Neuropeptide Y drives vitiligo progression via NPYR-mediated NF-κB activation: a novel therapeutic target

Abstract

Neuropeptide Y (NPY) is a neurotransmitter involved in stress response and immune modulation and its dysregulation may contribute to the pathogenesis of vitiligo by influencing melanocyte function and autoimmune responses. This study aimed to explore the role and molecular mechanism of NPY in the progression of vitiligo. In vitro, human immortalized keratinocyte (HaCaT) cells were treated with H₂O₂ to simulate oxidative stress conditions observed in vitiligo. Through specific knockdown of the NPY receptor (NPY2R or NPY4R) achieved by siRNA transfection, combined with techniques such as Western blot (WB), ELISA and RT-qPCR, we found that NPY primarily activated the NF-κB signaling pathway via the NPY2R, thereby inducing the expression of key inflammatory cytokines, including CXCL9, CXCL10, and IL-15. Concurrently, NPY reduced intracellular ROS levels and partially inhibited H₂O₂-induced keratinocyte apoptosis. In vivo, a H₂O₂-induced vitiligo mouse model revealed that NPY exacerbated skin depigmentation, promoted NF-κB activation, increased expression of IL-15, and enhanced CD8 + T cell infiltration. In contrast, NPY2R inhibitors effectively reversed these phenotypes. Further assays indicated that NPY treatment decreased MDA content in skin tissue while elevating the activities of SOD and GSH-Px. Moreover, TUNEL staining and WB analysis of cleaved caspase-3 demonstrated that NPY could inhibit melanocyte apoptosis. Overall, this study was the first to demonstrate that NPY promoted the expression of CXCL9, CXCL10, and IL-15 by activating the NPY2R-mediated NF-κB signaling pathway in keratinocytes, thereby synergizing with oxidative stress to recruit CD8 + T cells and drive vitiligo progression.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49493-0.

Introduction

Vitiligo is an autoimmune-mediated dermatological disorder characterized by the selective destruction of epidermal melanocytes, resulting in well-circumscribed, depigmented macules on the skin¹. The prevalence of vitiligo is approximately 0.5-2%², and the disease typically necessitates long-term management strategies, with around 40% of patients experiencing relapse within a year of treatment cessation³. Vitiligo can appear at any age, particularly affecting visible areas like the face and scalp, often resulting in substantial psychological stress. This chronic mental burden can lead to feelings of inferiority, low mood, and even depression, posing a serious threat to the mental and physical well-being of patients⁴. Phototherapy and topical or oral immunomodulators such as corticosteroids, calcineurin inhibitors and JAK-inhibitors, are common treatments for vitiligo⁵. While these interventions can induce repigmentation in some patients, significant challenges persist, particularly in sustained efficacy and relapse prevention. A retrospective study showed that the overall recurrence rate observed in cured vitiligo patients was 34.2%, with both age of onset and distribution of lesions being risk factors for vitiligo recurrence⁶. Additionally, psychological stress is also a precursor factor in the onset and progression of vitiligo⁷. Current research primarily focuses on autoimmune or oxidative stress mechanisms⁸, yet the exploration of driving factors behind vitiligo recurrence remains insufficient.

Human neuropeptide Y (hNPY), a highly potent 36-amino-acid neurotransmitter and bioactive peptide, exhibits extensive distribution throughout the central nervous system (CNS), peripheral nervous system (PNS), and their associated tissues, organs, and glands⁹. NPY plays pivotal roles in diverse physiological and pathological processes, including the regulation of energy metabolism, modulation of neuronal excitability and synaptic transmission, control of stress responses, as well as participation in inflammation and immune regulation¹⁰. In addition, accumulating evidence implicates NPY as a critical neuroimmune modulator in autoimmunity. In patients with rheumatoid arthritis (RA), serum NPY levels revealed significant correlations with both TNF-α levels and disease activity¹¹. Studies demonstrated that NPY levels in lesional skin tissue fluid were significantly elevated compared to unaffected skin in vitiligo patients, suggesting that NPY may play a pivotal role in vitiligo pathogenesis through neuroimmune mechanisms acting on melanocyte’s destruction^12,13. The NF-κB signaling pathway plays extensive regulatory roles in several physiological and pathological processes, including immune responses, inflammation, cell proliferation, and apoptosis. A bioinformatics study investigating vitiligo-related biomarkers revealed that certain biomarkers may participate in vitiligo pathogenesis by modulating the NF-κB pathway to mitigate melanocyte damage¹⁴. Moreover, in mouse model of dextran sulfate sodium colitis treated with NPY antisense oligonucleotides, researchers observed decreased TNF-α levels and reduced NF-κB phosphorylation¹⁵. Therefore, we hypothesized that in vitiligo patients, NPY may activate NF-κB signaling, thereby modulating immune cell function and promoting the release of inflammatory cytokines, ultimately contributing to disease pathogenesis.

The pathogenesis and recurrence of vitiligo are linked to dysregulated inflammatory and immune mechanisms, with a central role attributed to T-cell-mediated immunity¹⁶. Studies have demonstrated that in vitiligo, IFN-γ upregulates the expression of C-X-C motif chemokine ligand-9 (CXCL9) and CXCL10 via the JAK/STAT signaling pathway, thereby promoting the recruitment of CD8 + T cells into the skin^17,18. These infiltrating CD8 + T cells recognize and attack melanocytes, ultimately leading to their destruction. The interleukin-15 (IL-15) signaling pathway is critically involved in regulating the development, homeostasis, and effector functions of tissue-resident memory T (TRM) cells¹⁹. Study has demonstrated that IL-15 mRNA is expressed across a range of cells and tissues. In vitiligo lesions, keratinocytes represent the primary cellular source of IL-15, where it is typically presented as a membrane-bound complex in association with CD215 (α chain)²⁰. On T cells, the IL-15 receptor is composed of a heterodimer containing CD122 (β chain) and CD132 (γ chain). The IL-15/IL-15Rα complex engages IL-15 receptors on CD8 + CD122+ T cells, driving their proliferation and activation²¹. This activation promotes the release of the effector molecule IFN-γ, which in turn stimulates keratinocytes to secrete the chemokines CXCL9 and CXCL10²¹. Ultimately, this cascade contributes to melanocyte apoptosis and drives the pathogenesis of vitiligo. These findings suggest that targeting IL-15 signaling may be an effective approach for treating vitiligo relapse. Therefore, we aimed to investigate whether and how the NPY/NPYR axis drives vitiligo progression by activating NF-κB to amplify the inflammatory loop mediated by CXCL9/10 and IL-15.

Materials and methods

Cell culture and vitiligo animal model construction

Human immortalized keratinocyte (HaCaT) cell line was purchased from Shanghai Fuheng Biotechnology Co., Ltd. The cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Solarbio, China) at 37 °C in an atmosphere with 5% CO₂. To investigate the effects of NPY and oxidative stress, HaCaT cells were treated with NPY at specified concentrations or H₂O₂. For H₂O₂ treatment, cells were incubated with 100 µmol/L H₂O₂ for 24 h to induce oxidative damage. NPY was applied at concentrations of 0, 1.6, 3.1, 6.3, and 12.5 µg/mL for a duration of 24 h.

The female C57BL/6 mice, 8-week-old, were purchased from Shanghai Slake Experimental Animal Co., Ltd., and raised at the SPF Laboratory Animal Center of Air Force Medical Center. The mice were fed adaptively for one week. All mice experiments were conducted according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85 − 23, revised 1996). The oxidative stress model of vitiligo mice was simulated by applying 5% H₂O₂ to the back of mice for 50 consecutive days. The experimental mice were randomly divided into 4 groups: blank group (Control), vitiligo model group (H₂O₂), vitiligo mice injected with NPY group (H₂O₂ + NPY), vitiligo mice injected with NPY+NPY2R inhibitor (H₂O₂ + NPY+NPY2R inhibitor), 6 mice in each group. Following the injection, mice were euthanized by cervical dislocation 24 h later. The dorsal skin was then collected, fixed, and embedded for further analysis. NPY was dissolved in normal saline and injected into the tail vein. To evaluate the long-term phenotype, an independent animal cohort was established in this study. After the vitiligo mouse model was stably established (designated as Day 50, D50), the experimental group received a single injection of the NPY2R inhibitor. During the subsequent 20-day observation period (until Day 70, D70), we monitored and recorded the hair phenotype changes in the mice. Subsequently, euthanasia was performed on the mice via cervical dislocation. The dosage of NPY was 10 mg/kg. BIIE 0246 (NPY2R inhibitor) reconstituted in a 12.5% DMSO solution to a final injectable dose of 10 mg/kg. All animal procedures were approved by the Ethics Committee of Air Force Medical Center (2021-29-YJ01) and adhered to the ARRIVE guidelines.

Cell transfection

HaCaT cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and cultured for 24 h to reach 70% confluency. Cells were transfected with specific siRNAs (NPY2R, Santa Cruz, sc-42101, USA; NPY4R, Santa Cruz, sc-42103, USA) using Lipofectamine RNAiMAX (Invitrogen, 13778150, USA) according to the manufacturer’s instructions. Briefly, for each well, 5 µL of siRNA (20 µM) and 5 µL of transfection reagent were separately diluted in 125 µL Opti-MEM (Gibco, 31985062, USA). The two dilutions were then combined, gently mixed, and incubated for 15 min at room temperature. The complexes were added dropwise to the cells. After 6 h, the medium was replaced with fresh complete medium. Cells were harvested 48 h post-transfection for analysis.

Reverse transcription quantitative PCR (RT-qPCR)

According to the manufacturer’s instructions, total RNA was extracted from cells with TRIzol reagent (Beyotime, R0017S), and then reverse transcribed into cDNA using the cDNA synthesis kit (Beyotime, D7180M). Quantitative PCR (qPCR) was performed using SYBR Premix Ex Taq Master Mix. The PCR cycling protocol consisted of: initial denaturation at 95 °C for 30 s; followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. Subsequently, a melt curve analysis was performed by gradually increasing the temperature from 65 °C to 95 °C with fluorescence acquisition at increments of 0.5 °C. β-actin was selected as endogenous control. The relative expression of targeted mRNA was calculated by 2^−ΔΔCt method. Relevant primer sequences are shown in Supplementary Table 1.

Western blot

The treated cells and tissues were lysed by RIPA solution. 1×SDS loading buffer was added the protein samples and denatured by boiling at 95℃ for 5 min. After cooling on ice, the equivalent amounts of protein samples were separated by 8%-10% SDS-PAGE gel and then transferred to PVDF membranes. The 5% skim milk powder was used to block the membrane for 1 h. The primary antibodies (anti-IL-15, rabbit, Bioss, bsm-63286R, 1:500; anti-IL-15Rα, mouse, Abcam, ab91270, 1:500; anti-CXCL9, rabbit, Abcam, ab290643, 1:500; anti-CXCL10, rabbit, Abcam, ab306587, 1:500; anti-p65, rabbit, Cell Signaling Technology, 8242, 1:500; anti-p-p65, rabbit, Cell Signaling Technology, 3033, 1:500; anti-NPY 2R, rabbit, ImmunoWay, YT3184, 1:500; anti-NPY 4R, rabbit, Bioss, bs-11522R, 1:500; anti-β-Tubulin, rabbit, CST, 2128 S, 1:1000; anti-caspase-3, Cell Signaling Technology, #9662s, 1:1000; anti-cleaved caspase-3, Cell Signaling Technology, #9661T, 1:1000) were incubated at 4 °C overnight. Then, the secondary antibodies (Goat Anti-Mouse IgG H&L/HRP, Bioss, bs-0296G, 1:1000; Goat Anti-Rabbit IgG H&L/HRP, Bioss, bs-0295G, 1:1000) were incubated at room temperature for 1 h. β-tubulin served as an internal reference. The protein samples for β-tubulin detection were loaded on a separate gel and processed in parallel. TBST buffer was used to wash PVDF membrane for 3 times, and the strips were visualized by chemiluminescence.

Detection of reactive oxygen species (ROS), malondialdehyde (MDA), super oxide dismutase (SOD) and glutathione peroxidase (GSH-Px)

The HaCaT cells were plated in 6-well plates at a density of 5 × 10⁵ per well and treated as described above. The levels of intracellular ROS were determined using a ROS assay kit (Beyotime, S0034S) following the manufacturer’s instruction. The cells were incubated in serum-free DMEM medium including 0.1% DCFH-DA at 37 °C for 20 min and then washed with serum-free DMEM three times. A fluorescence microscope (ZEISS) was used to photograph. The ROS fluorescence was quantitatively analyzed by ImageJ v1.8.0 software.

For cell samples, HaCaT cells were treated as described, harvested, and lysed. For tissue samples, approximately 0.1 g of mouse skin tissue was homogenized on ice in 1 mL of cold PBS, and the supernatant was collected after centrifugation (8,000 × g, 10 min, 4 °C). All sample supernatants were kept on ice for immediate analysis. According to the manufacturers’ protocols, the levels of MDA, SOD, and GSH-Px were detected using specific commercial assay kits (MDA colorimetric assay kit, Thermo, EEA015; SOD activity kit, Thermo, EIASODC; GSH-Px assay kit, Thermo, EEA010). Use an enzyme-labeled instrument to detect absorbance and calculate sample concentration based on the labeled curve.

Enzyme-linked immunosorbent assay (ELISA)

The human CXCL9, CXCL10 and IL-15 Quantitative ELISA Kit (Bioss, bsk11033, bsk11036, bsk11029) was used to analyze CXCL9, CXCL10 and IL-15 concentration in cell culture supernatant according to the manufacturer’s instructions.

Crystal violet staining

The culture plate was removed from the CO₂ incubator. The supernatant was then discarded, and the cells were washed three times with pre-cooled PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 15–20 min. After discarding the fixative, the cells were washed three times with PBS. Then, 0.5 mL of 0.1% crystal violet solution was added to each well and incubated at room temperature for 5 min. Following the removal of the staining solution, the cells were washed three times with PBS. Finally, the stained cells were observed and photographed under a microscope at 100× magnification.

Immunofluorescence assay

After collecting the cells, fix them with formaldehyde, permeabilize them with a crosslinker, and wash them with PBS three times for 5 min. Block the cells with BSA, which is usually 30 min. Incubate the primary antibody (anti-IL-15, rabbit, Bioss, bsm-63286R, 1:50; anti-IL-15Rα, mouse, Abcam, ab91270, 1:50) at room temperature for 1 h or at 4 °C overnight, rinse three times with PBST, and incubate the secondary anti-body (Goat Anti-Rabbit IgG, BF488 conjugated, Bioss, bs-60295G-BF488, 1:100; Goat Anti-Mouse IgG, APC conjugated, Bioss, bs-0296G-APC, 1:100) at room temperature in the dark for 1 h, rinse three times with PBST. Seal the slides and examine with a fluorescence microscope. The average fluorescence intensity of three random fields per sample was calculated and data expressed in fold of the control group.

Immunohistochemical staining

Serial sections were cut from the paraffin-embedded blocks. Subsequent deparaffinization and rehydration in xylene and graded series of alcohol were performed. The tissue was treated with 3% Triton X-100 for 20 min. The slides were incubated overnight at 4 °C with anti-rabbit Phospho-NF-κB p65 antibody (Bioss, bs-3543R, 1:100), IL-15 antibody (Bioss, bs-1829R, 1:100), CD8 + antibody (Bioss, bsm-61078R, 1:200). A secondary antibody (Goat Anti-Rabbit IgG H&L, HRP conjugated, Bioss, bs-40295G-HRP, 1:1000) was then added to sections. Normal mouse skin was used as a control group.

Apoptosis detection by flow cytometry

An annexin V-FITC cell apoptosis detection kit (Beyotime) was used to identify apoptotic cells. Briefly, cells were harvested by trypsinization and centrifuged at 1000 g for 5 min. The pellet was resuspended in PBS and counted. Then, 10 × 10³ cells were centrifuged again at 1000 g for 5 min, and the supernatant was discarded. Cells were resuspended in 195 µL of Annexin V-FITC binding buffer, followed by the addition of 5 µL of Annexin V solution and 10 µL of propidium iodide staining solution. After gentle mixing, the cells were incubated in the dark at room temperature for 15 min and then placed on ice. Finally, apoptosis was analyzed immediately using flow cytometry.

TUNEL assay

Paraffin-embedded skin sections were processed for TUNEL staining using a commercial kit according to the manufacturer’s instructions. Following staining, sections were counterstained with DAPI. Fluorescence images were captured under a confocal microscope. The number of TUNEL-positive cells was counted in at least five random fields per sample using ImageJ software, and the apoptotic index was calculated as the percentage of TUNEL-positive nuclei relative to the total DAPI-stained nuclei.

Statistical analyses

All statistical analyses were performed using GraphPad Prism software, Version 7.0 (GraphPad Software, San Diego, CA, USA). Normality tests were performed using the Shapiro-Wilk’s normality test. Dual comparisons were analyzed with the unpaired student’s t-test. Groups of three or more were analyzed by using one-way analysis of variance (ANOVA) with Tukey post-tests. P-values less than 0.05 were considered statistically significant. Data are presented as mean ± standard deviation (SD) from at least three independent experiments, each performed in triplicate.

Results

NPY promoted the expression and secretion of CXCL9, CXCL10 and IL-15 in HaCaT cells

To investigate the role of NPY in immune regulation during vitiligo, we analyzed the expression of key cytokines related to vitiligo in HaCaT cells following NPY treatment. Following treatment of HaCaT cells with NPY at concentrations of 0, 1.6, 3.1, 6.3, and 12.5 µg/mL, the protein expression levels of CXCL9, CXCL10, IL-15, and IL-15Rα were assessed by Western blot. Compared to control group, the expression of CXCL9, CXCL10, IL-15, and IL-15Rα was up-regulated in a concentration-dependent manner with increasing NPY concentrations (Fig. 1A). ELISA results further confirmed that the secretion levels of CXCL9, CXCL10, and IL-15 levels were also elevated in response to increasing concentrations of NPY (Fig. 1B). Additionally, RT-qPCR demonstrated a significant upregulation in the mRNA levels of CXCL9, CXCL10, IL-15, and IL-15Rα compared with the control group (Fig. 1C).

Fig. 1.

NPY modulated the expression of CXCL9, CXCL10, and IL-15 in HaCaT cells under oxidative stress. (A-C) Dose‑dependent effects of NPY on protein (analyzed by Western Blot and ELISA) and mRNA (analyzed by RT-qPCR) levels. (D-F) Expression levels after 12.5 µg/mL NPY treatment for different durations on protein (analyzed by Western Blot and ELISA), and mRNA (analyzed by RT-qPCR) levels. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Original blots/gels are presented in Supplementary materials (Original Uncropped Blots). Statistical analysis was performed using the independent sample t-test. ^****P < 0.0001.

Then we investigated the sustained effect of 12.5 µg/mL NPY on cytokine expression in HaCaT cells. The expression of CXCL9, CXCL10, IL-15, and IL-15Rα protein were elevated at 24 h after NPY exposure, and remained highly expressed (Fig. 1D). Consistently, the elevated secretion levels of CXCL9, CXCL10, and IL-15 levels persisted up to 72 h after NPY treatment (Fig. 1E). Similar phenomenon was obtained with those cytokine mRNA levels (Fig. 1F). Based on these findings, a concentration of 12.5 µg/mL NPY with 24 h of treatment was chosen for subsequent experiments.

NPY and H₂O₂ synergistically enhance the expression and secretion of CXCL9, CXCL10 and IL-15

To investigate cytokine expression in HaCaT cells under oxidative stress conditions, we used immunofluorescence to semiquantitatively assess IL-15 levels in the control, NPY-treated, H₂O₂-treated, and H₂O₂+NPY co-treated groups. Compared to the control group, fluorescence intensities of IL-15 were significantly higher in NPY-treated and H₂O₂-treated HaCaT cells. In addition, NPY in combination with H₂O₂ treatment had synergistic effects on IL-15 expression (Fig. 2A). Western blot and ELISA results showed that compared with control group, both NPY and H₂O₂ treatment increased the expression and secretion of IL-15, IL15Rα, CXCL9, and CXCL10. Moreover, NPY further enhanced the cytokine expression and secretion induced by H₂O₂, indicating a synergistic effect (Fig. 2B-C). Similar phenomenon was observed at the mRNA level for these cytokines (Fig. 2D).

Fig. 2.

Effects of NPY and H₂O₂ co-treatment on the expression of CXCL9, CXCL10 and IL-15 in HaCaT cells. (A) The expression levels of IL-15 and IL-15Rα in various groups were assessed by immunofluorescence. (B-D) The expression levels of CXCL9, CXCL10, IL-15 and IL-15Rα were measured using Western Blot, ELISA and RT-qPCR. H₂O₂ group of HaCaT cells was treated with 100 µmol/mL for 24 h. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Original blots/gels are presented in Supplementary materials (Original Uncropped Blots). Statistical analysis was performed using the independent sample t-test. ^**P < 0.01 ^*** P < 0.001 ^**** P < 0.0001.

NPY partially mitigates H₂O₂-induced apoptosis in keratinocytes

In vitiligo, keratinocytes secreting pro-inflammatory cytokines contribute to CD8 + T cell infiltration and melanocyte death. To further investigate the functional effect of NPY on oxidative stress in keratinocytes, we treated HaCaT cells with NPY, H₂O₂, and H₂O₂+NPY, followed by crystal violet staining and microscopic observation of cell viability. The result showed that, compared with control, NPY treatment alone had no significant impact on cell morphology or necrosis, while H₂O₂ treatment alone increased the proportion of dead cells. Moreover, NPY could largely abolish H₂O₂-induced cell necrosis (Fig. 3A). Flow cytometry analysis also revealed that NPY had ability to mitigates H₂O₂-promoted cell apoptosis (Fig. 3B).

Fig. 3.

Effect of NPY on H₂O₂-induced keratinocyte apoptosis. (A) After crystal violet staining, the survival of HaCaT cells was observed under an inverted microscope (100×) (B) HaCaT cells were stained with Annexin V-FITC and propidium iodide (PI). Apoptotic cells were quantified by flow cytometry. The scatter-plot shows Annexin V-FITC staining on the x-axis and PI staining on the y-axis. The lower right quadrant (Annexin V+/PI-) represents cells in early apoptosis. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Statistical analysis was performed using the independent sample t-test. ^*P < 0.05 ^****P < 0.0001.

NPY eliminates ROS levels in keratinocytes

H₂O₂-increased intracellular ROS could activate cell apoptosis. To determine whether NPY influences this process, we measured intracellular ROS levels. Compared with control group, H₂O₂ treatment significantly increased ROS levels. Notably, NPY largely abolished the H₂O₂-induced ROS accumulation (Fig. 4A). We further evaluated the effect of NPY on oxidative stress induced by H₂O₂ in HaCaT cells by assessing the activity of SOD and GSH-Px, and the level of MDA. H₂O₂ treatment alone increased MDA content, but decreased both SOD and GSH-Px activity. However, NPY largely reversed these H₂O₂-induced effects in HaCaT cells (Fig. 4B-D). Taken together, these results suggested that NPY reduced ROS accumulation and suppressed apoptosis in oxidatively stressed keratinocytes.

Fig. 4.

Effect of NPY on ROS levels in keratinocytes. (A) ROS levels were measured by the fluorescent probe in various groups (B-D) Cellular content of MDA and enzymatic activities of SOD and GSH-Px in various groups of HaCaT cells. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Statistical analysis was performed using the independent sample t-test. ^**P < 0.01 ^****P < 0.0001.

Activation of the NF-κB pathway via NPY receptor NPY2R promotes the expression of CXCL9, CXCL10 and IL-15

As NF-κB signaling is one of most crucial pathways involved in immune response, we investigated p65 phosphorylation, a subunit of the NF-κB transcription factor complex, in HaCaT cells with NPY treatment. Compared to control group, p65 phosphorylation were upregulated in a concentration-dependent manner with increasing NPY concentrations (Fig. 5A). We also investigated the sustained effect of NPY on p65 phosphorylation in HaCaT cells. Following NPY exposure, p65 phosphorylation was elevated at 24 h, and remained highly expressed (Fig. 5B). Moreover, NPY in combination with H₂O₂ treatment exhibited synergistic effects on p65 phosphorylation. H₂O₂ treatment also promoted NPY receptor NPY2R and NPY4R expression (Fig. 5C-D). BAY11-7082, an irreversible inhibitor of IκB-α phosphorylation and NF-κB signaling, could abolish NPY-promoted p65 phosphorylation and the expression of CXCL9, CXCL10, IL-15, and IL-15Rα in HaCaT cells (Figs. 5E-G). To identify the specific receptor mediating the effects of NPY, we transfected HaCaT cells with siRNAs targeting NPY2R and NPY4R, respectively. In NPY2R-knockdown cells, NPY-induced phosphorylation of p65 was almost completely inhibited. In contrast, knockdown of NPY4R only slightly attenuated the effect of NPY (Fig. 5H). Furthermore, specific knockdown of NPY2R effectively blocked the NPY-induced upregulation of CXCL9, CXCL10, and IL-15, whereas knockdown of NPY4R had limited effects on the expression of these factors. These findings demonstrated that NPY activated the NF-κB pathway and drove the expression of inflammatory cytokines in HaCaT cells, a process largely dependent on the NPY2R.

Fig. 5.

NPY upregulates inflammatory factors expression via the NF-κB pathway in HaCaT cells. (A-B) Relative protein expression level of p-p65 in HaCaT cells were measured under different NPY concentrations and 12.5 µg/mL NPY treatment for different durations. (C-D) Relative protein and mRNA expression levels of NPY2R, NPY4R and p-p65 in HaCaT cells from different treatment groups. (E-F) Relative protein and mRNA expression levels of inflammatory factors in each group of HaCaT cells following NPY inhibitor treatment. (G) The protein expression levels of p-p65 in each group of HaCaT cells following NPY inhibitor treatment. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Original blots/gels are presented in Supplementary materials (Original Uncropped Blots). Statistical analysis was performed using the independent sample t-test. ^****P < 0.0001.

NPY inhibitor ameliorates skin depigmentation and reduces CD8 + T lymphocyte infiltration in a vitiligo mouse model

We conducted pathological, depigmentation, and immunohistochemical analyses on a stress-induced vitiligo mouse model treated with NPY or NPY2R inhibitor. Phenotypic results revealed that the H₂O₂-treated group exhibited significant pigment loss compared to the control group. Co-treatment with H₂O₂ and NPY resulted in more severe pigment loss. However, when an NPY2R inhibitor was added concurrently, cellular pigmentation was significantly restored (Fig. 6A). Moreover, compared to normal skin, lesional skin from vitiligo mice exhibited significantly elevated expression of IL-15 and phosphorylation of p65. NPY treatment further increased IL-15 expression and p65 phosphorylation, while administration of the NPY2R inhibitor abolished these effects (Fig. 6B-C). Additionally, CD8 + T lymphocyte infiltration was markedly increased in vitiligo lesions relative to normal skin. NPY treatment increased CD8⁺ T lymphocyte infiltration and NPY2R inhibitor treatment abolished this effect (Fig. 6B and D). These findings indicated that NPY2R inhibitor can reduce CD8 + T lymphocyte infiltration in a stress-induced vitiligo mouse model.

Fig. 6.

Effect of NPY treatment on vitiligo progression in mice, including p-p65 and IL-15 expressions, and CD8 + cells infiltration. (A) Macroscopic phenotype of vitiligo in mice. (B-D) The expression levels of p-p65, IL-15 and CD8 + in various groups were assessed by immunohistochemical staining. Scale bars: 50 μm. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Statistical analysis was performed using the independent sample t-test. ^**P < 0.01 ^***P < 0.001 ^****P < 0.0001.

The anti-apoptotic and antioxidant effects of NPY in the in a vitiligo mouse model

To further investigate the effect of NPY in vitiligo progression, we systematically evaluated the influence of NPY on skin oxidative stress and cell apoptosis in a stress-induced vitiligo mouse model. As shown in Fig. 7A, a large number of TUNEL-positive apoptotic cells could be observed in the lesion area of the model group, and NPY treatment significantly reduced the number of TUNEL-positive cells. This finding was also confirmed by Western blot analysis. The expression of cleaved caspase-3 in the skin of the model group was significantly upregulated, while NPY treatment effectively inhibited its expression (Fig. 7B). Notably, BIIE-0246 inhibited the anti-apoptotic effect of NPY. In addition, we also detected the oxidative stress levels in the skin tissue. The results showed that compared with the control group, the malondialdehyde level in the lesion area of the model group mice was significantly increased, and the activities of SOD and GSH-Px were significantly decreased. NPY treatment partially reversed this inhibition and increased the activities of SOD and GSH-Px (Fig. 7C-E).

Fig. 7.

The anti-apoptotic and antioxidant effects of NPY in the in a vitiligo mouse model. (A-B) The anti-apoptotic effect of NPY was analyzed through TUNEL assay and western blot of cleaved caspase-3. Scale bars: 100 μm. (C-E) Content of MDA and enzymatic activities of SOD and GSH-Px in vitiligo mouse model. Data are presented as mean ± SD from at least three independent experiments, each performed in triplicate. Original blots/gels are presented in Supplementary materials (Original Uncropped Blots). Statistical analysis was performed using the independent sample t-test. ^*P < 0.05 ^**P < 0.01 ^***P < 0.001.

Discussion

This study was the first to reveal that NPY mediated its effect through its receptor NPY2R, activating the NF-κB signaling pathway in HaCaT cells, thereby promoting the expression and secretion of key inflammatory cytokines (CXCL9, CXCL10, IL-15). This mechanism may be an important driving factor for the progression of vitiligo. Additionally, NPY exhibited a synergistic effect with oxidative stress and mitigated apoptosis and ROS levels in keratinocytes. In vivo experiments also confirmed that inhibiting NPY signaling effectively alleviates vitiligo (Fig. 8). This study established a crucial link between the nervous system, keratinocytes, and adaptive immunity, providing novel perspectives and experimental evidence for understanding the molecular mechanisms underlying vitiligo progression.

Fig. 8.

The schematic diagram for mechanism of NPY-induced vitiligo progression via the NPY2R/NF-kB pathway.

Our findings demonstrated that the treatment with NPY alone was sufficient to significantly increase the mRNA and protein levels of CXCL9, CXCL10 and IL-15 in HaCaT cells. CXCL9 and CXCL10, as ligands of CXCR3, guide CD8 + T cells to migrate to inflammatory sites²², while IL-15 provides survival and proliferation signals for these cells, maintaining their effector functions²³. Together, these cytokines form the core cytokine network responsible for recruiting and activating cytotoxic CD8 + T cells. This indicated that under the stimulation of NPY, keratinocytes actively participated in the immune response process of destroying their own melanocytes. This study also discovered that NPY and H₂O₂ have a synergistic effect in inducing the expression of inflammatory factors. In the skin of vitiligo patients, the widespread pathological oxidative stress (represented by H₂O₂) forms a persistent microenvironmental background²⁴, while NPY acts as a key molecular mediator of acute relapse triggers. Their synergistic effect indicates that in skin with a pre-existing vitiligo pathological basis, a single stress event is sufficient to trigger an intense inflammatory response far exceeding expectations, leading to rapid disease recurrence and sharply accelerated lesion spread. As a study had shown, oxidative stress could contribute to the pathogenesis of vitiligo by inducing the expression of IL-15 and its trans-presentation in keratinocytes, thereby activating CD8 + T cells²⁵. This further supported our study findings.

Moreover, we demonstrated that NPY activated the NF-κB signaling pathway through NPY2R. Using the NF-κB inhibitor, BAY11-7082, we further validated the specific signaling pathway through which NPY promotes cytokines related to vitiligo progression expression. Our results showed that inhibiting NF-κB with BAY11-7082 reduced the expression levels of IL-15, IL-15Rα, CXCL9, and CXCL10. NF-κB signaling serves as a critical link between external stressors and immune responses, playing a key role in cell activation and a variety of autoimmune diseases²⁶. Study has shown that oxidative stress activated NF-κB via Toll-like receptor 2 (TLR2) and TLR4, thereby enhancing cytokine expression²⁷. Additionally, oxidative stress can induce melanocytes to produce and secrete heat shock protein 70 (HSP70), which is associated with the activation of dendritic cells (DCs) and the induction of proinflammatory cytokines via TLR2, TLR4, or TLR2-TLR4^28,29. Therefore, oxidative stress may regulate IL-15 trans-presentation by inducing HSP70 production and secretion, which then binds to TLR2 and TLR4 in keratinocytes and activates the NF-κB signaling pathway to promote the transcription and expression of IL-15 and IL-15Rα in keratinocytes. Chen et al. further demonstrated that NF-κB signaling mediates CXCL9/CXCL10 chemokine upregulation and secretion induced by oxidative stress, ultimately leading to CD8 + T cell recruitment and melanocyte damage²⁵. Additionally, NF-κB signaling enhanced CD8 + TEM cell activation and cytotoxic protein expression by promoting IL-15 expression and trans-presentation. During trans-presentation, IL-15 and IL-15Rα appeared as a stable complex on the keratinocyte cell membrane, presenting IL-15 to T cells expressing the IL-15Rβ/γ heterodimer²⁵. Although both NPY2R and NPY4R were upregulated under oxidative stress, our loss-of-function experiments using specific siRNA revealed that knockdown of NPY2R potently suppressed NPY-induced NF-κB activation and the subsequent expression of CXCL9, CXCL10, and IL-15. This genetic evidence established a clear hierarchy, positioning NPY2R as the primary signaling driver for inflammation. Consequently, we rationally selected the NPY2R-specific antagonist BIIE0246 for in vivo validation. Our animal experiments confirmed that NPY2R can promote IL-15 release and recruit CD8 + T cells via the NF-κB pathway. These results indicate that NPY regulates cytokines related to vitiligo progression expression in keratinocytes under oxidative stress by activating the NPY2R/NF-κB signaling pathway.

A seemingly paradoxical yet crucial finding was that NPY, while promoting inflammation, also reduced H₂O₂-induced keratinocyte apoptosis and decreases intracellular ROS levels. In vitiligo lesions, the extensive apoptosis of keratinocytes leads to the disruption of the skin barrier³⁰. However, excessive apoptosis may also reduce the sustained secretion of pro-inflammatory factors, thereby limiting the amplification of the immune response. Therefore, the partial anti-apoptotic effect of NPY enables keratinocytes to maintain a certain survival rate under oxidative stress damage, allowing them to continuously secrete CXCL9, CXCL10 and IL-15. These cytokines ensure that CD8 + T lymphocytes are effectively recruited to the lesion sites and exert sustained immune effects³¹. This also explains the phenomenon that inflammation persists during the recurrence of vitiligo, while the skin lesions progress slowly. ROS not only act as direct mediators of oxidative damage but also serve as signaling molecules that activate inflammatory pathways such as NF-κB and AP-1, promoting the expression of pro-inflammatory factors³². However, the antioxidant effect of NPY did not suppress inflammation, suggesting that NPY may activate NF-κB via NPY2R, thereby bypassing the ROS-dependent checkpoint. This is supported by our data showing that specific NPY2R knockdown abolish NF-κB activation without reversing the reduction in ROS. This direct receptor-driven pathway may involve canonical GPCR effectors such as Gβγ subunits activating PI3K/Akt or directly engaging IKK complexes, thereby overriding the loss of the ROS signal and sustaining inflammation even in a lowered oxidative environment^33,34. This dual role aligns with the concept of the stress-skin axis, where neuropeptides like NPY act as key integrators, translating psychological or local stress into sustained skin inflammation independent of initial oxidative triggers.

This study had several limitations. First, our in vitro findings were primarily based on the HaCaT cells which might not fully replicate the behavior of primary keratinocytes within the complex microenvironment of vitiligo skin. Future studies using primary human keratinocytes from both healthy and vitiligo patients will be essential to validate and extend these results. Second, as our study aimed primarily to investigate the upstream pathological processes of vitiligo, namely oxidative stress, we employed a hydrogen peroxide-induced vitiligo mouse model. Future research will incorporate classical immune-mediated models to verify the role of NPY and its receptors in the complete oxidative stress-immune attack pathological cycle. Finally, the conclusions of this study were derived from preclinical models, and the pathological role of the NPY pathway in vitiligo patients, as well as the clinical feasibility and safety of its intervention strategies, required future investigation for validation.

In summary, our study revealed the central role of the NPY/NPY2R signaling axis in driving vitiligo progression. It activated the NF-κB pathway in keratinocytes, inducing a key inflammatory environment that recruited and activated CD8 + T cells, and synergized with oxidative stress to amplify this effect. Additionally, NPY ensured the prolonged survival of these activated keratinocytes within the inflammatory environment by suppressing oxidative damage and apoptosis. This dual pro-inflammatory and pro-survival action provided a novel mechanistic explanation for the persistent inflammation observed in vitiligo lesions.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Q.L. conceptualized the study; Y.Z. and W.H. developed the methodology; B.L. performed the software; B.T., J.G., J.S. and B.Z. conducted the validation; B.T. carried out the formal analysis and investigation; Q.L. provided the resources; H.L. and A.J. handled the data curation; Y.W. wrote the original draft; B.T. reviewed and edited the manuscript; J.C. supervised the project; Q.L. administered the project and acquired the funding. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Beijing Natural Science Foundation (grant number 7222186) and National Natural Science Foundation of China (grant number 5227030162).

Data availability

Data supporting the results of this article will be provided on request, by the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics statement

The animal study protocol was approved by the Ethics Committee of Air Force Medical Center (2021-29-YJ01) and adhered to the ARRIVE guidelines.