Photinia glabra-derived exosome-like nanovesicles mitigate skin inflammaging via dual regulation of inflammatory signaling and calcium homeostasis

ABSTRACT

Aims

Chronic low-grade inflammation accelerates skin aging, termed inflammaging. This study investigates whether Photinia glabra-derived exosome-like nanovesicles (PgELNs) can alleviate inflammaging by modulating inflammatory signaling and calcium homeostasis.

Materials and methods

PgELNs were isolated using tangential flow filtration and characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Human keratinocytes (HaCaT) were stimulated with tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to model inflammaging. PgELN uptake, cell viability, senescence markers, cytokine expression, tight junction proteins, and calcium levels were assessed via flow cytometry, quantitative Real-Time Polymerase Chain Reaction (RT-PCR), immunoblotting, and transcriptomic profiling.

Results

PgELNs were efficiently internalized without cytotoxicity. In stimulated cells, PgELNs reduced proinflammatory cytokines, senescence-associated secretory phenotype (SASP) markers, and restored IL-10 and tight junction proteins. Transcriptome and pathway analyses revealed suppression of JAK/STAT signaling via STAT1 and ISG15 downregulation, reducing CXCL9/10 expression. This led to normalization of intracellular and extracellular Ca²⁺ levels.

Discussion

PgELNs mitigate skin inflammaging by dual regulation of inflammation and calcium homeostasis. Targeting the STAT1–CXCL9/10 axis, PgELNs reduce senescence and preserve barrier integrity. These findings highlight PgELNs as a promising plant-derived nanotherapeutic for managing inflammation-associated skin aging.

GRAPHICAL ABSTRACT

1. Introduction

Exosomes are extracellular vesicles ranging from 30 to 150 nm in diameter, secreted by both eukaryotic organisms and plants, and are typically observed in cup-shaped or disc-like morphologies [1]. Initially regarded as carriers of cellular waste, exosomes have, since the 2000s, been recognized, through advances in molecular biology and bioinformatics, as pivotal mediators of intercellular communication. These vesicles transport fragmented nucleic acids, lipids, and various proteins that modulate the activity of recipient cells [2].

Among these, plant-derived exosome-like nanovesicles (P-ELNs) have garnered increasing interest due to their low toxicity, minimal immunogenicity, and high biocompatibility relative to exosomes derived from animal cells [3,4]. Furthermore, unlike exosomes obtained from animal tissues or cultured cells, which are typically limited in yield, P-ELNs can be extracted in large quantities given sufficient plant biomass. These attributes have accelerated research into the use of P-ELNs as bioactive agents in therapeutic applications. To date, investigations have primarily focused on edible plants such as ginger, grapes, and blueberries, which have demonstrated beneficial effects on skin health, immune regulation, and gastrointestinal function [5]. However, future studies should expand beyond edible species to include medicinal and stress-resistant plants, which may possess unique therapeutic properties and facilitate more targeted interventions [6].

Inflammaging describes a state of chronic, low-grade inflammation that arises with aging and plays a substantial role in the onset of age-associated diseases [7]. In the skin, inflammaging is exacerbated by both intrinsic factors, such as immunosenescence and hormonal alterations, and extrinsic stressors including ultraviolet radiation, environmental pollutants, and smoking [8]. These stimuli enhance oxidative stress in keratinocytes, increase intracellular reactive oxygen species (ROS), and disrupt key signaling pathways. In particular, persistent secretion of proinflammatory cytokines, including TNF-α and IFN-γ, contributes to cellular senescence and tissue degeneration, manifesting as cutaneous aging phenotypes such as wrinkling, thinning of the dermis, and hyperpigmentation [9].

An emerging mechanistic link between inflammation and cellular aging is calcium ion (Ca²⁺) homeostasis [10,11]. Ca²⁺ is a critical secondary messenger involved in regulating cellular proliferation, differentiation, apoptosis, and stress responses [12]. Under physiological conditions, calcium flux between the extracellular matrix, cytoplasm, and intracellular organelles is tightly regulated. However, aging and inflammatory insults disrupt this equilibrium, resulting in elevated intracellular Ca²⁺ concentrations, impaired calcium efflux, and depletion of extracellular calcium stores [13]. This dysregulation activates stress signaling cascades, induces mitochondrial dysfunction, and promotes the expression of SASPs. When combined with oxidative stress, these disturbances accelerate inflammaging and impair the structural and functional integrity of the skin. Accordingly, the combined effects of oxidative damage, proinflammatory cytokines, and calcium imbalance not only expedite skin aging but also exacerbate chronic dermatological conditions such as atopic dermatitis and psoriasis [14,15].

Photinia glabra, commonly known as red robin, is an evergreen shrub belonging to the family Rosaceae. Although traditionally used in herbal medicine, P. glabra remains underexplored in scientific literature. Preliminary studies suggest that the plant contains polyphenolic compounds and antioxidant constituents with antimicrobial and anti-inflammatory activities, indicating potential efficacy in managing inflammatory skin diseases and promoting tissue repair [16].

Whether PgELNs can attenuate epidermal inflammaging by modulating the calcium – JAK/STAT – chemokine axis in keratinocytes, and thereby restore barrier integrity, remains unknown. Using a TNF-α/IFN-γ–stimulated keratinocyte model, we investigated this hypothesis by assessing whether PgELNs exert anti-inflammaging effects through suppression of STAT1/ISG15 signaling, rebalancing of intracellular Ca²⁺, normalization of tight junction proteins, and reduction of SASP-associated chemokines. By elucidating the mechanistic relationship between PgELNs and the calcium – JAK/STAT – chemokine axis in the epidermis, this study identifies a barrier-focused anti-inflammaging pathway and positions PgELNs as a plant-derived nanomaterial with translational relevance for restoring epidermal homeostasis.

2. Materials and methods

2.1. Isolation of PgELNs

P. glabra leaves were procured on five separate occasions from HYUN-Flower Wholesale Shop (Seo-gu, Daejeon, Republic of Korea). Only leaves, with stems removed, were used. These were thoroughly rinsed under running water more than three times. A total of 100 g of washed leaves was homogenized in 1 L of phosphate-buffered saline (PBS) filtered through a 0.2 µm membrane (1:10 w/v). The homogenate was centrifuged at 10,000 rpm for 30 min, and the resulting supernatant was filtered through a 0.45 µm membrane. Exosome-like nanovesicles were isolated using a tangential flow filtration (TFF) system equipped with a 500 kDa molecular weight cutoff (MWCO) cartridge (Cytiva, MA, USA). The eluate was further purified and concentrated using an ultrafiltration module (XOSOM-C 100; Metapore, Suwon-si, Gyeonggi-do, Republic of Korea).

TFF operating parameters. Unless otherwise specified, TFF was conducted at 25°C, with a crossflow rate of ~20 mL/min and a transmembrane pressure (TMP) of 2–4 psi. Samples were washed by diafiltration with ≥10 diavolumes of 0.2 µm-filtered PBS, followed by a final 0.2 µm filtration (“polishing”). The final yield was approximately 8 × 10¹² particles/100 g of fresh leaves, as determined by nanoparticle tracking analysis (NTA). Isolated vesicles were aliquoted at 1 × 10¹² particles/mL, stored at −80°C in single-use aliquots, and thawed immediately before use. For all experiments, PgELNs were diluted to the indicated concentrations in culture medium to ensure consistency and reproducibility.

Batch definition and quality control (QC). Each isolation from 100 g of leaves was designated as one batch. Prior to use, batches were qualified by NTA (mode size, concentration), TEM (vesicle integrity), and particle-to-protein ratio. Only batches that met predefined QC criteria were used. Unless otherwise noted, all results were confirmed using ≥2 independent batches.

2.2. Particle counting and size distribution

Particle concentration and size distribution of PgELNs were assessed using a nanoparticle tracking analyzer (NS300; Malvern Panalytical, UK). Samples were diluted in 0.2 µm-filtered PBS to achieve ~20–80 particles/frame and loaded into the flow cell using a 1 mL syringe. For each sample, three 60-second videos (technical replicates) were acquired and averaged. Acquisition settings were standardized across all runs: camera level 13 for extracellular vesicle samples and 200 nm polystyrene standards, and level 8 for 100 nm standards; the detection threshold was set to 3. Daily instrument performance was verified using 100 nm and 200 nm polystyrene beads under identical conditions. The hydrodynamic diameter was calculated from the diffusion coefficient using the Stokes – Einstein equation, and particle concentration (particles/mL) was derived from valid tracks normalized to the analyzed volume and dilution factor. Results are reported as mode size (nm) and concentration (mean ± SD of triplicates). (All NTA measurements were performed at Metapore, Suwon-si, Gyeonggi-do, Republic of Korea.)

2.3. Transmission electron microscopy

For negative-stain TEM, 5 µL of PgELNs (~1 × 10¹² particles/mL) was applied to glow-discharged, carbon-coated 200-mesh copper grids. After 1–2 min of adsorption, grids were wicked, rinsed once with distilled water, and stained with 1% uranyl acetate for 30–60 s, then air-dried. Images were acquired using a Tecnai 10 transmission electron microscope (FEI) operating at 100 kV and equipped with a 2k × 2k CCD camera (UltraScan; Gatan). Representative fields were captured at ~10,000× magnification (≈1.0 nm/pixel). Vesicle morphology (e.g., cup-shaped profiles) and membrane integrity were assessed across ≥5 fields/sample. (TEM analysis was conducted at Metapore, Suwon-si, Gyeonggi-do, Republic of Korea.)

2.4. Cell culture and reagents

HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wellgene, Gyeongsangbuk-do, Republic of Korea) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin (Thermo Fisher Scientific, MA, USA).

2.5. Immunofluorescence staining

For PgELN uptake assays, 3 × 10⁴ HaCaT cells were seeded onto confocal culture slides (Sigma-Aldrich, St. Louis, MO, USA). PgELNs (1 × 10³ particles/mL) were labeled with DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indodicarbocyanine) and incubated in the dark for 5 min prior to co-culture with cells for 3 h. Cellular uptake was analyzed by flow cytometry. For tight-junction staining, cells were seeded at 3 × 10⁴ cells/well and cultured to full confluence. Immunofluorescence followed a standard protocol: cells were fixed in 4% paraformaldehyde for 10 min at 25°C, permeabilized with 0.1% Triton X-100 for 10 min, and blocked for 15 min with a commercial blocking solution (Thermo Fisher Scientific). Cells were incubated with a primary antibody against ZO-1 (1:100 in antibody diluent) at 4°C for 24 h, followed by a secondary antibody (1:100) at room temperature for 1 h. Nuclei were counterstained with DAPI during the secondary step. Cells were washed three times with PBS containing 0.05% Tween-20 (PBS-T) between steps.

2.6. Senescence-associated β-galactosidase activity

Senescence-associated β-galactosidase (SA-β-gal) activity was evaluated using a commercial kit (Cell Signaling Technology, Danvers, MA, USA). Cells were fixed for 10 min, then incubated with X-gal staining solution at 37°C for 48 h in a parafilm-sealed plate to prevent evaporation. Blue-stained senescent cells were visualized under a phase-contrast microscope. For quantitative assessment, SA-β-gal activity was measured using a fluorescence-based senescence assay kit (Abcam, Cambridge, MA, USA). A total of 5 × 10⁶ cells were seeded in 12-well plates and cultured to full confluence. Cells were stained at 37°C for 2 h, washed three times, detached by trypsinization, and analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA; excitation 490 nm, emission 514 nm).

2.7. Cell viability assays

Cell viability was determined using an MTS assay. Cells (1 × 10³/well) were seeded in 96-well plates and treated with the indicated PgELN concentrations. Viability was measured every 24 h for three days. Following addition of the MTS reagent, plates were incubated for 1 h at 37°C, and absorbance was read at 490 nm.

2.8. Western blotting

Total protein was extracted from HaCaT cells using a mammalian protein extraction buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were resolved on 4–20% SDS – PAGE gels and transferred to PVDF membranes (Bio-Rad, CA, USA). Membranes were blocked for 1 h at room temperature and incubated overnight at 4°C with primary antibodies diluted in antibody diluent (Nacalai Tesque, Kyoto, Japan). After three washes in TBS-T, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using a digital imaging system (Bio-Rad) and quantified with ImageJ software. Detailed antibody information is provided in Supplementary Table S2.

2.9. Quantitative real-time polymerase chain reaction (RT-PCR)

Total RNA was isolated using a phenol/guanidinium-based reagent (APBio, Namyangju-si, Gyeonggi-do, Republic of Korea). First-strand cDNA was synthesized from 1 µg of total RNA using a cDNA synthesis kit (Thermo Fisher Scientific). Quantitative PCR was performed with a SYBR Green-based master mix (Biofact, Daejeon, Republic of Korea) on a real-time PCR system (CFX96 Dx; Bio-Rad). Primer sequences are listed in Supplementary Table S3. The Raw Ct values are provided in Supplementary Table S4.

2.10. Measurement of RANTES and MDC expression

The expression levels of RANTES (Cat# DY278) and MDC (Cat# DY336) were measured using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) in accordance with the manufacturer’s instructions. Fluorescence intensity was detected using an ELISA microplate reader (Synergy HTX Multi-Mode Reader, BioTek, Winooski, VT, USA).

2.11. 3${ }^{\mathrm{\prime }}$ mRNA sequencing-based gene expression analysis

Gene Transcriptomic profiling was performed using 3$\mathrm{\prime }$ mRNA sequencing. The 3${ }^{\mathrm{\prime }}$ untranslated regions (UTRs) of transcripts were amplified into a cDNA library using the QuantSeq 3$\mathrm{\prime }$ mRNA-Seq Library Prep Kit FWD (Lexogen GmbH, Vienna, Austria). Sequencing was performed on the Illumina NextSeq 550 platform (Illumina, CA, USA) with single-end reads. Data were processed using the nf-core/rnaseq pipeline, which employed STAR for alignment, Salmon for quantification, and GRCh38 as the reference genome [17,18]. Principal component analysis (PCA) and differential gene expression analysis were performed using DESeq2 [19]. Functional enrichment analysis was conducted with ClueGO [20], and pathway activity inference was performed using a multivariate linear model (MLM) implemented in the decoupleR package [21].

2.12. Measurement of free Ca²⁺ and fluo-4 AM–positive cells

Extracellular free Ca²⁺ concentrations were quantified using a commercial calcium ELISA kit (MAK022; St. Louis, MO, USA), following the manufacturer’s protocol. To prevent interference from divalent ions, all rinsing, sample handling, dilution steps, and standard-curve preparations were performed using Ca²⁺/Mg²⁺ -free PBS (pH 7.4), matrix-matched to the sample matrix. No chelating agents (e.g., EDTA, EGTA) were present in any buffer solutions. Assays were conducted in parallel with appropriate blanks and standards. Intracellular Ca²⁺ accumulation was evaluated by staining cells with fluo-4 AM (Thermo Fisher Scientific), followed by flow cytometric analysis (excitation: 494 nm; emission: 506 nm).

2.13. Statistical analysis

Statistical All statistical analyses were conducted using GraphPad Prism 9.0 software. One-way analysis of variance (ANOVA) was used for comparisons involving multiple groups. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Results

3.1. PgELNs are efficiently internalized by keratinocytes without affecting cell viability

To isolate intact PgELNs, a TFF system was employed, followed by final filtration through a 0.2 µm membrane to obtain nanovesicles of uniform size (Figure 1(A)). NTA confirmed that the majority of PgELNs exhibited an average diameter of approximately 100 nm, while TEM revealed their characteristic cup-shaped morphology with a well-defined lipid bilayer and internal vesicular structures (Figure 1(B)). To assess cellular uptake, PgELNs were labeled with DiD dye and co-incubated with keratinocytes for 3 h. Fluorescence microscopy demonstrated efficient cytoplasmic internalization of PgELNs (Figure 1(C)). Flow cytometry analysis based on DiD fluorescence showed that over 95% of keratinocytes internalized PgELNs after 24 h of incubation (Figure 1(D)). Importantly, PgELN treatment did not compromise cell viability, indicating the absence of cytotoxic effects on keratinocytes (Figure 1e).

Figure 1.

PgELNs isolated from “Photinia glabra” exhibit no cytotoxicity. (A) Schematic overview of the PgELN isolation procedure using tangential flow filtration (TFF). (B) Size distribution of PgELNs analyzed by nanoparticle tracking analysis (NTA); the x-axis is scaled in 100 nm intervals, and the y-axis in 0.5 particles/mL intervals. Inset: representative transmission electron microscopy (TEM) image of PgELNs. Scale bars = 200 nm (C) fluorescence microscopy image showing DiD-labeled PgELNs (red) and DAPI-stained nuclei (blue). Scale bars = 10 µm (D) quantification of intracellular PgELN uptake via flow cytometry based on panel C. (E) assessment of cell viability across time points and PgELN concentrations. Concentrations are indicated in particles/mL.

3.2. PgELNs suppress inflammaging in keratinocytes via anti-inflammatory activity

Persistent secretion of TNF-α and IFN-γ is a major contributor to skin inflammaging [22,23]. To investigate whether PgELNs modulate inflammatory responses induced by combined TNF-α and IFN-γ treatment (TI), keratinocytes were co-treated with TI and PgELNs, and the expression of inflammatory cytokines was analyzed. TI-induced upregulation of IL-1β, IL-6, and IL-8 was dose-dependently suppressed by PgELNs, while IL-10, an anti-inflammatory cytokine downregulated by TI, was restored in a dose-dependent manner. In addition, expression of COX2 and MMP1, enzymes associated with cytokine-driven inflammation and extracellular matrix degradation, was effectively reduced by PgELNs (Figure 2(A)). As TI-mediated inflammation accelerates cellular senescence and impairs proliferation, we next assessed whether PgELNs could restore keratinocyte viability under inflammatory conditions. Co-treatment with PgELNs increased cell viability in a dose- and time-dependent manner over a 3-day period (Figure 2(B)), suggesting protective effects against inflammation-induced cellular stress.

Figure 2.

PgELNs suppress inflammation and cellular senescence. (A) Quantitative RT-PCR analysis of IL1B, IL6, IL8, IL10, COX2, and MMP1 mRNA expression (n = 4). (B) cell viability following TNF-α and IFN-γ (TI) treatment with or without PgELNs. (C, upper) SA-β-galactosidase staining identifying senescent (blue) and non-senescent (unstained) cells. (C, lower) flow cytometric quantification of senescent cells. Scale bars = 50 µm. (n = 4) (D) Western blot analysis of aging-associated proteins. Tubulin and GAPDH served as loading controls; lamin A/C was used as a nuclear marker. (n = 3) (E) densitometric analysis of protein expression from panel D.

To evaluate the anti-senescent effects of PgELNs, SASP markers were analyzed. Cells exposed to TI displayed pronounced SA-β-galactosidase activity, evidenced by blue staining, which was reduced in a dose-dependent manner upon PgELN treatment. In addition to restoring reduced cell density, PgELNs significantly decreased the proportion of SA-β-gal-positive cells (Figure 2(C)). Flow cytometry-based quantification of SA-β-gal activity corroborated these findings. Protein-level analyses revealed that PgELNs suppressed the TI-induced elevation of p21^Waf1/Cip1 and phosphorylated H2A.X, both of which are hallmarks of cellular senescence. Furthermore, Lamin B1 levels, which are typically reduced during senescence, were restored by PgELN treatment (Figure 2(D,E)). Collectively, these results suggest that PgELNs mitigate TI-induced inflammaging and may alleviate inflammation-driven skin pathologies.

3.3. PgELNs protect skin barrier integrity by attenuating inflammaging

Senescence-induced dysfunction of keratinocytes compromises skin barrier integrity, increasing susceptibility to environmental stressors [24,25]. To determine whether PgELNs confer protective effects on the skin barrier by modulating inflammaging, we examined the expression of tight junction proteins. TI exposure increased nuclear p21^Waf1/Cip1 expression while reducing the expression of Occludin, a key tight junction component. PgELN treatment significantly reduced nuclear p21^Waf1/Cip1 levels and restored Occludin expression (Figure 3a). Similar restorative effects were observed for Claudin-1 and ZO-1, as demonstrated by Western blot analysis (Figure 3(A,C)). These findings indicate that PgELNs ameliorate TI-induced barrier disruption by suppressing inflammation and promoting the recovery of tight junction integrity, thereby supporting their potential role in preserving epidermal homeostasis.

Figure 3.

PgELNs protect against tight junction disruption in keratinocytes. (A) confocal microscopy images showing p21^Waf1/Cip1 (red), Occludin (green), and nuclear staining (blue). Scale bars = 10 µm (B) Western blot analysis of tight junction-associated proteins, with GAPDH and Tubulin as internal controls. (n = 3) (C) quantitative analysis of protein expression from panel B.

3.4. Transcriptomic analysis reveals JAK/STAT-mediated inflammation and calcium homeostasis as key mechanisms of PgELNs

Transcriptome-wide gene expression profiling provides a comprehensive snapshot of cellular regulatory states. To elucidate the mechanisms underlying the effects of PgELNs, we performed 3$\mathrm{\prime }$ mRNA sequencing and transcriptome analysis. PCA revealed that the three experimental groups formed distinct clusters, indicating that both TI and PgELN treatments produced discrete transcriptomic landscapes (Figure 4(A)). We subsequently identified a subset of ‘compensated genes,’ defined as differentially expressed genes (DEGs) that were dysregulated by TI treatment but reversed in the opposite direction upon PgELN treatment (Supplementary Table S5). A heatmap of the top 200 compensated genes (ranked by fold change between the TI and PgELN groups) exhibited clear symmetry, indicating that PgELNs counteracted inflammation-induced transcriptional changes (Figure 4(B)). To investigate the functional relevance of these genes, we conducted a gene ontology (GO) enrichment analysis. The resulting network of enriched GO terms revealed a downregulation of inflammation-related pathways (blue cluster) and an upregulation of calcium ion (Ca²⁺) homeostasis-related pathways (red cluster) (Figure 4(C,D)). The observed suppression of inflammatory responses was consistent with previous findings in this study. To further dissect the signaling mechanisms, we performed pathway activity prediction based on the expression profiles of downstream target genes. This analysis revealed a marked downregulation of JAK/STAT target genes and substantial inhibition of JAK/STAT signaling activity (Figure 4(E)). These data corroborate the PgELN-mediated suppression of JAK/STAT signaling shown in Figure 4. Collectively, these results suggest that PgELNs mitigate inflammaging by repressing JAK/STAT-mediated inflammatory signaling and restoring Ca²⁺ homeostasis.

Figure 4.

Transcriptome analysis reveals PgELN-mediated modulation of cellular activity in keratinocytes. (A) Principal component analysis (PCA) of control (yellow), TI-treated (red), and PgELN-treated (blue) keratinocytes. (B) heatmap of the top 200 compensated genes. ‘Compensated genes’ are defined as those differentially expressed following TI treatment and exhibiting inverse expression trends upon PgELN treatment. (C) gene ontology (GO) network diagram of significantly enriched terms among compensated genes. (D) top 10 differentially expressed genes involved in inflammation and Ca²⁺ homeostasis. Genes with expression levels above the mean were ranked by absolute Fold change (TI vs. PgELN). (E) predicted signaling pathway activity and expression patterns of JAK/STAT target genes.

3.5. STAT1 and chemokines mediate the crosstalk between inflammation and calcium homeostasis in the anti-inflammaging effects of PgELNs

Bioinformatic analyses identified two major biological processes underlying the anti-inflammaging effects of PgELNs: suppression of inflammation and restoration of Ca²⁺ homeostasis. To determine the regulatory axis linking these processes, we conducted a Gene-GO network analysis using ClueGO and CluePedia [20,26]. In this network, nodes represent enriched GO terms and their associated compensated genes, while edges denote gene-GO annotations, shared genes between GO terms, and coexpression data from the STRING database [27]. We identified the shortest path linking ‘cellular response to type I interferon’ (reflecting TI-induced inflammation) to ‘calcium ion homeostasis.’ This analysis revealed that downregulation of inflammation-associated genes STAT1 and ISG15 led to reduced expression of their downstream chemokines CXCL9 and CXCL10, which in turn modulate calcium homeostasis (Figure 5(A)). A literature review supported this regulatory cascade, indicating that PgELN-mediated suppression of STAT1 and ISG15 reduces CXCL9 and CXCL10 expression, thereby limiting Ca²⁺ influx into neighboring cells and contributing to the restoration of intracellular Ca²⁺ homeostasis [28,29]. While ISG15 has also been linked to CXCL10, this association is currently supported only by correlative data and warrants further validation. To experimentally confirm these predictions, we quantified mRNA levels of STAT1, ISG15, CXCL9, and CXCL10 by RT-PCR. All four genes were significantly upregulated following TI treatment but were attenuated in a dose-dependent manner upon PgELN administration (Figure 5(B)). Inflammatory stimulation led to a marked reduction in extracellular free Ca²⁺ and a concomitant accumulation of intracellular Ca²⁺, indicative of disrupted calcium homeostasis and cellular stress. Measurement of free Ca²⁺ concentrations in the culture medium confirmed a substantial decline after TI treatment, which was effectively reversed by PgELN exposure (Figure 5c). Similarly, flow cytometric analysis of Fluo-4 AM-positive cells showed a > 30% increase in intracellular Ca²⁺ levels following TI treatment, which was significantly reduced upon PgELN treatment (Figure 5d). These findings validate our transcriptomic predictions and demonstrate that PgELNs reverse inflammation-induced calcium dysregulation. Together, the results suggest that PgELNs contribute to tissue homeostasis under inflammatory stress by coordinately regulating inflammatory signaling and calcium ion dynamics.

Figure 5.

STAT1 and downstream chemokines may link inflammation and Ca²⁺ homeostasis. (A) Proposed mechanistic pathway linking interferon signaling to calcium homeostasis, based on gene ontology (GO) network analysis. (B) quantitative RT-PCR analysis of STAT1, ISG15, CXCL9, and CXCL10 expression. (C) ELISA-based quantification of intracellular free Ca²⁺ levels in keratinocytes. (D) flow cytometric analysis and quantification of cells with elevated intracellular Ca²⁺. (E) schematic model illustrating the regulatory role of PgELNs in mitigating skin inflammaging through coordinated modulation of inflammatory signaling and Ca²⁺ homeostasis.

4. Discussion

Chronic inflammation has emerged as a fundamental contributor to aging and age-related pathologies, a process collectively termed “inflammaging” [7,22]. A critical but often underrecognized aspect of inflammaging is the disruption of calcium (Ca²⁺) homeostasis. Calcium ions function as ubiquitous second messengers that regulate diverse cellular processes, including gene expression, proliferation, differentiation, and apoptosis [12]. Under physiological conditions, calcium signaling is tightly regulated through coordinated flux between intracellular stores and the extracellular environment. However, persistent inflammatory stimuli, such as elevated levels of TNF-α, IFN-γ, and other proinflammatory cytokines, disrupt calcium channels and pumps, resulting in aberrant intracellular Ca²⁺ accumulation and extracellular calcium depletion. This dysregulation activates cellular stress responses, including mitochondrial dysfunction and oxidative stress, ultimately promoting cellular senescence. Emerging evidence suggests that calcium imbalance is not merely a downstream consequence of aging but may act as a causal mediator linking chronic inflammation to tissue dysfunction [30,31]. Thus, the interplay between inflammatory signaling and calcium homeostasis constitutes a critical axis in the pathogenesis of inflammaging and represents a promising therapeutic target.

To model the epidermal barrier – centered inflammaging axis, we employed HaCaT keratinocytes. This cell type was selected based on three key attributes: (i) keratinocytes are first responders to TNF-α/IFN-γ stimulation [32]; (ii) they are principal producers of SASP-associated chemokines (e.g., CXCL9, CXCL10, RANTES, MDC) that amplify inflammatory signaling [33]; and (iii) they regulate tight junction integrity and maintain the epidermal calcium gradient. This model aligns with our mechanistic endpoints – namely, JAK/STAT – mediated chemokine regulation and calcium homeostasis [34]. Within this framework, we demonstrate that PgELNs attenuate skin inflammaging by concurrently modulating inflammatory signaling and intracellular calcium dynamics. TNF-α/IFN-γ–stimulated keratinocytes exhibited hallmark senescence features, including elevated SASP markers and disrupted Ca²⁺ homeostasis, both of which were reversed by PgELN treatment [35]. Mechanistically, PgELNs downregulated STAT1 and ISG15, and reduced phosphorylation of JAK and STAT proteins induced by inflammatory stimulation, indicating inhibition of JAK/STAT signaling at both transcriptional and post-translational levels (Supplementary Fig. S1). Consistent with the established role of dysregulated JAK/STAT signaling in inflammatory skin disorders, PgELNs also decreased levels of RANTES and MDC, two chemokines implicated in atopic skin inflammation [36,37] (Supplementary Fig. S2).

The skin serves as a primary barrier against environmental insults, providing essential physical, chemical, and immunological protection [38]. However, chronic inflammation and aging compromise this barrier, increasing susceptibility to external stressors such as pollutants, pathogens, ultraviolet radiation, and allergens. This vulnerability perpetuates a cycle of tissue damage and sustained inflammation, ultimately impairing epidermal homeostasis and regenerative function. Notably, PgELN treatment restored the expression of tight junction proteins, Occludin, Claudin-1, and ZO-1, which are typically downregulated under chronic inflammatory conditions. By preserving tight junction integrity, PgELNs enhanced intercellular adhesion and stabilized the epidermal structure, contributing to the maintenance of skin barrier function under aging-related stress. To rule out the possibility that the observed biological effects were attributable to residual soluble phytochemicals from aqueous processing, we conducted an early head-to-head comparison of density-gradient ultracentrifugation (DG-UC) and TFF. Preparations obtained via both methods exhibited comparable bioactivity across key endpoints, including cell viability and SASP protein levels, thereby validating TFF as the preferred manufacturing approach due to its superior reproducibility and scalability (Supplementary Fig. S3).

Although our findings are consistent with previous studies on plant-derived nanovesicles, this work is the first to establish a mechanistic link between anti-inflammaging activity and calcium homeostasis via STAT1-mediated chemokine regulation. This supports the hypothesis that chronic inflammation and calcium imbalance are interdependent drivers of skin aging and barrier dysfunction. By targeting both inflammatory and calcium signaling pathways, PgELNs offer a promising natural strategy for restoring skin health and counteracting age-related inflammatory decline. Nevertheless, several limitations should be acknowledged. First, although we observed consistent transcript-level changes, further validation at the protein level, via immunoblotting, immunostaining, or phospho-proteomics, and in vivo functional assays is necessary to strengthen causal inference. Second, while our keratinocyte-centric model allowed focused interrogation of the epidermal barrier axis, it did not capture the role of human dermal fibroblasts (HDFs), which are key regulators of inflammaging through extracellular matrix (ECM) synthesis, turnover, and remodeling; TGF-β/MMP signaling; and secretion of SASP factors such as IL-6, IL-8, and various chemokines. Through paracrine signaling and mechano-responsive interactions linked to matrix stiffness, HDFs can also indirectly modulate tight-junction organization and the epidermal calcium gradient. Future studies should therefore assess PgELN effects in HDF monocultures, keratinocyte – fibroblast co-cultures, 3D skin equivalents, and ex vivo human skin to capture epidermal – dermal crosstalk in a more physiologically relevant context. Third, the specific molecular cargo responsible for these effects, such as miRNAs or lipids, remains to be elucidated. Isolating and characterizing these bioactive components, and mapping their molecular targets, will be essential. Fourth, while STAT1 and CXCL10 were identified as key mediators, broader profiling of the calcium-regulatory network (including channels, transporters, and pumps) modulated by PgELNs is warranted. From a translational perspective, development of an optimized topical delivery system will be critical for clinical application. Prior studies have shown that nanoparticle-loaded hydrogels enhance ROS scavenging, anti-inflammatory activity, and wound healing [39–41]. Given their biocompatibility, moisture retention, and sustained-release properties, hydrogels represent a promising platform for PgELN-based formulations. Future research should aim to optimize PgELN – hydrogel compositions, focusing on stability, release kinetics, and skin permeation/retention, while establishing dosing regimens in disease-relevant models (e.g., dermatitis, photoaging) and defining GMP-compliant TFF scale-up and QC parameters. Together, these efforts would facilitate the commercial translation of PgELNs within the cosmeceutical sector, synergizing with existing skin therapeutics.

5. Conclusion

In summary, our findings demonstrate that PgELNs possess potent anti-inflammaging activity by concurrently suppressing proinflammatory cytokine signaling and restoring calcium ion homeostasis. Mechanistically, PgELNs attenuate JAK/STAT pathway activation and reduce the expression of downstream chemokines, thereby mitigating inflammation-induced calcium dysregulation and cellular senescence. These effects collectively contribute to the preservation of skin barrier integrity and the reversal of senescence-associated phenotypes in keratinocytes (Figure 5e). This study provides the first evidence that PgELNs function at the intersection of inflammatory and calcium signaling pathways in the context of skin aging. Owing to their plant origin, biocompatibility, and scalable production, PgELNs represent a promising platform for the development of next-generation, nature-derived therapeutics targeting chronic skin inflammation and age-associated degeneration.

Funding Statement

This work was financially supported by grants from the Korean Institute of Oriental Medicine, Ministry of Science and ICT, Republic of Korea [grant number KSN2224011].

Article highlights

• PgELNs demonstrate potent anti-inflammaging activity in a TNF-α/IFN-γ-stimulated keratinocyte model, showing efficient cellular uptake without detectable cytotoxicity.

• PgELNs inhibit JAK/STAT signaling, downregulate SASP-associated chemokines, restore intracellular Ca²⁺ homeostasis, and normalize tight junction proteins, thereby reinforcing epidermal barrier integrity.

• PgELNs attenuate senescence-associated phenotypes and promote the restoration of epidermal homeostasis, as evidenced by transcriptome-wide analysis.

• PgELNs represent a promising plant-derived nanomaterial with translational potential for anti-inflammaging skin therapies.

Author contributions

Kang-In Lee: Conceptualization, Methodology, Software, Data curation, Validation, Writing – original draft. Yousang Jo: Visualization, Investigation, Validation, Writing – original draft. Hyungjun Kim: Supervision, Funding acquisition, Writing – review & editing. Hye Jin Kim: Investigation, Validation, Writing – review & editing Ki-Sun Park: Conceptualization, Methodology, Supervision, Writing – original draft, Writing – review & editing, Project administration. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary Information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2025.2572991