Purification and Anti-Inflammatory Activity of Walnut Exosome-like Nanoparticles

Abstract

This study reports the first successful isolation and characterization of exosome-like nanoparticles from walnut kernels (WELNs). The isolated WELNs exhibited a typical cup-shaped morphology with an average diameter of 139.7 ± 67.5 nm, a concentration of 7.4 × 10¹¹ particles/mL, and a zeta potential of −17.47 ± 4.06 mV. Proteomic and small RNA sequencing analyses confirmed the presence of diverse proteins and microRNAs within WELNs. In vitro assays demonstrated their potent antioxidant capacity, with radical scavenging rates of 67.54% against ABTS⁺ and 48.59% against DPPH⁺ at 102 μg/mL and IC₅₀ values of 89.7 μg/mL and >102 μg/mL for scavenging of ABTS⁺ and DPPH⁺ radicals, respectively. Cytotoxicity assays indicated no adverse effects on RAW264.7 macrophage viability at concentrations up to 60 μg/mL. In LPS-stimulated RAW264.7 macrophages, WELN treatment (20–60 μg/mL) dose-dependently mitigated oxidative stress by reducing intracellular ROS levels (down to 81.22% of the control at 60 μg/mL) and malondialdehyde (MDA) content while restoring the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Furthermore, WELNs significantly suppressed the production of nitric oxide (NO) and pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (reduced to approximately 30.8%, 22.7%, and 23.6% of LPS-induced levels, respectively, at 60 μg/mL). Mechanistic investigation revealed that the anti-inflammatory effect was mediated through the inhibition of the MAPK signaling pathway, as evidenced by decreased phosphorylation of p38, ERK, and JNK. In conclusion, WELNs exhibit dual anti-inflammatory and antioxidant properties. This study provides the first evidence of bioactivity for walnut-derived exosome-like nanoparticles, advancing the mechanistic understanding of walnuts’ health benefits and highlighting their potential as a natural component for functional food applications.

1. Introduction

Chronic inflammation and oxidative stress are central pathophysiological processes that drive the development of numerous metabolic and age-related diseases, with their interplay serving as a critical target for nutritional intervention [1]. Macrophages, as key sentinel cells of innate immunity, play a central regulatory role in this process [2]. Upon activation by stimuli such as bacterial lipopolysaccharide (LPS), macrophages polarize toward a pro-inflammatory (M1) phenotype. This shift is characterized by the robust secretion of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 (IL-1β), accompanied by a burst of reactive oxygen species (ROS) generation [3]. The resulting oxidative stress not only causes direct cellular damage through lipid peroxidation, protein carbonylation, and DNA injury but also perpetuates and amplifies inflammatory signaling via the activation of redox-sensitive pathways like nuclear factor-kappa B (NF-κB), creating a self-sustaining inflammatory–oxidative vicious cycle [4]. Therefore, dietary strategies capable of simultaneously intervening at both ends of this cycle are of significant importance.

Conventional pharmacological approaches for managing inflammation, such as the use of non-steroidal anti-inflammatory drugs (NSAIDs), are often associated with adverse effects, including gastrointestinal and renal toxicity [5]. Although numerous plant-derived bioactive compounds possess promising anti-inflammatory and antioxidant properties, their efficacy is frequently limited by poor bioavailability and rapid metabolism [6]. This underscores the importance of developing safer and more bioavailable delivery systems derived from natural dietary sources, which can simultaneously address both inflammation and oxidative stress. These considerations highlight the growing potential of targeted nutritional strategies and food-based interventions as effective means to support health through daily dietary intake.

In this context, plant-derived exosome-like nanoparticles (PELNs) have emerged as a promising natural platform [7]. Exosomes are nanoscale extracellular vesicles, typically ranging from 30 to 150 nm in diameter, characterized by a phospholipid bilayer membrane. They carry a diverse cargo of bioactive molecules, including proteins and microRNAs, and are recognized as essential mediators of intercellular communication [8]. In recent years, plant-derived exosome-like nanoparticles (PELNs), as natural evolutionary products, have offered a novel strategy to address these delivery challenges [9]. While similar in morphology and size to their animal-derived counterparts, plant exosomes offer distinct advantages: they are more accessible, present fewer safety concerns, and are abundantly present across a wide variety of plant species [10]. Furthermore, plant exosomes can be internalized by mammalian cells and participate in cross-kingdom signaling [11]. Notably, exosomes derived from plants such as ginger [12], grapefruit [13], and bitter melon [14] have demonstrated anti-inflammatory and antioxidant bioactivities. These findings underscore the potential of plant exosomes as a valuable alternative therapeutic strategy for inflammation, potentially overcoming limitations associated with conventional anti-inflammatory drugs and mammalian cell-derived exosomes. However, research on plant-derived exosome-like nanoparticles remains limited, and exosomes from many plant sources have yet to be systematically investigated.

Among functional foods, walnuts are a nutritional powerhouse, renowned for their high contents of polyunsaturated fatty acids (notably α-linolenic acid), polyphenols (e.g., ellagitannins), and γ-tocopherol [15]. Epidemiological and preclinical studies consistently link walnut consumption to improved cardiovascular, cognitive, and metabolic health, benefits largely attributed to their anti-inflammatory and antioxidant properties [16]. However, conventional research has almost exclusively focused on the bioactivity of solvent-extractable compounds or isolated fractions. The potential existence and functional significance of a sophisticated, natural nano-delivery system within walnuts—walnut-derived exosome-like nanoparticles (WELNs)—have been entirely overlooked. We hypothesize that WELNs constitute a unique, evolutionarily conserved “nano-food” compartment that may mediate the beneficial effects of walnuts in a more potent and bioavailable manner than isolated compounds. To test this hypothesis, the present study aimed to: (1) isolate and physically and chemically characterize WELNs from common walnut kernels; (2) evaluate their capacity to mitigate oxidative stress by modulating intracellular ROS levels, lipid peroxidation, and the endogenous antioxidant defense system; (3) investigate their effects on LPS-induced pro-inflammatory cytokine production and associated signaling pathways in RAW264.7 macrophages; and (4) preliminarily explore the involvement of key regulatory pathways such as MAPK. Our findings provide the first evidence for the anti-inflammatory and antioxidant bioactivity of WELNs, opening a new frontier in understanding the health-promoting mechanisms of walnuts through the lens of food nanoscience and offering a novel platform for the development of advanced nutraceuticals.

2. Materials and Methods

2.1. Materials and Reagents

RAW 264.7 macrophage was extracted by EallBio Co. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM; high glucose) and penicillin–streptomycin (PS) were obtained from Gibco (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Vazyme (Nanjing, China). LPS from Escherichia coli O111:B4 was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Phospho-p38 (#4511), p38, (#8690), phospho-SAPK/JNK (#4668), SAPK/JNK (#9252), phospho-ERK (#4370), ERK (#4695), β-actin (#4970), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (#7074) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

2.2. Isolation and Purification of WELNs from Walnut

Fresh walnut kernels (without pellicle) were homogenized with phosphate-buffered saline (PBS, pH 7.0) at a 1:5 (w/v) ratio using a Joyoung L6-L621 commercial juicer (160 W, Joyoung Co., Ltd., Hangzhou, China) for 3 min per batch at room temperature. The resulting homogenate was subjected to sequential centrifugation at 4 °C to remove large particles and cellular debris: first at 1200× g for 30 min, then at 3000× g for 30 min, and finally at 10,000× g for 60 min. The supernatant was subsequently ultracentrifuged at 100,000× g for 60 min (Hitachi CP100NX ultracentrifuge, P70AT fixed-angle rotor, k-factor ~ 145, HITACH, Tokyo, Japan) to pellet the crude exosome fraction. The pellet was resuspended in PBS and further purified by sucrose density gradient centrifugation. The resuspended sample was layered onto a discontinuous sucrose gradient (8%, 30%, 45%, and 60%) and centrifuged at 100,000× g for 60 min (Hitachi CP100NX ultracentrifuge, P40ST fixed-angle rotor). The fraction collected from the 30–45% interface was diluted with PBS and ultracentrifuged again at 100,000× g for 60 min to remove sucrose. Finally, the purified exosome preparation was sequentially filtered through 0.45 μm and 0.22 μm polyethersulfone (PES) membrane filters and stored at −80 °C for future use.

2.3. Characterization of WELNs

The WELN suspension was diluted before measurements using PBS for 1:10,000 dilution. The particle size and concentration of the sample were determined using nanoparticle tracking analysis (NTA). Concurrently, surface charge profiling via a Zetasizer Nano ZS (Malvern, UK) and ultrastructural morphology assessment by transmission electron microscopy (TEM, HT7800, HITACH, Tokyo, Japan) were performed. Total protein quantification utilized a bicinchoninic acid (BCA) assay kit (Biorigin, Beijing, China), with WELN purity calculated as the particle-to-protein ratio. All characterization measurements (NTA and DLS) were performed on at least three independently prepared batches of WELNs to ensure reproducibility. Total protein quantification of WELNs was performed using a BCA assay kit. Throughout this study, all reported WELN concentrations (expressed as µg/mL) refer to the total protein concentration determined by this method. The purity of WELN preparations was also assessed by calculating the particle-to-protein ratio.

2.4. Small RNA Sequencing and miRNA Identification

Total RNA was isolated from WELNs. The concentration and purity of the extracted RNA were assessed using a NanoDrop 2000 spectrophotometer, and the RNA quality number (RQN) (Thermo Fisher Scientific, Waltham, MA, USA) was determined using an Agilent 5300 system. Quality assessment and preprocessing of the raw sequencing data for each sample were performed using FASTP software (0.23.4). This step included: (1) removal of 3′ adapter sequences, (2) elimination of reads without insert fragments due to adapter self-ligation, (3) trimming of low-quality bases (quality score < 20) from the 3′ ends, (4) removal of reads containing unknown bases (N), and (5) discarding of reads that were too short (<18 nt) or too long (>32 nt). To filter out non-coding RNAs (e.g., rRNA, tRNA, and snRNA), the clean small RNA tags were aligned against the Rfam database. Subsequently, the remaining clean reads were mapped to the reference genome sequence using Bowtie. The mapped reads were then used to identify known miRNAs by alignment with the miRBase database (release 21.0). For the prediction of novel miRNAs, the miREvo (v3.3a) and miRDeep2 (Mackowiak S D, 2011) software packages were employed.

2.5. Proteomic Analysis

Total protein was isolated from WELNs, denatured, loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and stained with Coomassie blue; then, the individual protein bands were cut for proteomic analysis by Majorbio BioPharm Technology Co., Ltd. (Shanghai, China). The acquired MS/MS spectra were searched against the Juglans regia (walnut) protein database (UniProt, release 2023_12) using Proteome Discoverer (version 2.5, Thermo Fisher Scientific, Waltham, MA, USA) with the Sequest HT search engine. The search parameters were set as follows: precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.02 Da, trypsin as the digestive enzyme with a maximum of two missed cleavages, carbamidomethylation of cysteine as a fixed modification, and oxidation of methionine as a variable modification. Peptide and protein identifications were filtered at a false discovery rate (FDR) of less than 1%. Only proteins identified with at least two unique peptides were considered for further analysis. No depletion of abundant walnut storage proteins (e.g., globulins and albumins) was performed prior to analysis, as the aim was to characterize the complete endogenous protein cargo of WELNs, including potentially bioactive proteins that may be naturally packaged into these nanoparticles.

2.6. In Vitro Antioxidant Ability of WELNs

ABTS assay: ABTS + radicals were first prepared by mixing ABTS (7 mM) with potassium persulfate (K2S2O8, 2.45 mM) at room temperature for 12 h. The corresponding concentrations (6.7, 12.75, 25.5, 51 and 102 µg/mL) of WELNs were taken and incubated with ABTS+, and the absorbance at 734 nm was recorded by a multifunctional microplate detector (Spark, Tecan, Männedorf, Switzerland).

DPPH assay: A volume of 10 µM DPPH was mixed with an equal volume of ethanol. Then, 50 µL of WELNs was added for 30 min, making the final concentrations of WELNs 6.7, 12.75, 25.5, 51 and 102 µg/mL. The absorbance was measured at 517 nm by a multifunctional microplate detector (TECAN, Männedorf, Switzerland).

Ferric reducing antioxidant power: The total reducing capacity was determined using a potassium ferricyanide reduction assay. Briefly, different concentrations of WELNs, PBS, and potassium ferricyanide (K₃ [Fe (CN)₆]) were mixed in test tubes at a volume ratio of 2:1:1. The mixture was incubated in a water bath at 50 °C for 20 min and cooled, then supplemented with 10% trifluoroacetic acid. After thorough mixing, the solution was centrifuged. The supernatant was collected, diluted with an equal volume of deionized water, and mixed with ferric chloride (FeCl₃) solution. Absorbance was measured at 700 nm using a multimode microplate reader.

Ascorbic acid (vitamin C) was used as a positive control and assayed in parallel under identical conditions. Due to its exceptionally high antioxidant potency, it exhibited near-saturation scavenging activity in the ABTS assay at the lowest tested concentration. In the DPPH assay, a classical dose response was observed, allowing for determination of the Half Maximal Inhibitory Concentration (IC₅₀).

2.7. Cell Culture and Viability Assay

RAW 264.7 cells line were cultured in DMEM supplement with 10% (v/v) FBS and 1% (v/v) PS at 37 °C under 5% CO₂ and 80% humidity. After the cells reached 80–90% confluency, they were harvested using a cell scraper and subculture.

RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well. Then, 24 h after seeding, cells were washed and incubated with 10, 20, 40, 60, 80, 100, and 120 µg/mL of WELNs for 24 h. To check the cell viability, 10 μL of CCK8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt) (Biorigin, Beijing, China) was added to each well, followed by incubation at 37 °C for 1 h. Optical density (OD) was measured at a wavelength of 450 nm using a multifunctional microplate detector, and the percentage of viable cells was calculated as $\mathrm{Type}\%\mathrm{viable}\mathrm{cells}={\displaystyle \frac{\left(\mathrm{OD}\mathrm{treatments}\right)\times 100}{\mathrm{OD}\mathrm{untreated}\mathrm{control}}}$.

2.8. Determination of ROS Generation

RAW 264.7 macrophage suspensions were seeded at a density of 1.3 × 10⁵ cells/mL in black-walled 96-well plates and cultured for 24 h to allow adherence. The treatment groups were incubated for 24 h in medium containing 1 µg/mL LPS and different concentrations of WELNs (0, 20, 40, or 60 µg/mL). Cells cultured in complete medium without LPS or WELNs served as the control group. Intracellular ROS levels were measured using fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). After the treatment period, the supernatant was removed from each well and replaced with 1 µM DCFH-DA (Beyotime, Shanghai, China) solution. The plates were then incubated for 25 min at 37 °C in a humidified atmosphere of 5% CO₂. Following incubation, cells were washed three times with PBS to thoroughly remove any extracellular DCFH-DA. Fluorescence intensity was quantified using a multifunctional microplate detector at excitation and emission wavelengths of 488 nm and 525 nm, respectively.

2.9. Antioxidant Enzyme Activities and Biomarkers of Oxidative Stress

Cell pretreatment followed the procedure described in Section 2.10. Following treatment, the supernatant was discarded, and the macrophages were washed with PBS. Cells were then lysed on ice for 20 min using RIPA lysis buffer and harvested with a cell scraper. The lysate was centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA assay kit to enable subsequent normalization of malondialdehyde (MDA) catalase (CAT) and superoxide dismutase (SOD) content relative to total protein. MDA, CAT and SOD (Beyotime, China) activities were measured according to the kit’s instructions.

2.10. Nitric Oxide Production Assay

RAW 264.7 macrophages were seeded into 6-well plates at a density of 5 × 10⁴ cells per well and allowed to adhere for 24 h [17]. The cells were then treated with 1 μg/mL lipopolysaccharide (LPS) alone or in combination with varying concentrations of WELNs (0, 20, 40, and 60 μg/mL) for an additional 24 h. Following treatment, culture supernatants were harvested by centrifugation at 500× g for 5 min at 4 °C to remove cellular debris. Nitric oxide (NO) levels in the supernatants were assessed using a Griess Reagent Kit (Biorigin, Beijing, China) according to the manufacturer’s protocol. In brief, 50 μL aliquots of each sample were mixed with an equal volume of Griess reagent and incubated for 5 min at room temperature in the dark. Absorbance was then measured at 540 nm using a microplate reader.

2.11. Cytokine Production Assay

The protein expression levels of cytokines were quantified by ELISA. RAW 264.7 macrophage culture supernatants for cytokine quantification (IL-1β, IL-6, and TNF-α) were collected following the same procedure described for the 2.10 production assay. Cytokine concentrations were measured using Mouse Uncoated ELISA Kits (IL-1β, Cat. # JL18442-96T; IL-6, Cat. # JL20268-96T; TNF-α, Cat. # JL10484-96T; Shanghai Jonlnbio Industrial Co., Ltd., Shanghai, China), following the manufacturer’s instructions for a standard sandwich ELISA. Briefly, 96-well ELISA plates pre-coated with capture antibodies were blocked for 10 min at RT. Culture supernatants were then added and incubated for 1 h at RT, followed by incubation with biotinylated antibody detection working solution. After that, enzyme conjugate working solution was added and incubated at 37 °C for 30 min. After 15 min of color development using a TMB substrate, the reaction was stopped by adding stop fluid. Absorbance was measured at 450 nm using a Synergy H1 microplate reader. Cytokine concentrations were calculated from standard curves generated using the corresponding recombinant cytokine standards.

The transcript levels of cytokines were assessed by qRT-PCR. After treating RAW264.7 cells for 24 h according to the different methods described above, the cells were collected. Total RNA was isolated from the harvested cells using an RNA Easy Fast Animal Tissue/Cell Total RNA Extraction Kit (Tiangen Biotech, Beijing, China; Cat. # DP451), strictly following the manufacturer’s protocol. RT-qPCR was performed using SYBR Green I (Tiangen Biotech, China) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each 20 μL reaction contained 10 μL of 2 × FastKing RT-qPCR Buffer (SYBR Green), 0.8 μL of 25 × RT-PCR Enzyme Mix, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 1.2 μL of total RNA template, and 7 μL of nuclease-free water. All reaction setups were carried out on ice. Gene-specific primers were designed based on the corresponding human mRNA sequences, following standard primer design principles (Supplementary Table S1). All primers were synthesized by Zhong mei Taihe Biotechnology Co., Ltd. (Beijing, China). The thermal cycling conditions were as follows: initial pre-denaturation at 50 °C for 30 min and 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s.

2.12. Western Blot Analysis

For the examination of signaling-pathway proteins, Western blotting was performed. Cells were first lysed in RIPA buffer for 30 min, after which the lysates were collected and centrifuged at 12,000× g for 20 min at 4 °C. Protein concentrations in the supernatants were determined using a BCA protein assay kit. For nuclear protein isolation, a Nuclear and Cytoplasmic Protein Extraction Kit was employed. The protein extracts were combined with 4× SDS loading buffer at a 3:1 ratio (v/v), denatured by heating at 100 °C for 10 min, and subsequently stored at −80 °C until analysis. Equal amounts of protein were resolved by 12% SDS-PAGE and electrotransferred onto PVDF membranes. Following blocking with 5% skim milk, the membranes were washed with TBST and incubated overnight at 4 °C with primary antibodies (diluted 1:1000). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:10,000) for 1.5 h at ambient temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Millipore, Burlington, MA, USA), and the signal intensity was quantified with ImageJ (1.53k) software (Image Quant LAS 500, GE) to evaluate relative protein expression levels [18].

2.13. Statistical Analysis

The data were analyzed using SPSS 13.0 software and GraphPad Prism 10.0 software. Each experiment was replicated at least three times in parallel, and data were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was performed, followed by Dunnett’s multiple-comparisons test, to compare the differences between groups, and the significance level was set at p < 0.05.

3. Results and Discussion

3.1. Extraction and Characterization of WELNs

WELNs were isolated from fresh walnut juice using differential centrifugation, followed by purification via sucrose density gradient centrifugation. The purified nanoparticles were primarily recovered at the interface corresponding to 30–45% sucrose (Figure 1A). After identification, the concentration of WELNs was 1470 μg/mL.

WELNs were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM analysis revealed that the WELNs displayed a characteristic cup-shaped morphology with a discernible bilayer membrane structure, consistent with the typical appearance of exosomes (Figure 1B). The results of the NTA showed that WELNs had an average diameter of 139.7 ± 67.5 nm and a concentration of 7.4 × 10¹¹ particles/mL (Figure 1C). WELNs had a zeta potential of −17.47 ± 4.06 mV (Figure 1D). These results demonstrated that we successfully extracted WELNs with uniform particle size, good dispersion, enriched contents and high purity.

Research on exosomes derived from walnuts has not been reported to date. In this study, WELNs were successfully isolated and purified for the first time using a combination of differential centrifugation and sucrose density gradient centrifugation. TEM revealed that the WELNs exhibited a typical cup-shaped morphology with a discernible bilayer membrane structure. NTA indicated an average particle size of 139.7 ± 67.5 nm and a concentration of 7.4 × 10¹¹ particles/mL. The observed morphology and size distribution align with the established characteristics of plant-derived exosomes. For comparison, ginger exosomes [19] have been reported with an average size of 161 nm, and Chinese yam exosomes [20] have been reported with an average size of 168 nm, both falling within the common 100–200 nm range for plant exosomes. Thus, the WELNs isolated in this work not only meet the standard criteria for plant exosomes but also possess a comparatively smaller average particle size.

3.2. Identification of Proteins Within WELNs

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed a broad distribution of protein molecular weights within the ranges of 17–25 and 63 kDa (Figure 1E). Given the diverse protein profile identified within WELNs, a comprehensive proteomic analysis was conducted to further characterize their molecular composition and explore their functional potential. Proteomic analysis identified a total of 231 proteins in WELNs. Among these were classical exosome marker proteins, confirming that the isolated vesicles conform to the definition of exosomes. The proteomic profile of walnut-derived exosomes was functionally annotated through integrated GO enrichment and KEGG pathway analyses. The GO analysis shown in Figure 2A revealed a significant enrichment of proteins associated with several discrete yet interlinked functional modules. These included: (1) exosome biogenesis and cargo sorting, as evidenced by key components of the ESCRT machinery and multiple RAB GTPases; (2) protein synthesis and quality control, comprising a near-complete suite of ribosomal subunits, translation factors, aminoacyl-tRNA synthetases, molecular chaperones, and the ubiquitin proteasome; (3) signal transduction, involving transmembrane receptors, signaling adaptors, and kinases; (4) RNA binding and metabolism, with proteins like polyadenylate-binding proteins and RNA helicases; and (5) cytoskeletal organization and energy metabolism. Complementing this, the KEGG pathway enrichment analysis shown in Figure 2B systematically contextualized these protein clusters within broader biological processes. The most significantly enriched pathways were “Translation” and “Transport and catabolism,” directly mapping onto the protein synthesis and vesicle biogenesis modules identified by GO analysis. Furthermore, pathways for “Folding, sorting and degradation”, “Signal transduction”, “Cell motility”, and “Energy metabolism” were also prominently enriched, providing a coherent metabolic and regulatory framework for the exosome protein inventory.

The synergistic findings from GO and KEGG analyses depict walnut exosomes not as mere cellular by-products but as sophisticated, multifunctional platforms primed for intercellular communication. The co-enrichment of a virtually complete translational machinery (ribosomal proteins, tRNA synthetases, and initiation/elongation factors) alongside RNA-binding proteins is particularly striking. This suggests that plant exosomes may function as “mobile translational units” or “translationally competent vesicles”, a feature less emphasized in animal exosome literature. They could potentially deliver both mRNA and the apparatus required for its localized translation in recipient cells. Among these proteins, we found the RAB GTPase family and ESCRT-related proteins, which play crucial roles in the formation and secretion of exosomes. RAB GTPases [21] regulate membrane trafficking, fusion, and endocytosis, ensuring the efficient transport of exosomes from the endosome to the extracellular space. Meanwhile, ESCRT proteins [22], such as CHMP2A and VTA1, regulate membrane deformation, cargo packaging, and endosome sorting. The collaboration between RAB GTPases and ESCRT proteins promotes important biological processes such as intercellular material exchange, signal transduction, and immune regulation. Besides these proteins, other proteins involved in stress-response processes include heat-shock proteins (HSP70), which are major proteins found in exosomes. HSP70 [23] assists in the folding and stabilization of exosome contents, and its release by tumor cells can facilitate immune evasion.

3.3. MicroRNA Identification Within WELNs

Through sequencing and analysis of microRNA in WELNs, we obtained the following quality control and identification results. Initial sequencing generated a total of 10,734,752 raw reads. After filtering out impurities and low-quality reads, an average of 4,835,816 high-quality clean reads per library was obtained for subsequent analysis (Supplementary Table S2). The length distribution of these clean reads was primarily concentrated in the range of 18–24 nt, consistent with the typical size characteristics of plant small RNAs. Following size selection, a total of 4,657,526 miRNA sequences were acquired from the samples. Among these, approximately 826,008 sequences (17.73% of the total) were successfully mapped to the reference genome (Supplementary Table S3). The filtered sequences were aligned against all known mature plant miRNAs in the miRBase database, leading to the identification of 2108 distinct known miRNAs in walnut exosomes. Furthermore, using miREvo (v3.3a) and miRDeep2 (Mackowiak S D, 2011) software for novel miRNA prediction, 67 novel miRNAs were discovered, with lengths ranging from 18 to 32 nucleotides.

MiRNAs are ubiquitously present within exosomes, and their functional roles are predominantly governed by the miRNAs they encapsulate. To characterize the RNA composition of WELNs, small RNA sequencing was performed. This approach enables the comprehensive profiling of all RNA species and their sequences within WELNs, followed by in silico target prediction [24]. The results indicated that the miRNAs identified in this study ranged from 18 to 22 nucleotides (nt) in length. Previous research has reported that over 60% of plant exosome miRNAs typically measure 21 nt [25]. The findings of this study are generally consistent with those earlier observations. After miRNA identification, we further analyzed the functions of their potential target genes and the involved regulatory networks. miRNAs primarily regulate gene expression by binding to target mRNAs and inducing their cleavage or translational inhibition.

3.4. Functional Analysis of Target Genes of the UPMs

To explore the potential physiological regulatory functions of WELNs in humans, we employed the Miranda algorithm to predict the targets of all upregulated miRNAs against the human genome. The results indicated that a majority of the up-miRNAs primarily target mRNAs involved in inflammation, immunity and metabolism. Furthermore, the analysis revealed a many-to-many regulatory relationship between miRNAs and target genes, where a single miRNA can regulate multiple mRNAs and a single mRNA can be targeted by multiple miRNAs. Subsequently, GO and KEGG enrichment analyses were performed. GO annotations are shown in Figure 3A,B, indicating that the predicted target genes were enriched in diverse functional categories. In the cellular-component domain, targets were mainly associated with the cell, cell part, intracellular region, and intracellular organelles. Within molecular function, the targets were primarily related to binding, such as protein binding, nucleic acid binding, and catalytic activity. Under biological process, the most significantly enriched terms included cellular process, metabolic process, cellular macromolecule metabolic process, and organic substance metabolic process. The KEGG pathway analysis is shown in Figure 3C,D, further highlighting the top 20 significantly enriched pathways associated with the target genes, including the cAMP signaling pathway, pathways in cancer, and the MAPK signaling pathway. These results suggest that the miRNAs carried by WELNs may possess immunomodulatory and metabolic regulatory capabilities, potentially acting through these keys signaling and metabolic pathways.

The MAPK pathway plays crucial roles in cellular proliferation, stress response, and immune regulation. Plant-derived exosomes, as natural bioactive molecule delivery systems, may regulate this pathway through their carried miRNAs. For instance, miRNA families identified in this study, such as miR156 and miR159, have been previously reported to participate in plant development and stress responses and are known to engage in cross-talk with the MAPK pathway. For example, in recent work [26], miR156 from ginseng was found to significantly enhance the proliferation capacity of macrophages, enhancing their phagocytosis ability and their immunomodulatory effect. A study conducted with plant miR159 [27] showed that the concentration of orally ingested miRNA was inversely related to the chances and progression of breast cancer. We speculate that WELN miRNAs may modulate the expression of MAPK-related genes in recipient cells by mimicking or interfering with endogenous RNA networks during cross-kingdom communication, thereby influencing their physiological states. This finding provides novel molecular insights for exploring the potential applications of walnut exosomes in functional foods or plant-based therapeutics. Future functional studies are warranted to validate the regulatory effects of specific miRNAs on key nodes within the MAPK pathway. In addition, miR-396 [28], which was identified in the present study, is among the most abundant miRNAs reported in garlic-derived exosomes. Previous work has demonstrated that miR-396 regulates metabolic reprogramming in macrophages, largely through modulation of PFKFB3.

3.5. In Vitro Antioxidant Capacity of WELNs

The free radical-scavenging capacities of WELNs against three different radicals are shown in Figure 4A–C. WELNs exhibited potent scavenging activity, which was positively correlated with concentration within the tested range. At a concentration of 102 μg/mL, the scavenging rates for ABTS⁺ and DPPH⁺ radicals reached 67.54 ± 4.56% and 48.59 ± 11.34%, respectively. WELNs also demonstrated strong ferric-reducing antioxidant power. A direct comparison with classic antioxidant ascorbic acid (vitamin C) revealed a distinct activity profile. In the ABTS⁺ assay, ascorbic acid showed exceptionally strong activity, exceeding 83% inhibition at the lowest concentration (6.7 μg/mL) and plateauing at higher doses, which precluded accurate IC₅₀ determination within the tested range. In contrast, WELNs produced a well-defined sigmoidal dose response, with a calculated IC₅₀ of 89.7 μg/mL (95% CI: 72.1–111.4 μg/mL). This marked difference in potency was further confirmed in the DPPH⁺ assay. Here, ascorbic acid yielded an IC₅₀ of 5.62 μg/mL (95% CI: 4.89–6.46 μg/mL), whereas the IC₅₀ for WELNs exceeded the maximum tested concentration (>102 μg/mL).

The assessment of in vitro antioxidant potential primarily focuses on the capacity to neutralize free radicals. Established methodologies, including the ABTS, DPPH, and FRAP assays, provide complementary measures of radical-scavenging efficacy, hydrogen-donating activity, and overall reducing power [29,30,31]. This observed antioxidant potency is consistent with the reported bioactivity of other plant-derived exosome-like nanoparticles. Notably, the free radical-scavenging profile of WELNs aligns with the robust effects documented for exosomes derived from Momordica charantia, substantiating the position of WELNs among plant exosomes with considerable in vitro antioxidant capacity [14]. The pronounced antioxidant activity of WELNs suggests a key functional role in redox modulation. WELNs may alleviate oxidative damage, subsequently influencing downstream inflammatory signaling and cellular homeostasis. These properties not only highlight the intrinsic bioactivity of WELNs but also establish a foundational rationale for their prospective development as natural, nanoparticle-based agents for antioxidant therapies and functional health products, warranting further investigation into their precise molecular mechanisms and in vivo efficacy.

3.6. RAW 264.7 Cell Viability

The cytotoxicity of WELNs was assessed by exposing RAW 264.7 cells to varying concentrations of WELNs (0, 10, 20, 40, 60, 80, 100, and 120 µg/mL) for 24 h, followed by the administration of CCK8 reagent. The results are shown in Figure 5A. In the concentration range of 10–60 μg/mL, cell viability remained above 80%. However, at concentrations exceeding 80 μg/mL, viability decreased to approximately 76%. These results indicate that WELNs exhibit no cytotoxicity toward macrophages within the 10–60 μg/mL range and do not interfere with normal cell growth. Based on this dose-response profile, concentrations of 20, 40, and 60 μg/mL were selected for subsequent experiments.

3.7. WELNs Reduced LPS-Induced Oxidative Stress in RAW 264.7 Macrophages

As shown in Figure 5B, LPS stimulation significantly increased intracellular ROS levels by 117% compared to the control group, confirming its effectiveness in inducing oxidative stress in macrophages. In contrast, co-treatment with WELNs (20, 40, and 60 μg/mL) dose-dependently attenuated LPS-induced ROS generation. The corresponding ROS levels were 105.27%, 100.36%, and 81.22% of the control value, demonstrating a concentration-dependent reduction in oxidative stress. As shown in Figure 5C, the MDA levels, reflecting the extent of lipid peroxidation, were significantly elevated upon LPS (1 μg/mL) stimulation, reaching 14 μmol/mL compared to the control. Treatment with WELNs dose-dependently attenuated this increase, reducing MDA levels to 11, 7, and 6 μmol/mL at concentrations of 20, 40, and 60 μg/mL, respectively. As shown in Figure 5D, LPS stimulation significantly suppressed CAT activity to 0.8 U/mg protein. Co-treatment with WELNs at 40 and 60 μg/mL restored CAT activity to 1.4 and 1.8 U/mg protein, respectively. Similarly, as shown in Figure 5E, SOD activity was markedly reduced to 0.5 U/mg protein after LPS exposure. WELNs at 40 and 60 μg/mL significantly increased SOD activity to 0.9 and 3.1 U/mg protein, respectively, demonstrating a concentration-dependent recovery of antioxidant enzyme function.

Building on previous in vitro evidence of radical-scavenging capacity, the present study evaluated the cellular antioxidant activity of WELNs using an LPS-induced macrophage model. Treatment with WELNs significantly reduced ROS and MDA levels while restoring CAT and SOD activities under LPS-induced oxidative stress. This suggests that WELNs mitigate oxidative stress not only by directly neutralizing radicals but also by protecting against LPS-induced suppression of antioxidant enzyme activities. The effects may be mediated by bioactive components carried within WELNs, such as plant miRNAs or proteins, which could modulate key regulatory pathways, including Nrf2/Keap1 and MAPK, thereby promoting the expression and activation of antioxidant enzymes. The antioxidant effects of WELNs observed in this study are consistent with those reported for other plant-derived exosome-like nanoparticles. For example, citrus-derived exosomes [32] and strawberry-derived exosomes [33] have been shown to enhance CAT and SOD activities in oxidative stress models, and WELNs demonstrated a similar capacity to restore these enzymes under LPS-induced oxidative conditions. While direct quantitative comparisons are limited by differences in experimental protocols, the magnitude of enzyme activity restoration observed with WELNs is within the range reported for other plant-derived systems. This suggests that WELNs mitigate LPS-induced oxidative stress by preserving antioxidant enzyme activities and reducing oxidative damage under inflammatory conditions. Notably, the present study focused specifically on the protective effects of WELNs against LPS-induced oxidative stress, in line with our primary objective of evaluating their potential as a functional food component under inflammatory conditions. Whether WELNs independently modulate basal redox homeostasis in resting macrophages was not within the scope of this investigation and remains a topic for future research aimed at understanding their broader biological interactions.

3.8. WELNs Reduced LPS-Induced Inflammatory Response in RAW264.7 Macrophages

3.8.1. Inhibition of NO Production by WELNs in LPS-Stimulated Macrophages

To evaluate the potential of WELNs to inhibit nitric oxide (NO) production, RAW 264.7 macrophages were treated with increasing concentrations of WELNs (0, 20, 40, and 60 μg/mL), then stimulated with LPS (1 μg/mL). NO levels in the culture supernatant were quantified by measuring nitrite accumulation. As shown in Figure 6A, compared to the untreated control, LPS stimulation significantly increased NO production to 44 μM. Co-treatment with WELNs resulted in a dose-dependent suppression of NO release. Notably, at concentrations of 40 and 60 μg/mL, WELNs significantly reduced NO levels to approximately 7 μM, indicating potent anti-inflammatory activity.

NO serves as a key inflammatory mediator that participates directly in inflammatory responses and whose production can be regulated to control inflammation [34,35]. Therefore, we used NO generation to verify the modeling effect of LPS and to evaluate the anti-inflammatory efficacy of WELNs. In this study, LPS induction significantly increased NO levels in macrophages, while intervention with WELNs markedly reduced NO content. The suppression of NO production by WELNs is comparable to that reported for other plant-derived exosome-like nanoparticles. For instance, papaya-derived exosomes have been reported to reduce NO levels by approximately 40% in LPS-stimulated macrophages [36], while WELNs at 60 μg/mL achieved a reduction of approximately 84% under our experimental conditions. Although methodological differences preclude direct quantitative comparison, these findings collectively support the anti-inflammatory potential of plant-derived exosomes, including WELNs.

3.8.2. Inhibition of Pro-Inflammatory Cytokine Expression and Secretion by WELNs

The expression of pro-inflammatory cytokine genes was analyzed using qRT-PCR, and the amount of protein secreted by cells was measured using ELISA. The ELISA results are shown in Figure 6B–D. The secretion levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) were quantified by ELISA. Compared with the control group, LPS stimulation significantly elevated the secretion of TNF-α, IL-6, and IL-1β to 37,000 pg/mL, 396 pg/mL, and 158 pg/mL, respectively. Treatment with WELNs notably attenuated this increase. In the 60 μg/mL WELNs group, the cytokine levels were reduced to 11,402 pg/mL (TNF-α), 90 pg/mL (IL-6), and 37.3 pg/mL (IL-1β), demonstrating a potent anti-inflammatory effect. The qRT-PCR results shown in Figure 7A–D indicate that LPS stimulation significantly upregulated the expression of inflammatory mediator genes, including IL-1β, IL-6, TNF-α, and iNOS, in RAW 264.7 cells. In contrast, treatment with WELNs markedly downregulated the transcript levels of these pro-inflammatory factors and iNOS. Notably, WELNs at a concentration of 60 μg/mL exerted the most pronounced inhibitory effect on LPS-induced inflammatory gene expression in macrophages.

This study confirms that WELNs exert distinct anti-inflammatory effects in an LPS-induced macrophage model. To further elucidate their molecular targets, we measured mediators occupying key nodes in the inflammatory cascade: early-response factor TNF-α [37]; core-effector cytokines IL-1β [38] and IL-6 [39]; and inducible enzyme iNOS [40], which governs NO synthesis. The expression levels of these molecules collectively reflect the inflammatory status. Results demonstrated that WELNs broadly suppressed the production of all these factors, with particularly pronounced inhibition of TNF-α and IL-6. The broad suppression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) by WELNs is consistent with the anti-inflammatory profiles reported for other plant-derived exosome-like nanoparticles. For example, exosomes from black nightshade berries [41] and red cabbage [42] have been shown to inhibit specific inflammatory mediators in macrophage models. Our findings extend these observations by demonstrating that WELNs concurrently suppress multiple cytokines and iNOS expression, suggesting a multifaceted anti-inflammatory action that aligns with the growing body of evidence on plant exosome bioactivity. The anti-inflammatory properties of WELNs likely stem from their cargo of plant-derived miRNAs and signaling proteins. These components may act through multiple synergistic mechanisms, including direct scavenging of reactive oxygen species, cross-kingdom modulation of inflammatory signaling pathways such as NF-κB and MAPK, and the potential influence on macrophage polarization. Together, these actions underscore the integrated value of WELNs as naturally derived bioactive agents for inflammatory intervention.

3.9. WELN Treatment Positively Affected Inflammation-Related Pathways

To investigate the anti-inflammatory signaling mechanisms of WELNs, the expression of MAPK signaling pathways (p38, ERK and JNK) was investigated by Western blotting. The results showed that after stimulation of RAW 264.7 macrophages with LPS, the expression of phosphorylated p38 (p-p38), ERK (p-ERK), and JNK (p-JNK) proteins increased (p < 0.05) in the cells. RAW 264.7 macrophages pretreated with WELNs had reduced levels of p-p38 (Figure 8A), p-ERK1/2 (Figure 8B), and p-JNK (Figure 8C), with the 60 μg/mL concentration inducing the most pronounced reduction. These results suggest that WELNs inhibit the LPS-induced inflammatory response in RAW 264.7 macrophages by suppressing the activation of the MAPK signaling pathway.

To elucidate the molecular mechanism by which walnut exosome-like nanoparticles (WELNs) exert their anti-inflammatory effects, we focused on analyzing the MAPK signaling pathway, as it is a central mediator of LPS-induced inflammatory responses and directly regulates the expression of key cytokines such as TNF-α, IL-1β, and IL-6 [43]. Our results show that WELNs significantly suppressed LPS-induced phosphorylation of ERK1/2, JNK, and p38, indicating that they attenuate inflammatory cytokine production by interfering with upstream MAPK signaling. This inhibition aligns with the observed anti-inflammatory phenotype of WELNs, suggesting that modulation of the MAPK pathway is a key mechanism through which WELNs act, potentially via their bioactive cargo, such as plant-derived miRNAs or lipid mediators, that may disrupt TLR4/MYD88 complex formation or downstream kinase activation [44,45]. Furthermore, the ability of WELNs to broadly inhibit MAPK activation is consistent with reports on other plant exosomes, including ginseng-derived exosomes [46], which similarly suppress ERK, JNK, and p38 phosphorylation to enhance anti-inflammatory efficacy, supporting the notion that MAPK pathway regulation represents a shared yet potentially source-specific mode of action among plant-derived exosomes. In the proteomics analysis of WELNs, our KEGG analysis revealed that WELNs’ mechanism of action is linked to the MAPK pathway, which validates our in vitro experiment from a different perspective. Together, these results demonstrated that WELN treatment diminished inflammation onset and development.

4. Conclusions

This study reports the first successful isolation and characterization of exosome-like nanoparticles from WELNs. The isolated WELNs exhibited typical nanovesicle morphology, with an average diameter of 139.7 ± 67.5nm, and were enriched with proteins and miRNAs. In vitro assays demonstrated their potent free radical-scavenging capacity. In an LPS-induced model of inflammation and oxidative stress in RAW 264.7 macrophages, WELNs dose-dependently mitigated oxidative stress by reducing intracellular ROS and MDA levels while restoring the activities of antioxidant enzymes CAT and SOD. Furthermore, WELNs significantly suppressed the production of pro-inflammatory mediators, including NO, TNF-α, IL-6, and IL-1β. Mechanistic investigation revealed that the anti-inflammatory effect was mediated through the inhibition of the MAPK signaling pathway, as evidenced by decreased phosphorylation of p38, ERK, and JNK. In conclusion, WELNs demonstrate both anti-inflammatory and antioxidant activities. This study provides the first evidence of the bioactivity of walnut-derived exosome-like nanoparticles, offering novel insights into the health-promoting mechanisms of walnuts and highlighting their potential as natural candidates for functional food development. Based on the isolation yield obtained in this study, the effective in vitro doses (20–60 μg/mL) correspond approximately to the consumption of 0.7–2.0 g of walnut kernels, though direct dietary extrapolation requires caution due to factors such as gastrointestinal digestion stability and bioavailability. Future research should focus on identifying the specific bioactive molecules within WELNs that mediate these effects and further evaluating their functionality in more complex in vitro and in vivo models.