Immunomodulatory effects of peptides on RAW264.7 cells

2.5

Cell culture

2.5.1

RAW264.7 cells were cultivated in a CO₂ incubator (Model 150i, Thermo Fisher Scientific, Waltham, MA, USA) using Dulbecco's Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Wei et al., 2022). The cells were kept at a temperature of 37 °C with 5% CO₂ and 95% humidity. The medium was replaced every two d, and subculturing occurred every three d. An inverted microscope (CKX41, Olympus, Tokyo, Japan) was employed to examine cell morphology, and experiments utilized cells that were in the logarithmic growth phase.

Cell proliferation assay

2.5.2

The assessment of cell proliferation was conducted using the CCK-8 assay. Cells were placed at a density of 5 × 10^4^ cells/mL in a 96-well plate. After a 24 h attachment period, 10 μL of filter-sterilized (0.22 μm) solution samples were introduced. The control groups included a complete medium (blank) and 1 μg/mL LPS (positive). After another 24 h of incubation, 10 μL of CCK-8 reagent was incorporated. The absorbance was measured at 450 nm after an incubation of 1.5 h using a microplate reader (1510, Thermo Fisher Scientific, Waltham, MA, USA) (Li, An, et al., 2024). The proliferation rates were determined by calculating the ratios of experimental OD to blank OD.

Cell morphology observation

2.5.3

Cells were plated in 6-well plates. Following 24 h of culture for adhesion, various concentrations of the sample solution (10, 100 μg/mL) were introduced. The negative control group received an equivalent amount of complete medium, whereas the positive control group was treated with an equal volume of 1 μg/mL LPS. After another 24 h of culture, cell morphology was observed and documented using an inverted microscope (Zhang et al., 2019).

Phagocytosis of neutral red assay

2.5.4

Following 24 h of adherence, cells were exposed to either the test samples or controls (LPS/blank) for an additional 24 h. A neutral red solution was applied for 2.5 h, and the absorbance at 540 nm was measured using an ELISA reader (Zhang et al., 2019). The phagocytic activity was quantified as the ratio of experimental OD to blank OD.

Detection of immunological markers

2.5.5

Initially, RAW264.7 cells were plated at a density of 5 × 10^4^ cells/cm^2^ in 75 cm^2^ cell culture flasks. Once 80%–90% confluence was attained, immunological assays along with subsequent iTRAQ-based proteomics analysis were conducted. The treatment groups consisted of the following: the CG1 group received 15 mL of a solution containing 10 μg/mL, the CG2 group received 15 mL of a solution containing 100 μg/mL, the control group was administered 15 mL of complete medium, and the LPS group was treated with a 1 μg/mL LPS solution. The cells were incubated for 24 h in an atmosphere containing 5% CO₂ at 37 °C. After 24 h, centrifuged at 4 °C for 10 min at 1500 r/min and collected the supernatant. Quantification of NO, TNF-α, IL-1β, and IL-6 was performed using commercially available ELISA kits (Zhang et al., 2019).

Proteomic investigation

2.6

iTRAQ Labeling

2.6.1

Following the methodology outlined in Section 2.5.5, after 24 h of incubation, RAW264.7 cells from each experimental group were collected through centrifugation and rinsed twice with PBS. The cells were then lysed in an 8 M urea buffer containing protease inhibitors, subjected to three cycles of sonication on ice, and subsequently digested with trypsin at a 30:1 protein-to-enzyme ratio at 37 °C for 4 h. The reaction was halted with 2 μL of 0.2% TFA, followed by desalting via Sep-Pak C18, vacuum drying, and labeling using an 8-plex iTRAQ kit (Shu et al., 2024).

High-resolution mass spectrometry analysis via LC-MS/MS

2.6.2

For the LC-MS/MS analysis, labeled peptides were separated through high-pH RP-HPLC and analyzed on a timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) utilizing an Eksigent C18 column (75 μm × 250 mm, 1.7 μm). The mobile phases employed were A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile, with a gradient transitioning from 5% B (0 min) to 22% B (48 min), then to 35% B (53 min), reaching 90% B (56 min), and finally back to 3% B (57–60 min) at a flow rate of 300 nL/min. The mass spectrometer was configured with a spray voltage of 1.5 kV and a scan range from 100 to 1700 m/z.

Protein identification and bioinformatic analysis

2.6.3

The data processing was conducted using Spectronaut Pulsar version 18.4 (Biognosys, Switzerland), where proteins were filtered to include those with at least one unique peptide (unused score exceeding 2). Proteins that exhibited differential expression (p < 0.05, FC ≥ 2.0) were annotated via DAVID 6.8, focusing on Gene Ontology (GO) terms and KEGG pathways. Additionally, interactions between proteins were examined using both STRING and Cytoscape software.

Western blot validation

2.7

Protein samples (10 μg each) were subjected to electrophoresis on an SDS-polyacrylamide gel. Subsequently, they were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore GmbH, Darmstadt, Germany), and the membrane's unbound surface was blocked with 5% skimmed milk powder for one hour. An anti-primary antibody (1:1000 dilution, Abcam, Cambridge, MA, USA) was then applied and incubated overnight at 4 °C. Following this, the membrane was treated with a secondary antibody (1:3000 dilution, Abcam, Cambridge, MA, USA) for two hours at room temperature, followed by three washing steps. The protein bands were visualized using Novex ECL (Thermo Fisher Scientific, Rockford, IL, USA). Finally, the results of the western blot bands were analyzed and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

2.8

The analysis of the data was carried out with SPSS version 22.0 (IBM, Armonk, NY, USA), employing one-way ANOVA and subsequently performing LSD post hoc tests. The results were illustrated using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as mean ± SD based on at least three independent experiments. For all analyses, statistical significance was determined at p < 0.05.

Results and discussion

3

Effect of oyster peptides on macrophage proliferation rate

3.1

Fig. 1A illustrates that oyster peptides created with various proteases (at a concentration of 100 μg/mL) influence the proliferation of RAW264.7 cells. When compared to the control group, there was a notable increase in cell proliferation rates for the oyster peptides generated using alkaline protease and papain, with those from papain showing the most significant enhancement. The increase was highly significant (p < 0.01) relative to the control group, reaching 115.47%. Previous research indicates that proteolytic peptides from ovalbumin and Nibea japonicus treated with papain also led to a marked rise in the proliferation of RAW264.7 cells, aligning with the results of this investigation (Cho et al., 2023; Zhang et al., 2019). At this stage, our primary objective was to efficiently identify a protease capable of producing the most potent immunologically active peptides. Cell proliferation serves as a prerequisite and an early indicator for the activation of immune cells. Extracts that effectively promote macrophage proliferation are more likely to contain components with immunostimulatory properties. Consequently, the papain-derived oyster peptides were chosen for additional research. Subsequently, we initiated in-depth screening and identification of active peptides using papain hydrolysate, as well as an investigation into their immunomodulatory mechanisms.Fig. 1. Results of RAW264.7 cell viability and immune index detection. (A)Effect of oyster peptides prepared by different proteases on the proliferation rate of RAW264.7 cells; (B) Effect of synthetic peptides on the proliferation rate of RAW264.7 cells; (C) Effect of synthetic peptides on the phagocytic ability of macrophages towards neutral red; Effects of SWDNFLQR on RAW 264.7 cells NO secretion(D), TNF-α, and IL-6(F)production. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Fig. 1

(Notes: Different letters indicate significant differences between different samples or between different samples under the same concentration conditions, p < 0.05).

Peptide identification screening and chemical synthesis

3.2

Following the LC-MS/MS analysis of oyster peptides, the raw data was analyzed using Byonic software. This process yielded 1665 peptide fragments, each with a score exceeding 200 and a high level of certainty. A bioinformatics approach, supported by databases, was utilized for screening and predicting peptide sequences. The tool PeptideRanker was used to assess the potential biological activity of the identified peptides, with scores above 0.5 suggesting possible biological effects. The evaluation of active peptide sequences indicated that low-molecular-weight peptides exhibited greater activity compared to their high-molecular-weight counterparts. Typically, these active peptides are composed of 2 to 10 amino acids. The research identified 29 peptides that had a confidence score of at least 300, a predicted activity score of 0.8 or higher, and a length of 10 amino acids or fewer. As detailed in Table 1, these peptides were assessed for their molecular weight, hydrophobicity, potential toxicity, and prior validation. While 29 peptides were determined to be non-toxic and lacked relevant literature, they may represent novel active peptides. Screening was continued according to the platform activity prediction results. Considering that IL-6 could only predict peptides consisting of 8–25 amino acids, the peptides with all five predicted activities and consisting of 8 amino acids or more were SWDNFLQR, VDWCPTGF, AGPAGPMGPN. The peptides with four activities, namely IL-4, IL-10, IFN-γ, and anti-cancer, and consisting of fewer than 8 amino acids were LRHF, LGSPW, LGAFP, LAGFP, RGPAGPM. All eight peptides demonstrated high safety, with no potential for sensitization or toxicity. Ultimately, eight peptides—LRHF, LGSPW, LGAFP, LAGFP, RGPAGPM, SWDNFLQR, VDWCPTGF, AGPAGPMGPN—were chosen for their predicted high activity.Table 1. Identification of Target Peptide Segments and Prediction of Biological Activity.Table 1. Serial No.Amino Acid SequencePredicted ScoreMolecular Mass(Da)Hydrophobicity/kcal/molIL-6IL-4IL-10IFN-γAnti-cancer1SGPGPHCPRC0.9491581009.4215.18−−−−−2VDWCPTGF0.932406923.388.80+++++3RSPPCICPR0.9244871027.5011.24+++−+4GFDPL0.920077547.269.87*+−++5IFGP0.919943432.246.36*+−+−6DWDYIPLPR0.8968231173.5812.10++−+−7SGPGPHCPR0.895327906.4115.20+−−−−8FPEPLPPPK0.8943651020.5612.07++−−+9SWIP0.890758501.265.29*+−++10PNSPF0.889047560.267.78*−+++11MQGWFQ0.885494795.346.12*+++−12AGPAGPMGPN0.882925867.3912.95+++++13LGSPW0.877397558.286.31*++++14SGSDPMPM0.868765820.3112.55++++−15SPWDAGRDPF0.8651831146.5115.58+−+−−16LAGFP0.864376503.276.73*++++17SFIP0.861563462.255.67*+−+−18SWDNFLQR0.8585461064.5010.38+++++19PANIPW0.85685696.366.32*+−++20SLYDRMFG0.850946987.4510.62+++−+21LGGMP0.844699473.238.42*−−++22TLNPCFPFPK0.8446381162.587.53++++−23SPPTGPLG0.837815724.3710.08+−−−+24DPDSVFPL0.833697888.4212.50+++−+25LGAFP0.833049503.276.73*++++26SGFGPA0.825162534.249.59*++−−27RGPAGPM0.82262684.3412.12*++++28LRHF0.815855571.329.08*++++29GCDIPDSPF0.804964949.3814.22++−−+(Notes: +Indicates active prediction, − indicates inactive prediction, * indicates restricted prediction)

The immunomodulatory activity of these peptides is closely related to the composition and sequence of their amino acids, as well as the ratio of hydrophobic and basic residues. Among the 29 high-scoring peptide segments, those predicted to possess broader immunomodulatory functions consistently exhibit hydrophobicity values in the medium-to-high range (>6.31 kcal/mol). Notably, peptides that demonstrate all five predicted activities share this hydrophobic characteristic. In contrast, peptides characterized by lower hydrophobicity or a high content of polar, uncharged amino acids such as Ser and Thr tend to exhibit fewer predicted activities. This observation aligns with existing literature, which suggests that hydrophobic amino acids facilitate binding to cell membranes, while basic amino acids can form salt bridges with acidic counterparts to enhance cellular uptake. The final peptide, SWDNFLQR, identified in this study, features both hydrophobic anchoring (via central Trp and Phe) and charge complementation (with acidic Asp and basic Arg). This structural composition may account for its broad predicted activities and the subsequent demonstration of superior functionality in experimental settings. The purity of the chemically synthesized peptides is 98% or greater. Future research will involve further testing of these synthesized peptides for their immunomodulatory properties.

Effects of synthetic peptides on macrophage proliferation rate

3.3

RAW264.7 cells serve as a widely utilized model for investigating the immunomodulatory properties of peptides derived from food sources (Paterson et al., 2023). In Fig. 1B, the effects of eight synthetic peptides on macrophage proliferation at varying concentrations are depicted. The data indicates a concentration-dependent relationship, where higher peptide concentrations tend to enhance macrophage proliferation, while lower concentrations may exhibit weaker or even inhibitory effects. As the peptide concentration increases, macrophage proliferation accelerates. At a concentration of 100 μg/mL, all peptides effectively stimulated macrophage proliferation, achieving percentages ranging from 102.78% to 136.93%. However, when the concentrations were reduced to 50 μg/mL and 10 μg/mL, the proliferation-promoting effects of certain peptides diminished, resulting in percentages falling below the control level of 100%. Additionally, the mechanisms by which different peptides influence macrophage proliferation varied significantly. SWDNFLQR consistently promoted macrophage proliferation across all tested concentrations, reaching a peak stimulation of 136.93% at 100 μg/mL, the highest among the peptides studied. Even at the lowest concentration of 5 μg/mL, SWDNFLQR maintained a proliferation rate of 109.84%, surpassing many other peptides. This pronounced effect on macrophage proliferation suggests that SWDNFLQR may possess enhanced immunomodulatory capabilities. Certainly, the enhanced cell proliferation may result from multiple factors. To investigate whether this proliferation is associated with a nonspecific mitotic response or the activation of specific immune programs, we examined the effects of the peptide on key immune functional indicators of macrophages and employed proteomic techniques to explore its underlying molecular mechanisms.

Effects of synthetic peptides on macrophage phagocytosis of neutral red

3.4

The effect of eight different peptides on the uptake of neutral red by RAW264.7 cells was assessed across various concentrations Fig. 1C. An increase in peptide concentration correlated with enhanced phagocytic activity in these macrophages. Specifically, as the peptide levels rose, the rate of phagocytosis also showed an upward trend. However, the impact varied among the peptides. Notably, SWDNFLQR exhibited the strongest influence on macrophage phagocytosis across all tested doses, achieving phagocytosis rates of 116.87% and 119.85% at concentrations of 50 μg/mL and 100 μg/mL, respectively, marking the highest rates among the peptides evaluated. Other peptides also facilitated phagocytosis at certain levels, but to a lesser degree. For instance, AGPAGPMGPN demonstrated comparable phagocytic activity to SWDNFLQR at some concentrations, although its effectiveness at 100 μg/mL was significantly lower. SWDNFLQR consistently enhanced macrophage phagocytosis across all concentrations, maintaining high phagocytosis rates, which suggests it may possess robust immunomodulatory properties. The unique structure of SWDNFLQR is crucial to its superior phagocytic promotion effect compared to other predicted active peptides such as VDWCPTGF and AGPAGPMGPN. Specifically, the amino acid composition of SWDNFLQR features a high proportion of hydrophobic residues (Trp, Phe, Leu) and key charged polar residues (Asp, Arg), resulting in a moderately amphiphilic cationic structure. This structural characteristic, particularly the positioning of Trp and Arg, confers stronger membrane affinity and potential receptor binding capabilities, facilitating a more stable binding and activation of macrophage membranes. Consequently, the unique structural pattern of SWDNFLQR is more conducive to the sustained promotion of phagocytosis when compared to cysteine-enriched peptides containing disulfide bonds (such as VDWCPTGF) or peptides rich in flexible Gly and Pro residues (such as AGPAGPMGPN). Previous studies have identified immunomodulatory peptides from species such as the Alaska walleye(Hou et al., 2012), Mytilus edulis (Wang et al., 2024), and various oysters (Hao et al., 2022). These peptides primarily function to boost macrophage phagocytic ability and stimulate the production of NO, the expression of iNOS, the generation of reactive oxygen species (ROS), and the secretion of cytokines from RAW264.7 cells. Based on these findings, SWDNFLQR has been preliminarily identified as a promising candidate for further investigation into its immune-enhancing potential.

Future studies will explore its role in macrophage immunoregulation and the underlying mechanisms involved.

Effects of SWDNFLQR on the morphology of RAW264.7 cells

3.5

A comprehensive examination of how SWDNFLQR influences the structure of RAW264.7 cells was conducted using an optical microscope to observe cellular growth states. Fig. 2 reveal that the control group cells have distinct edges and are either round or oval, indicating they remain unactivated. In contrast, the CG1 group cells began to show alterations in their appearance, with some exhibiting increased size, a few vacuoles, and shorter pseudopodia, suggesting a mild effect of SWDNFLQR without full activation. The CG2 group displayed a further increase in cell size, a greater number of vacuoles, and longer, more numerous pseudopodia, leading to a transformation into spindle-shaped or irregular forms. At this concentration, SWDNFLQR effectively activates RAW264.7 cells, boosting their phagocytic ability and immune responses. The findings indicate that macrophages are activated through the development of longer and more numerous pseudopodia, which also improve cellular adhesion and phagocytic function (Zhang et al., 2019). This aligns with morphological changes in RAW264.7 cells induced by sturgeon cartilage immunomodulatory peptide (Li et al., 2024). The LPS group cells exhibited significantly larger sizes, numerous vacuoles, elongated pseudopodia, and highly irregular shapes, confirming that stimulation with agents like LPS leads to macrophage activation and morphological alterations.Fig. 2. Effects of SWDNFLQR fraction on RAW 264.7 cells morphology.Fig. 2

Effects of SWDNFLQR on immunological markers in RAW264.7 cells

3.6

Macrophages primarily secrete cytokines to interact with other immune cells. One of the most significant inflammatory cytokines produced by macrophages is tumor necrosis factor (TNF-α), which plays a crucial role in encouraging cell growth and differentiation; it is also recognized as one of the most effective cytokines exhibiting strong antitumor properties to date (Huang et al., 2025). Additionally, macrophages generate substantial amounts of IL-6, a vital cytokine that influences inflammation, immune responses, and tumor progression by modulating macrophage polarization, metabolism, and functionality (Kang et al., 2020). This study aimed to investigate the specific immunomodulatory effects of SWDNFLQR on RAW264.7 cells by assessing the release of (NO) and the concentrations of TNF-α and IL-6 (Fig. 1D, E，F). The findings revealed a notable increase in NO release in the CG1 and CG2 groups compared to the control group, suggesting that SWDNFLQR enhances NO production in macrophages. The LPS group, serving as the positive control, demonstrated the highest NO levels, confirming the validity of the experiment.

Both CG 1 and CG 2 groups show increased levels of TNF-α when compared to the control group, although they are not as high as those in the LPS group. While SWDNFLQR does stimulate TNF-α production, its effect is notably less potent than that of LPS. TNF-α levels in CG2 were marginally elevated compared to CG1, but this difference was not statistically significant. The current research indicates that IL-6 levels in both CG1 and CG2 are significantly elevated compared to the control group, suggesting that SWDNFLQR effectively enhances IL-6 secretion from macrophages. In contrast, the LPS group displayed a markedly higher increase in IL-6 levels compared to those treated with SWDNFLQR. The activation of the Toll-like receptor and NF-kB signaling pathways by LPS results in the secretion of a substantial amount of proinflammatory cytokines, as previously documented (Luo et al., 2020). Collectively, these results indicate that SWDNFLQR exerts considerable immunomodulatory effects, promoting the release of NO, TNF-α, and IL-6 from RAW264.7 cells. The activities of these cytokines can enhance macrophage immunological functions by modulating inflammatory responses and intercellular communication.

Immunomodulatory peptides are gaining importance in the fields of food and pharmaceuticals due to their potential bioactive properties. The focus of this study is on SWDNFLQR, derived from oysters, which exhibits notable immunomodulatory effects, contributing to the variety of such peptides. The complete amino acid sequence corresponding to SWDNFLQR is Ser-Trp-Asp-Asn-Phe-Leu-Gln-Arg. With a molecular weight of 1064.50 Da, a hydrophobicity of 10.38 kcal/mol, and a pI of 6.42, these physicochemical characteristics enhance its specific and effective immunomodulatory capabilities. SWDNFLQR significantly enhances the proliferation, phagocytic activity, and release of immune factors in RAW264.7 cells, indicating that its effects are linked to specific amino acid sequences and the formation of its secondary structure. Research indicates that the structural activity of bioactive peptides is crucial for their biological functions, particularly for smaller peptides (2–10 amino acids) that exhibit high bioavailability and effectively modulate immune responses. The biological efficacy of SWDNFLQR, which has the amino acid sequence SWDNFLQR, is influenced by the type and proportion of its amino acids, particularly the hydrophobic and basic ones. The peptide comprises 37.5% hydrophobic amino acids, including phenylalanine(F), leucine(L), and tryptophan(W), which may facilitate its attachment to the membranes of RAW264.7 cells, thereby activating its immunomodulatory properties (Hou et al., 2012; Mansour et al., 2015). Additionally, SWDNFLQR contains 12.5% of the basic amino acid arginine(R). Aspartic acid(D), being charged, may interact with basic amino acids through salt bridges or hydrogen bonds, aiding SWDNFLQR's binding to negatively charged regions on cell membranes, promoting its cellular entry and signal transduction (Buck et al., 2021). This aligns with the research by Li (Li et al., 2019), who identified immunomodulatory peptides from oyster hydrolysates, demonstrating that DNSIAMESMK and LLQLGSGR can enhance macrophage proliferation, TNF-α and NO production, IL-6 levels, and phagocytic capacity. Peptides with a higher hydrophobic amino acid content exhibited stronger effects, although their specific immunomodulatory mechanisms and signaling pathways remain to be further explored through proteomics and bioinformatics studies.

Proteomics-based investigation of the immunomodulatory mechanism of SWDNFLQR

3.7

Overview of differentially expressed proteins

3.7.1

The analysis of proteomics in RAW264.7 cells cells utilized iTRAQ labeling for quantitative assessment. The experimental setup included a control group alongside CG1 (low SWDNFLQR concentration), CG2 (high SWDNFLQR concentration), and LPS, each with three biological replicates. Mass spectrometry analysis identified 7343 proteins that met the screening criteria: Score Sequest HT > 0, unique peptide count ≥1, Localization probability ≥0.75, or Delta score ≥ 8. PCA analysis, illustrated in Fig. 3 A, demonstrated clear clustering of replicates within their respective groups. Differentially expressed proteins were identified based on a fold change of ≥2.0 and a p-value of <0.05. The Venn diagram and volcano plot indicated that, relative to the control group, the CG1 group exhibited 100 proteins upregulated and 157 downregulated; the CG2 group had 102 upregulated and 112 downregulated; while the LPS group showed 302 upregulated and 950 downregulated proteins. In total, 47 proteins were differentially expressed across the three groups, comprising 23 upregulated and 25 downregulated. The LPS group had a significantly higher number of differentially expressed proteins (1252), indicating a stronger activation of macrophages (Luo et al., 2020). This activation led to changes in transcription factors such as NF-κB and MAPK, which in turn affected the expression of numerous inflammation-related proteins. The notable secretion of TNF-α and IL-6 by LPS-stimulated macrophages supports previous research, highlighting significant alterations in protein expression due to various stimuli in the LPS-induced group.Fig. 3protein expression profile in RAW264.7 cells. (A) PCA based on quantitative data of the selected proteins in each group (n = 3 for each group). (B)Differential protein statistical chart. (C) Venn diagram of three groups (Different colors represent different groups, and the numbers in the figure represent the number of intersections and the unique quantity of each group.). The volcano map of CG1-vs-Control (D), CG2-vs-Control(E), LPS-vs-Control(F) (Notes: blue and red dots represent upregulated and downregulated proteins, respectively, darker colors indicate more significant differences, and gray dots represent proteins with p-value≥0.05).Fig. 3

Gene ontology (GO) functional annotation enrichment analysis

3.7.2

In order to gain a deeper understanding of the functional roles of differentially expressed proteins (DEPs), we conducted a Gene Ontology (GO) functional annotation analysis utilizing the bioinformatics platform DAVID 6.8. This analysis was carried out across three distinct levels: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). As illustrated in Fig. 4, the five most significantly enriched terms (p < 0.05) in each category demonstrated notable enrichment. The GO enrichment analysis of iTRAQ-labeled proteomics data unveiled the regulatory mechanisms by which immunomodulatory bioactive peptides influence RAW264.7 cells. A comparison between the low-concentration sample group CG1 and the normal control group revealed that molecular function analysis indicated a significant upregulation in protease binding, signal receptor binding, and SMAD binding due to the active peptides. These molecular functions are crucial for sensing and transmitting immune signals. Furthermore, treatment with these active peptides resulted in contrasting effects on ubiquitin binding and ubiquitin-proteasome activity, suggesting a selective modulation of macrophage immune functions. At the cellular component level, the increased presence of fibrinogen complexes, collagen-rich extracellular matrix, and platelet α-granules suggests that bioactive peptides may influence macrophage activity by altering the extracellular matrix composition and platelet-derived elements. The findings at the biological process level, which include the upregulation of acute phase response, cellular response to IL-1, and positive regulation of chemotaxis, ndicate that bioactive peptides modulate immune responses and the migration of immune cells.Fig. 4GO function annotation enrichment analysis diagram. (A) CG1-vs-Control UP. (B) CG1-vs-Control Down. (C) CG2-vs-Control UP. (D) CG2-vs-Control Down. (E) LPS-vs-Control UP. (F) LPS-vs-Control Down.Fig. 4

When comparing the CG2 sample group to the normal control group and examining molecular functions, CG2 demonstrated a significantly stronger capacity to enhance translation repressor activity and DNA-(apurinic or apyrimidinic site) endonuclease activity than the control group. This indicates that CG2 might affect macrophage functionality by stabilizing protein synthesis and initiating DNA repair mechanisms. Additionally, there was a notable increase in the activities of ubiquitin-protein transferase and glutathione synthase, implying a contribution to glutathione production. Each of these proteins exhibited apyrimidinic site endonuclease activity, suggesting that CG2 could influence macrophage function through its effects on protein synthesis and DNA repair. A decrease in ubiquitin-protein transferase activity and glutathione peroxidase activity indicates that CG2 might impact pathways related to protein degradation and responses to oxidative stress. Treatment with CG2 led to a significant increase in proteins linked to the nucleus and adhesion junctions at the cellular level, implying that CG2 could affect transcription processes within macrophages and intercellular communication. Analysis of biological processes revealed that CG2 treatment resulted in the most significant upregulation of cell response to interleukin-1 and negative regulation of p53-like signaling. Concurrently, the substantial downregulation of regulation of canonical NF-kappa B signal transduction suggests that CG2 may have the ability to mitigate abnormal inflammatory responses (Li et al., 2024). Notably, both concentration groups showed an increase in biological processes such as cellular response to interleukin-1 and negative regulation of signal transduction by p53-like mediators, indicating that these pathways might be common mechanisms through which bioactive peptides exert their immunomodulatory effects. The findings presented here could facilitate macrophage proliferation and enhance their phagocytic abilities.

The LPS-vs-Control group acted as a positive control, exhibiting typical features of an inflammatory response (Kim et al., 2016). At the molecular level, LPS significantly boosts the binding of proteases and chemokine receptors (CCR5/CCR1), indicating the initiation of the inflammatory cascade and the recruitment of leukocytes (Qi et al., 2017). There was an increase in the extracellular space, cell surface, and Golgi membrane, which points to cytokine release and enhanced transport of membrane receptors. LPS triggers a robust inflammatory reaction in cells subjected to lipopolysaccharide and interleukin-1. Additionally, LPS inhibits apoptosis by negatively regulating extrinsic apoptosis signaling pathways. One of the most notable impacts of LPS is its ability to hinder fibrinolysis while promoting coagulation, creating a pro-survival and pro-coagulant environment during inflammation. Conversely, LPS reduces energy-consuming processes, as evidenced by diminished ATP-binding and protein kinase activities. The reduced availability of cellular components like the nucleus and cytoplasm leads to a downregulation of non-immune gene transcription. During immune activation, cell cycle arrest occurs, obstructing essential biological functions such as DNA repair, cell division, and chromosome segregation.

KEGG pathway enrichment analysis

3.7.3

To elucidate the mechanism of action of SWDNFLQR，we utilized the KEGG database to conduct an enrichment analysis on the differentially expressed proteins (DEPs) associated with selected metabolic pathways. We specifically focused on metabolic terms with a p-value of less than 0.05. The results indicated notable variations in the enrichment of metabolic pathways when comparing the sample to the positive controls. Fig. 5 indicate that the majority of proteins with altered expression in the CG1-vs-Control comparison were significantly associated with pathways involving TLRs, PI3K-Akt, complement and coagulation cascades, as well as cytokine-cytokine receptor interactions. The increased activity in the complement and coagulation pathways suggests that peptides can modify macrophage polarization, thereby influencing the adaptive immune response. Concurrently, the engagement of the cytokine-cytokine receptor interaction pathway implies that bioactive peptides might enhance macrophage immune surveillance through the release of proinflammatory cytokines like IL-6 and TNF-α. Toll-like receptors (TLRs) play a crucial role in detecting both external and internal danger signals, triggering a series of immune responses that range from activating innate immunity to influencing adaptive immunity (Najah et al., 2021). The heightened activity within the TLR signaling pathway supports the idea that bioactive peptides stimulate MyD88-dependent NF-κB activation via pattern recognition receptors such as TLR4, aligning with the previously noted increase in macrophage phagocytic ability. The PI3K-Akt pathway is essential for regulating cell growth, survival, metabolism, and proliferation. Akt influences cell survival, growth, and metabolism through its downstream targets (Manning & Toker, 2017). Immunomodulatory peptides may boost macrophage survival and proliferation by activating the PI3K-Akt pathway while simultaneously inhibiting the mTOR pathway, which mitigates excessive inflammatory responses.Fig. 5KEGG enrichment analysis diagram. (A) CG1-vs-Control UP. (B) CG1-vs-Control Down. (C) CG2-vs-Control UP. (D) CG2-vs-Control Down. (E) LPS-vs-Control UP. (F) LPS-vs-Control Down.Fig. 5

KEGG enrichment analysis revealed that the treatment of RAW264.7 cells with high concentration CG2 group immunomodulatory bioactive peptides exhibits a partially overlapping yet distinct immunoregulatory pattern compared to the low concentration CG1 group. The research identified significant co-enrichment in the TLR and PI3K-AKT signaling pathways. Previous studies indicate that certain bioactive peptides derived from the ocean have a strong affinity for toll-like receptors 2 and 4 (TLR2 and TLR4/MD-22180), leading to immunomodulatory effects on cells (Jiang et al., 2022). Notably, in the CG2-vs-Control comparison, there was an increase in toll-like receptor signaling alongside a decrease in NF-κB signaling, which was particularly noteworthy. The activation of the TLR pathway likely triggers the release of early inflammatory mediators, such as IL-1β, through MyD88-dependent mechanisms. The suppression of the NF-κB pathway suggests that these bioactive peptides may mitigate excessive inflammatory responses by disrupting the phosphorylation of IκBα or its degradation via ubiquitination. The downregulation of both the TNF signaling pathway and the T cell receptor signaling pathway further supports the notion that high concentrations of bioactive peptides exert an inhibitory influence on adaptive immunity, potentially modulating the Th1/Th2 balance. Additionally, pathways related to lipid metabolism, including lipid and atherosclerosis, were found to be upregulated, while glutathione metabolism was downregulated. This suggests that macrophages in the CG2 group may enhance short-term activation of the pro-inflammatory M1 phenotype by increasing lipid uptake and reducing antioxidant defenses.

The group exposed to LPS exhibited a characteristic inflammatory reaction as indicated by the KEGG enrichment analysis (LPS-vs-Control). The innate immune response was more pronounced in the LPS group compared to CG1/CG2, leading to a higher release of pro-inflammatory factors. This distinction highlights the differing impacts of external immune triggers versus the regulation by endogenous bioactive peptides. In the LPS-vs-Control comparison, LPS directly stimulated the MyD88-dependent classical NF-κB pathway through TLR4 (Ciesielska et al., 2021), which in turn activated the TNF and IL-17 signaling pathways in a cascading manner. A robust inflammatory response was noted, consistent with observations that experimental groups exposed to LPS released a significant quantity of cytokines. Notably, genes such as IL6, IL1B, and TNF were markedly upregulated within the cytokine-cytokine receptor interaction pathway, reinforcing the traditional model of LPS-induced inflammation.

Peptides that affect immune system functionality, particularly at elevated levels, may reduce the activity of the key nuclear transcription factor NF-κB through various mechanisms. These mechanisms could involve the stimulation of negative feedback regulators for NF-κB, increased levels of anti-inflammatory cytokines such as IL-10, and disruption of the TLR4-MyD88 signaling pathway. Studies indicate that many peptides derived from food sources can inhibit components of either the NF-κB or MAPK signaling pathways, exhibiting anti-inflammatory properties(Guha & Majumder, 2019). Numerous low-molecular-weight peptides extracted from thick-shelled mussels demonstrate significant immunomodulatory effects in RAW264.7 cells via NF-κB and MAPK signaling pathways (He et al., 2021). SWDNFLQR operates through a unique mechanism of action. The cellular response CG2 enhances immune activity while also curbing excessive immune reactions, similar to the effects of certain synthetic peptides. In research conducted by Yu (Yu et al., 2023), peptide sequences VQLSGEEK and GFSGLDGAKG were identified from gelatin hydrolysates, both of which displayed anti-inflammatory and immunomodulatory effects in RAW264.7 cells. Likewise, the peptide NVMEERKIK, isolated from ovalbumin by Li (Li, Abou-Elsoud, et al., 2024), was found to induce dual immunomodulatory effects in RAW264.7 cells.

Protein interaction network analysis

3.7.4

Cellular protein activity is predominantly regulated through protein-protein interactions. PPI interaction results of the three sample groups were shown in Fig. 6. In the comparison of CG1 and the control group, a distinct regulatory trend regarding the extracellular matrix (ECM) was observed. The proteins Fn1 and thrombospondin-1 (Thbs1) emerged as the most interconnected nodes, each exhibiting a connectivity degree of 22. Fn1 plays a crucial role in the activation and movement of immune cells (Longstreth & Wang, 2024). Their simultaneous upregulation suggests a potential role in facilitating similar immune adhesion mechanisms via integrin receptor activation. Additionally, the levels of Smad3 and Tgfbr1, which are vital components of the TGF-β signaling pathway, displayed contrasting regulatory behaviors. Furthermore, the significant upregulation of the chemokines Ccl5 and Ccl4 was noted. A lower concentration of peptides may promote immune activation by altering the TGF-β and Smad equilibrium, leading to the release of inflammatory cytokines. The synergistic relationship between ECM and chemokines aligns with the activation characteristics of TLR signaling.Fig. 6PPI network analysis of SWDNFLQR. (A) CG1-vs-Control group's top 23 connectivity protein interaction network diagram. (B) The highest-scoring sub-network in the CG1-vs-Control group was elected using the MCODE algorithm. (C) CG2-vs-Control group's top 22 connectivity protein interaction network diagram. (D) The highest-scoring sub-network in the CG2-vs-Control group was elected using the MCODE algorithm. (E) LPS-vs-Control group's top 23 connectivity protein interaction network diagram. (F) The highest-scoring sub-network in the LPS-vs-Control group was elected using the MCODE algorithm.Fig. 6

When comparing CG2 to the control group, notable proteins include Prkacb, Fn1, and Tlr6. Prkacb, a recognized modulator of cAMP signaling, influences immune cell activity and inflammation (Zhang et al., 2024), exhibiting a fold change of 2.1 and substantial connectivity (22), thereby establishing a new central node. The pronounced presence of Fn1, which demonstrates a dose-dependent response, suggests it could play a pivotal role in immunomodulation. Concurrently, the downregulation of NF-κB inhibitors Nfkbib and Traf3 indicates that samples with higher concentrations may influence NF-κB activity within the redox system. Furthermore, the coordinated upregulation of Tlr6 and Cd40, along with the notable expression of Serpinb8, may create a negative feedback regulatory loop, offering a molecular framework for understanding bidirectional immunomodulation.

The LPS-vs-Control group revealed distinct features of an inflammatory response. A network of various cyclin proteins (such as Cdk1, Cdk4, and Ccnd1) showed a collective downregulation, impacting apoptosis-related proteins like Casp3 and Tp53, which indicates a typical halt in the cell cycle. Although TNF levels remained elevated, its connectivity was lower (degree 23) compared to the cyclins, while MAPK signaling proteins (Mapk3 and Mapk14) faced inhibition. This implies that LPS may promote inflammation by targeting cell cycle checkpoints rather than activating the MAPK pathway, contrasting with the mechanism of polypeptide (TLR-PI3K/Akt activation).

Western blot validation

3.8

Western blot results (Fig. 7) demonstrated that SWDNFLQR, at both concentrations, significantly upregulated TLR4 protein expression and enhanced Akt phosphorylation (p-Akt) compared to the control (p < 0.01 or p < 0.05). This finding corroborates the proteomics predictions of TLR4/PI3K-Akt pathway activation. Notably, SWDNFLQR exhibited a dose-dependent bidirectional effect on NF-κB signaling. While the low concentration (CG1) had no significant impact, the high concentration (CG2) significantly downregulated both total NF-κB and its phosphorylated form (p-NF-κB) (p < 0.01). This protein-level finding supported the earlier KEGG-based prediction that SWDNFLQR suppresses NF-κB signaling at high doses, revealing a unique “activate TLR4/inhibit NF-κB” mode. Additionally, Western blot analysis confirmed the significant upregulation of Fn1 and Prkacb, two hub proteins identified in the PPI network, further solidifying their key roles in the SWDNFLQR-mediated immunoregulatory network.Fig. 7. The effect of SWDNFLQR on the expression of signal pathway-related proteins in RAW264.7 Cells.Fig. 7

(Notes: Compared to the control group, * p < 0.05, ** p < 0.01)

Analysis of the immunomodulatory structure-activity relationship and mechanism of action of SWDNFLQR

3.9

The peptide SWDNFLQR exhibits a significant promoting effect on macrophage proliferation, indicating its potential immune activity. However, an increase in cell proliferation is not synonymous with the positive regulation of specific immune functions. In our study, SWDNFLQR-induced macrophage proliferation was accompanied by a concurrent and significant enhancement in phagocytic activity (Fig. 1C) and the secretion of key immune mediators (NO, TNF-α, and IL-6) (Fig. 1D-F). Furthermore, our iTRAQ-based proteomics analysis revealed that this proliferation coincided with the specific activation of the Toll-like receptor (TLR) and PI3K-Akt signaling pathways (Fig. 5A, C). This profile indicates a coordinated, target-specific immune activation program rather than a nonspecific mitogenic effect. The enhanced phagocytosis induced by SWDNFLQR is accompanied by an increase in the secretion of NO, TNF-α, and IL-6. However, despite the increase in cytokine secretion levels, they remain significantly lower than those observed in the LPS positive control group. This indicates that peptide induction leads to a relatively mild and controllable activation, rather than a severe and explosive inflammatory response akin to that triggered by LPS.

SWDNFLQR identified in this study comprises 37.5% hydrophobic amino acids, including Phe, Leu, and Trp. These hydrophobic residues, particularly the centrally located Trp and Phe within the sequence, may function as “hydrophobic anchors” to enhance the interaction between the peptide and the membrane of RAW264.7 cells, thereby activating its immunomodulatory properties (Mansour et al., 2015). The C-terminal segment features an alkaline residue (R), which may contribute to electrostatic interactions. As reported two peptides, PRRTRMMNGGR and GPR, identified in cooked tuna juice and amaranth protein respectively, contain Arg at their C-terminals and exhibit anti-inflammatory activity in LPS-induced murine macrophages (Elass-Rochard et al., 1995). Additionally, the peptide incorporates the acidic amino acid Asp and the basic amino acid Arg. The coexistence of these oppositely charged amino acids may facilitate the peptide's binding to negatively charged regions on cell membranes through the formation of salt bridges or hydrogen bonds, thereby promoting cellular entry and signal transduction. This sequence simultaneously contains polar, charged, and hydrophobic amino acids. Such amphipathic characteristics, that is, possessing both hydrophilic and hydrophobic regions, provide a common structural basis for many bioactive peptides, including immunomodulatory and antimicrobial peptides, thereby facilitating the formation of stable interactions with lipid bilayers or receptor binding.

In this research, the proteomics assessment and western blot validation uncovered significant immunomodulatory signaling pathways and essential proteins associated with SWDNFLQR. The mechanisms involved are depicted in the accompanying Fig. 8. The low concentration SWDNFLQR (CG1) activates the TLR4-PI3K/Akt axis to promote macrophage proliferation and functional enhancement. The substantial increase in the level of the key ECM protein Fn1 also supports this finding. High concentration treatment (CG2) elicits an important regulatory change, whereby the TLR4-PI3K/Akt signaling remains active, but the downstream NF-κB signaling is attenuated. The simultaneously upregulated Prkacb (cAMP-PKA catalytic subunit) and downregulated NF-κB levels imply that SWDNFLQR may utilize the cAMP-PKA signaling axis to exert a negative-feedback brake on inflammation. The dual-mode “activate TLR4/inhibit NF-κB” operation may explain how SWDNFLQR promotes macrophage phagocytosis and proliferation yet avoids LPS-like hyperinflammation, consistent with the moderate cytokine profile (Fig. 1E-F). The combined actions of the hub proteins (Fn1 and Prkacb) and a bifurcated signaling pathway provide a coherent mechanistic explanation for the balanced immunomodulatory effects of SWDNFLQR. In brief, TLR4/PI3K-Akt signaling pathway is the major axis of SWDNFLQR operation. The mechanism behind its bidirectional regulation is that high doses activate the Prkacb/cAMP-PKA axis which in turn causes negative feedback specifically to inhibit NF-κB. The functionality of SWDNFLQR aligns with previous findings on immunomodulatory peptides derived from marine sources (Yu et al., 2023; Li et al., 2024). Proteins, peptides, or hydrolysates from marine origins primarily exert their effects through macrophage activation, phagocytosis stimulation, increased leukocyte counts, enhanced production of NO, immunoglobulins, and cytokines, as well as splenocyte proliferation, NK cell activation, and the engagement of NF-κB and MAPK pathways. Further research is necessary to elucidate how SWDNFLQR modulates these pathways to gain deeper insights into its immunomodulatory mechanisms.Fig. 8. Possible immunomodulatory mechanism diagram of SWDNFLQR.Fig. 8

Conclusions

4

This study identified a novel octapeptide, SWDNFLQR, derived from a papain hydrolysate of Crassostrea gigas, utilizing LC-MS/MS and computational methods. In experimental assays, SWDNFLQR significantly enhanced the proliferation and phagocytic activity of RAW264.7 cells, while also elevating the secretion of NO, TNF-α, and IL-6. Proteomic analysis and western blot validation revealed a dual immunomodulatory mechanism: at low concentrations, SWDNFLQR primarily functions as an immunomodulator by activating the Toll-like receptor and PI3K-Akt signaling pathways. At higher concentrations, it engages the same pathways to modulate key proteins such as Prkacb and Fn1, while simultaneously suppressing the NF-κB signaling pathways, thereby providing both immune activation and an anti-inflammatory response. This unique bidirectional activity positions SWDNFLQR as a promising food-grade additive for promoting immune balance or anti-inflammatory applications, particularly suitable for functional foods targeting populations with chronic inflammation. Future research will concentrate on optimizing its structure-activity relationship and evaluating its compatibility within food matrices.

CRediT authorship contribution statement

Gongming Wang: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Chunna Jiao: Writing – review & editing, Resources. Weijun Wang: Supervision, Funding acquisition. Jianqiang Chen: Supervision, Investigation. Huawei Qin: Funding acquisition, Investigation, Writing – review & editing. Jian Zhang: Writing – review & editing, Supervision. Yingjiang Xu: Supervision, Investigation. Yunping Zhao: Validation, Data curation. Hui Huang: Writing – review & editing, Supervision, Resources. Yuexin Jing: Validation, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.