Exosomes derived from AHR-overexpressing human umbilical cord mesenchymal stem cells attenuate H/R injury via AHR/NLRP3 pathway

ABSTRACT

Myocardial hypoxia-reoxygenation (H/R) injury is a frequently observed pathological event in various cardiovascular conditions. Despite the therapeutic promise of human umbilical cord mesenchymal stem cells (hUC-MSCs) in alleviating myocardial damage, their clinical use faces obstacles such as limited implantation efficiency, poor retention, and reduced post-transplantation viability. Exosomes secreted by hUC-MSCs have emerged as a viable alternative, potentially addressing these challenges. Nonetheless, the underlying mechanisms through which these exosomes confer cardioprotection have yet to be fully elucidated. This study aims to explore the protective effect of hUC-MSCs exosomes on myocardial H/R injury via the aryl hydrocarbon receptor (AHR)/NOD-like receptor family pyrin domain containing 3 (NLRP3) pathway and to assess their impact on immune cell phenotype conversion. hUC-MSCs exosomes significantly upregulated AHR expression, inhibited NLRP3-related inflammatory protein expression, enhanced myocardial cell survival, and reduced apoptosis. The protective effect of hUC-MSCs exosomes was abolished following AHR knockdown. Additionally, exosomes from AHR-overexpressing hUC-MSCs promoted the conversion of macrophages, dendritic cells (DCs), and T cells to an anti-inflammatory phenotype, thereby further enhancing myocardial protection. These findings indicted that exosomes from AHR-overexpressing hUC-MSCs protect myocardium via AHR/NLRP3 signaling, improving immune microenvironment and offering new therapeutic potential.

1. Introduction

Cardiovascular diseases are the leading cause of death worldwide, with ischemic heart disease remaining a major contributor [1,2]. Myocardial hypoxia – reoxygenation (H/R) injury, which arises during ischemic events and subsequent reperfusion therapy, triggers excessive oxidative stress, inflammation, and cardiomyocyte apoptosis [3,4]. These pathological processes aggravate cardiac dysfunction and hinder recovery, underscoring the urgent need for novel approaches to protect the myocardium from H/R-induced injury.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) possess potent self-renewal, multilineage differentiation, and immunomodulatory abilities, positioning them as attractive tools for regenerative medicine [5]. By secreting anti-inflammatory cytokines and trophic factors, hUC-MSCs promote angiogenesis, reduce fibrosis, and enhance tissue repair. These paracrine effects rather than direct differentiation are now recognized as the primary mechanisms underlying their therapeutic potential [6,7].

Despite encouraging preclinical results [8–10], the clinical translation of hUC-MSC therapy has been disappointing. Although animal and cell models show significant cardioprotective effects, recent clinical trials revealed minimal benefit in ischemic heart disease, largely due to the poor engraftment and survival of transplanted cells within cardiac tissue [11,12]. These limitations highlight the need to develop alternative, cell-free therapeutic approaches to achieve consistent and durable myocardial repair.

Exosomes, the principal mediators of the paracrine effects of hUC-MSCs, have emerged as a promising cell-free therapeutic approach [13]. These nanosized vesicles carry bioactive proteins, lipids, and RNAs that regulate target cell functions and promote tissue repair [14]. These exosomes can transfer signals between cells, regulating the function of target cells and playing a crucial role in injury repair [15–21]. hUC-MSC – derived exosomes have shown cardioprotective effects comparable to their parent cells, reducing inflammation and cardiomyocyte death by modulating macrophage polarization in models of myocardial ischemia – reperfusion injury [22,23]. Thus, exosome-based therapy may overcome the limitations of direct hUC-MSC transplantation and provide a more effective strategy for treating ischemic cardiac diseases [24].

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that senses endogenous and environmental signals to regulate immune and inflammatory responses [25]. AHR activation suppresses pro-inflammatory signaling and promotes anti-inflammatory immune cell polarization [26,27]. In cardiovascular contexts, AHR has been shown to protect against ischemic injury by reducing cardiomyocyte apoptosis and modulating immune responses, suggesting its potential role in stem cell – based therapies [28].

The NLRP3 inflammasome is a central regulator of inflammation and pyroptosis during myocardial hypoxia – reoxygenation (H/R) injury [29,30]. Excessive activation of NLRP3 promotes IL-1β and IL-18 release, exacerbating myocardial damage [31–33]. Emerging evidence suggests a reciprocal regulatory relationship between AHR and NLRP3, whereby AHR activation restrains inflammasome assembly and dampens inflammatory responses [34–36]. We therefore hypothesized that exosomes derived from AHR-overexpressing hUC-MSCs (AHR-exo) alleviate H/R-induced myocardial injury by modulating the AHR/NLRP3 signaling axis and promoting anti-inflammatory immune polarization.

To test this hypothesis, we employed an in vitro myocardial hypoxia – reoxygenation model to examine whether exosomes derived from AHR-overexpressing hUC-MSCs (AHR-exo) mitigate cardiomyocyte injury. We further explored how AHR-exo modulates AHR/NLRP3 signaling and inflammatory cytokine expression in cardiac and immune cells. These findings aim to clarify the molecular mechanisms underlying AHR-mediated cardioprotection and to inform the development of novel cell-free therapies for ischemic heart disease.

2. Materials and methods

2.1. Reagents and chemicals

Fetal bovine serum (FSD500) was provided by Excell Bio (Shanghai, China). Penicillin-streptomycin solution (100X, Cat. No. C0222), Annexin V-FITC apoptosis detection kit (Cat. No. C1062L), LDH Cytotoxicity Assay Kit (Cat. No. C0017), IL-1β ELISA Kit (Cat. No. PI305), IL-10 ELISA Kit (Cat. No. PI528), IL-18 ELISA Kit (Cat. No. PI558), TNF-α ELISA Kit (Cat. No. PT518), and Exosome Isolation Reagent (Cat. No. C3620) were purchased from Beyotime Biotechnology (Shanghai, China). DMEM high-glucose medium (Cat. No. 11,965,118), RPMI 1640 medium (Cat. No. 11,875,093), trypsin (Cat. No. R001100), CTS OpTmizer T-cell expansion SFM medium (Cat. No. A1048501), LipofectamineTM 3000 (Cat. No. L3000015), Opti-MEM™ I reduced-serum medium (Cat. No. 31,985,070), CD86 Monoclonal Antibody (IT2.2, Cat. No. 16–0869-85), CD80 Monoclonal Antibody (MEM-233, Cat. No. MA1-19590), CD163 Monoclonal Antibody (eBioGHI/61, Cat. No. 12–1639-42), CD206 Monoclonal Antibody (19.2, Cat. No. 53–2069-42), CD40 Monoclonal Antibody (5C3, Cat. No. 17–0409-42), CD103 Monoclonal Antibody (B-Ly7, Cat. No. 12–1038-42), HLA-DR/DP Monoclonal Antibody (MEM-136, Cat. No. MA1-19227), PD-L1 Polyclonal Antibody (Cat. No. PA5-18337), FOXP3 Monoclonal Antibody (236A/E7, Cat. No. 53–4777-42), CD3 Monoclonal Antibody (OKT3, Cat. No. 16–0037-81), and CD8a Monoclonal Antibody (RPA-T8, Cat. No. 12–0088-80) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Human umbilical cord mesenchymal stem cell serum-free complete medium (Cat. No. IMP-H122-1), AC16 human cardiomyocyte-specific medium (Cat. No. IM-H478-1), THP-1-specific medium (Cat. No. IM-H260-1), and human dendritic cell-specific medium (Cat. No. IMP-H018-1) were supplied by IMMOCELL (Xiamen, China). Cell/Tissue Total RNA Isolation Kit V2 (Cat. No. RC112), HiScript III 1st Strand cDNA Synthesis Kit (Cat. No. R312), and Taq Pro Universal SYBR qPCR Master Mix (Cat. No. Q712) were obtained from Vazyme Biotechnology (Nanjing, China). Prestained Protein Marker II (Cat. No. G2058-250 UL) was purchased from Servicebio (Wuhan, China). NLRP3 Rabbit mAb (D4D8T, Cat. No. 15,101), Cleaved Caspase-1 Rabbit mAb (D57A2, Cat. No. 4199), Bax Antibody (Cat. No. 2772), AHR Rabbit mAb (D5S6H, Cat. No. 83,200), CYP1A2 Mouse mAb (D2V7S, Cat. No. 14,719), CD81 Rabbit mAb (D3N2D, Cat. No. 56,039), Calnexin Antibody (Cat. No. 2433), and GAPDH Rabbit mAb (14C10, Cat. No. 2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). NF-κB p65 Recombinant Antibody (Cat. No. 80,979–1-RR) and CYP1A1 Polyclonal Antibody (Cat. No. 13,241–1-AP) were obtained from Proteintech (Wuhan, China). Goat Anti-Rabbit IgG H&L/HRP (Cat. No. bs-0295 G-HRP), Goat Anti-Mouse IgG H&L/HRP (Cat. No. bs-0296 G-HRP), and CCK-8 Cell Proliferation Assay Kit (Cat. No. BA00208) were supplied by Bioss (Beijing, China). Human TGF-β ELISA Kit (Cat. No. CB11786-Hu) was purchased from COIBO BIO (Shanghai, China).

2.2. Cell preparation

hUC-MSCs (IMP-H122), AC16 human myocardial cells (IM-H478), human peripheral blood dendritic cells (DCs, IMP-H018), and THP-1 monocytes were obtained from IMMOCELL (Xiamen, China). T lymphocytes (Delf-24623) were purchased from Hefei Wanwu Biotechnology Co., Ltd. (Hefei, China).

hUC-MSCs were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin – streptomycin (P/S; Vazyme, China). AC16 cardiomyocytes were maintained in DMEM (Gibco, USA) containing 10% FBS and 1% P/S. THP-1 monocytes were cultured in RPMI-1640 medium (Gibco, USA) with 10% FBS and 1% P/S. Human peripheral blood DCs and T lymphocytes were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% P/S, and 20 ng/mL recombinant human IL-2 (PeproTech, USA). All cells were maintained at 37°C in a humidified incubator with 5% CO₂.

2.3. Cell models

2.3.1. Establishment of the myocardial H/R model in AC16 cells

AC16 cells were subjected to hypoxia (1% O₂, 5% CO₂, 37 °C) for 6 h, followed by reoxygenation for 24 h to simulate ischemia/reperfusion (H/R) injury. After treatment, cell viability was assessed using the CCK-8 assay [37].

2.3.2. Induction of M1 macrophages

THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h to induce differentiation into M0 macrophages. These cells were subsequently stimulated with 100 μg/mL lipopolysaccharide (LPS) for 24 h to generate the M1 phenotype [38].

2.3.3. DC cell and T lymphocyte co-culture model construction

Dendritic cells and T lymphocytes were co-cultured at a 1:1 ratio in RPMI-1640 medium. Exosomes derived from AHR-overexpressing hUC-MSCs (AHR-exo) or control hUC-MSCs (NC-exo) were added to the co-culture at 50 μg/mL. To simulate immune activation, LPS (5 μg/mL) was added and cells were incubated for 48 h before analysis [39].

2.4. Cell transfection and overexpression

2.4.1. siRNA plasmid construction and transfection

The coding sequence of the human AHR gene (NCBI accession number: NM_001621.5) was obtained to design three small interfering RNA (siRNA) sequences targeting AHR (si-AHR-1, si-AHR-2, and si-AHR-3). A non-targeting siRNA (si-NC), confirmed to have no significant homology with human genes, served as a negative control. The sequences of all siRNAs are provided in Supplementary Table S1 and S2.

For siRNA transfection, 2 μL of 20 μM siRNA duplex was added to 200 μL of serum-free medium and gently mixed. Then, 10 μL of RNAFit transfection reagent (HB-RF-1000, Hanbio Biotechnology, China) was added, followed by vortexing for 10 seconds to form the siRNA – RNAFit complex. After a 10-minute incubation at room temperature (not exceeding 30 minutes), the transfection complex was added to cells that had been pre-cultured in 1.8 mL of fresh complete medium (containing 10% FBS). The final volume per well was adjusted to 2 mL, with a siRNA final concentration of 20 nM. Cells were incubated at 37°C with 5% CO₂ for 24 hours prior to further analysis.

2.4.2. Plasmid construction and AHR overexpression

To overexpress AHR, the full-length AHR coding sequence was cloned into the pcDNA3.1 vector (constructed and validated by Sangon Biotech, Shanghai, China) to generate the pcDNA3.1-AHR plasmid. The vector map is shown in Supplementary Figure S1, and the AHR gene sequence used is detailed in Supplementary Table S3.

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were seeded into 6-well plates and cultured until approximately 80% confluency. Transfection complexes were prepared as follows: (1) 3.75 μL of Lipofectamine 3000 reagent was diluted in 125 μL of Opti-MEM medium. (2) 5 μg of pcDNA3.1-AHR plasmid and 10 μL of P3000 reagent were diluted in another 125 μL of Opti-MEM medium. The two mixtures were combined, gently mixed, and incubated at room temperature for 15 minutes to form the transfection complex. This mixture was then added to the hUC-MSCs in each well. After 48 hours of incubation, transfection efficiency was evaluated, and cells with stable AHR overexpression were selected for downstream experiments.

2.5. Exosome isolation and characterization

2.5.1. Exosome isolation

hUC-MSCs with AHR overexpression (hUC-MSCs-AHR) and negative control (hUC-MSCs-Control) were cultured in medium supplemented with exosome-depleted fetal bovine serum. After 48 hours, the conditioned medium was collected and sequentially centrifuged at 4°C to remove cells and debris: 300 × g for 20 minutes, 800 × g for 20 minutes, 2000 × g for 20 minutes, and finally 10,000 × g for 30 minutes. The resulting supernatant was then subjected to ultracentrifugation at 100,000 × g for 70 minutes. The exosome pellet was resuspended in phosphate-buffered saline (PBS) and stored at −80°C until further analysis.

2.5.2. Transmission electron microscopy (TEM)

For morphological assessment, the exosome suspension was diluted with PBS at ratios of 1:10, 1:30, and 1:50. A 20 μL aliquot of each dilution was dropped onto a piece of Parafilm. A carbon-coated copper grid (200 mesh) was placed face-down onto the droplet and allowed to adsorb exosomes for 10 minutes at room temperature in a dry environment. Excess liquid was removed with filter paper, and the grid was then washed twice by floating on drops of PBS for 2 minutes each.

Next, 30 μL of 4% uranyl acetate was dropped onto Parafilm, and the grid was floated on the droplet for negative staining for 3 minutes. After staining, the grid was washed twice in PBS, blotted with filter paper, and air-dried at room temperature. The stained grids were observed under a transmission electron microscope (TEM) at an accelerating voltage of 80–120 kV. Exosomes were visualized and imaged at a magnification of 20,000× to assess their size and morphology.

2.6. CCK-8 assay

Cell viability was assessed using the Cell Counting Kit-8 according to the manufacturer’s instructions. Briefly, logarithmically growing cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and incubated at 37°C with 5% CO₂. After 6 hours of incubation to allow cell adherence, treatments were applied according to experimental grouping, with six replicate wells per group (n = 6).

Following 24 hours of treatment, 10 μL of CCK-8 reagent was added to each well 2 hours before the end of the incubation period. After the additional 2-hour incubation, absorbance at 450 nm was measured using a microplate reader (BioTek Instruments, USA) to determine cell viability.

2.7. Lactate dehydrogenase (LDH) cytotoxicity assay

Cytotoxicity was assessed using an LDH release assay kit (Beyotime, China) following the manufacturer’s protocol. Briefly, cells were seeded into 96-well culture plates at a density appropriate to achieve 80–90% confluence at the time of treatment. After adherence, cells were treated with different experimental conditions, and appropriate controls were included. After drug exposure, plates were centrifuged at 400 × g for 5 minutes using a plate centrifuge, and the culture medium was carefully removed. Subsequently, 150 μL of diluted LDH release reagent (prepared by mixing 1 volume of LDH release reagent with 10 volumes of PBS) was added to each well. The plate was gently shaken to ensure uniform distribution and incubated at 37°C in a 5% CO₂ incubator for 1 hour. After incubation, the plate was centrifuged again at 400 × g for 5 minutes, and 120 μL of the supernatant from each well was transferred to a new 96-well plate. For detection, 60 μL of LDH detection working solution (prepared freshly according to the kit instructions) was added to each well containing the transferred supernatant. The plate was gently mixed and incubated in the dark at room temperature (~25°C) for 30 minutes. Absorbance was measured at 490 nm using a microplate reader, with 600 nm as the reference wavelength. Background absorbance (from blank wells) was subtracted from all values prior to analysis.

2.8. Reverse transcription quantitative PCR (RT-qPCR)

2.8.1. Rna extraction

Total RNA was extracted from hUC-MSCs in the logarithmic growth phase using the FastPure® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, China) according to the manufacturer’s instructions. Briefly, 1 × 10⁶ cells were seeded per well in 6-well plates, serum-starved for 12 h, and treated according to experimental grouping. After 24 h of incubation, the culture medium was removed, and 500 μL of Buffer RL was added to lyse cells in each well. Lysates were transferred to gDNA-Filter Columns and centrifuged at 12,000 × g for 30 seconds. The flow-through was mixed with 0.5 volumes of absolute ethanol and loaded onto RNA binding columns, followed by sequential washes with Buffer RW1 and Buffer RW2. Finally, RNA was eluted with 30 μL RNase-free water. RNA concentration and purity were assessed using a NanoPhotometer (NanoDrop, Thermo Fisher Scientific, USA).

2.8.2. Reverse transcription

First-strand cDNA was synthesized using the HiScript® III RT SuperMix for qPCR Kit (Vazyme, China) in a 20 μL reaction system. A total of 500 ng RNA was preincubated with 2 μL 5×gDNA wiper mix at 42°C for 2 min to remove genomic DNA contamination. Subsequently, reverse transcription was performed by adding 10× RT mix, Hiscript enzyme mix, oligo(dT)₍₂₀VN₎ primers, and random hexamers, followed by incubation at 37°C for 15 min and 85°C for 5 sec. Synthesized cDNA was stored at −20°C until use.

2.8.3. Quantitative PCR

RT-qPCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with Taq Pro Universal SYBR Green qPCR Master Mix (Vazyme, China) in a 20 μL reaction volume containing 5 μL of fivefold diluted cDNA, 10 μL of 2× Master Mix, 0.4 μL each of 10 μM forward and reverse primers, and 4.2 μL nuclease-free water. The amplification conditions were as follows: (1) Initial denaturation at 95°C for 30 seconds; (2) 40 cycles of: 95°C for 10 seconds, 60°C for 10 seconds; (3) Melting curve analysis: 95°C for 15 seconds, 65°C for 60 seconds, and 95°C for 15 seconds. Relative gene expression was calculated using the 2^–ΔΔCt method. GAPDH was used as the internal control for normalization. Primer sequences, expected amplicon sizes, annealing temperatures, GenBank accession numbers, and primer-BLAST verification results are listed in Supplementary Table S4. Specificity of the primers was confirmed using NCBI Primer-BLAST, and all primers were verified to have no significant off-target amplification.

2.9. Western blot

2.9.1. Total and exosomal protein extraction

For total protein extraction, hUC-MSCs in the logarithmic growth phase were seeded at a density of 1 × 10⁶ cells per well in 6-well plates. After cell adherence, cells were treated according to experimental grouping and cultured at 37°C with 5% CO₂ for 24 hours. After incubation, cells were washed twice with pre-chilled phosphate-buffered saline (PBS), and 500 μL of RIPA lysis buffer supplemented with 1 mM PMSF (Beyotime, China) was added per well to lyse the cells on ice. Lysates were centrifuged at 12,000 × g for 5 minutes at 4°C, and the supernatants were collected and stored at −80°C for later use.

Exosomal proteins were extracted following ultracentrifugation, as described in Exosome isolation and characterization, and lysed with RIPA buffer containing PMSF on ice for 30 minutes, followed by centrifugation. Supernatants containing exosomal proteins were stored at −80°C.

2.9.2. Protein quantification and sample preparation

Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Beyotime, China). BCA working reagent was prepared by mixing Reagent A and Reagent B at a 50:1 ratio. Protein standards were prepared by serial dilution of a 0.5 mg/mL stock solution. Standards and samples (20 μL each) were loaded into a 96-well plate, followed by 200 μL of BCA working solution. The plate was incubated at 37°C for 30 minutes, and absorbance was measured at 562 nm using a microplate reader. Protein concentrations were calculated based on the standard curve. Each sample was mixed with 5× SDS-PAGE loading buffer (final concentration 1×), boiled at 100°C for 10 minutes, and stored at −20°C.

2.9.3. SDS-PAGE and immunoblotting

Proteins (20 μg per lane) were separated on 12% SDS-PAGE gels and transferred onto methanol-activated PVDF membranes (Millipore, USA) using a wet transfer system. After transfer, membranes were washed with TBST (Tris-buffered saline with 0.1% Tween-20) and blocked with rapid blocking buffer (Beyotime, China) for 30 minutes at room temperature on a shaker. Membranes were incubated overnight at 4°C with primary antibodies diluted in TBST (see antibody list Table S5). After three washes with TBST (10 min each), membranes were incubated with HRP-conjugated secondary antibody for 2 hours at room temperature. Following another three washes with TBST, the bands were visualized using enhanced chemiluminescence (ECL) reagent (JP-K6000, FDbio Science, China).

Signal intensities were quantified using ImageJ software (NIH, USA), and relative protein expression levels were calculated by normalizing the band intensity of the target protein to that of the internal control (GAPDH for total proteins; CD81 or Calnexin for exosomal markers).

2.10. Enzyme-linked immunosorbent assay (ELISA)

Cytokine levels in cell culture supernatants were measured using commercial ELISA kits following the manufacturer’s instructions.

2.10.1. Sample preparation

Logarithmically growing hUC-MSCs were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. After cell adherence, cells were serum-starved for 12 hours, followed by co-culture treatments with or without exosomes, according to the experimental design. Cells were incubated at 37°C in a humidified incubator containing 5% CO₂ for 24 hours. After treatment, 1 mL of culture medium was collected from each well and centrifuged at 1000 × g for 20 minutes at 4°C. The supernatants were carefully collected for ELISA.

2.10.2. ELISA procedure

Standard reagents were prepared by serial dilution in 96-well ELISA plates according to the manufacturer’s instructions. For sample detection, 50 μL of either standard or sample was added to the wells of pre-coated ELISA plates, ensuring direct pipetting to the bottom center of each well without touching the side walls. The plates were gently shaken to mix the contents, sealed with adhesive film, and incubated at 37°C for 30 minutes.

After incubation, the plates were washed five times with diluted wash buffer (prepared by diluting the 30× concentrate with distilled water). During each wash step, wells were filled, left to stand for 30 seconds, and emptied completely before blotting dry.

Next, 50 μL of enzyme-conjugated detection reagent was added to each well, except the blank controls. Plates were resealed and incubated again at 37°C for 30 minutes. After repeating the washing procedure, 50 μL of Chromogenic Substrate A and 50 μL of Chromogenic Substrate B were sequentially added to each well. The plate was gently shaken to mix and incubated at 37°C in the dark for 15 minutes.

The reaction was terminated by adding 50 μL of stop solution to each well, resulting in a color change from blue to yellow. Absorbance at 450 nm was measured immediately using a microplate reader (BioTek, USA). Sample concentrations were calculated from the standard curve generated in parallel.

2.11. Flow cytometry for detection of cell apoptosis, macrophage M1/M2 markers, DC cell surface molecules, and T cell subpopulations

Cells from each experimental group were seeded into 6-well plates and maintained at 37°C in a 5% CO₂ atmosphere for 24 hours. Following incubation, the culture medium was removed, and cells were harvested using trypsin. After centrifugation at 800 rpm for 5 minutes, the supernatant was discarded. Cells were washed once with 2 mL of PBS, resuspended in 500 μL PBS, and adjusted to a final concentration of 1 × 10⁶ cells/mL. A 50 μL aliquot of the suspension (approximately 1 × 10⁶ cells) was used for subsequent staining with fluorescent antibodies.

For the purpose of apoptosis analysis, 5 μL each of Annexin V-FITC and PI solution were added to the cell suspension, then incubated in the dark at room temperature for 15 minutes. For macrophage polarization markers, cells were stained with 10 μL each of CD86, CD80, CD163, and CD206 antibodies. To assess dendritic cell surface proteins, 10 μL of CD40, MHCII, OX62, and CD274 antibodies were applied. For T cell subpopulation analysis, 10 μL of CD3, CD4, CD8, CD25, and Foxp3 antibodies were used. All staining procedures were carried out at room temperature, protected from light, for 60 minutes.

Following antibody incubation, the cells were rinsed with 200 μL of PBS and spun at 800 rpm for 5 minutes to eliminate leftover reagents. They were subsequently washed two more times with 2 mL of PBS and ultimately resuspended in 300 μL of PBS. Flow cytometry was employed to evaluate apoptosis rates, macrophage polarization (M1/M2), dendritic cell surface markers, and T cell subset distributions.

2.12. Data analysis

Statistical analyses and graph generation were performed using GraphPad Prism 9.4.0. Data are presented as mean ± standard deviation (SD). Each experimental condition was tested in at least three independent biological samples, with a minimum of three technical replicates per sample to ensure reproducibility. For comparisons between two groups, normality of data distribution was assessed using the Shapiro – Wilk test. If the data conformed to normal distribution and exhibited homogeneity of variance, an unpaired two-tailed Student’s t-test was applied. Otherwise, the Mann – Whitney U test was used as a non-parametric alternative. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test for multiple comparisons. In cases where normality or homogeneity assumptions were violated, the Kruskal – Wallis test with Dunn’s multiple comparisons test was applied. Due to limited sample sizes (n = 3) in some assays, non-parametric tests were prioritized to avoid inappropriate use of parametric methods. A p-value of < 0.05 was considered statistically significant.

3. Results

3.1. hUC-MSCs improve survival and reduce apoptosis of AC16 cells after H/R injury

To evaluate the protective effect of hUC-MSCs against hypoxia/reoxygenation (H/R)-induced injury, AC16 cells were subjected to H/R and co-cultured with hUC-MSCs. CCK-8 assay showed that cell viability decreased by approximately 45.6% ± 3.8% after H/R compared with the normoxic control, whereas co-culture with hUC-MSCs restored viability to 89.2% ± 4.1% of control levels (p < 0.001). Consistently, LDH release increased 2.7-fold following H/R but was reduced by nearly 55% in the hUC-MSCs co-culture group (p < 0.001) (Figure 1(A,B)). Flow cytometry analysis further revealed that H/R increased the apoptotic rate from 14.68% ± 0.91% to 44% ± 1.03%, which was markedly decreased to 30.74% ± 0.69% by hUC-MSC co-culture (p < 0.001) (Figure 1(C)). These findings demonstrate that hUC-MSCs effectively protect AC16 cells from H/R-induced injury by restoring cell viability, reducing cytotoxicity, and inhibiting apoptosis.

Figure 1.

Protective effect of hUC-MSCs on myocardial cells subjected to hypoxia/reoxygenation (H/R) injury. (A) The viability of myocardial cells was evaluated through the CCK-8 assay (biological replicates, n = 6). (B) LDH release levels to assess myocardial cell damage (biological replicates, n = 6). (C) Apoptosis rate of myocardial cells analyzed by flow cytometry (biological replicates, n = 3). Groups: AC16 (AC16 cells cultured under normal conditions); AC16-H/R (AC16 cells subjected to hypoxia/reoxygenation); hUC-MSCs+AC16-H/R (co-culture of AC16 cells (lower chamber) and hUC-MSCs (upper chamber)); hUC-MSCs+AC16-H/R (co-culture of AC16-H/R cells (lower chamber) and hUC-MSCs (upper chamber)). “***” indicates p < 0.001.

3.2. hUC-MSCs suppress NLRP3 inflammasome and activate AHR signaling

We next investigated the molecular mechanisms underlying hUC-MSC-mediated protection, focusing on the NLRP3/AHR signaling pathway. Western blot results revealed that the corresponding protein levels of NLRP3, Bax, NF-κB, and caspase-1 were elevated by 5-fold after H/R treatment but were markedly downregulated in the hUC-MSC co-culture group (p < 0.001) (Figure 2(A)). Consistently, qRT-PCR analysis showed that H/R markedly increased the mRNA levels of NLRP3, Bax, NF-κB, and caspase-1 by approximately 8- to 10-fold compared with the control, whereas co-culture with hUC-MSCs significantly reduced these transcripts to near-baseline levels (p < 0.001) (Figure 2(B)).

Figure 2.

hUC-MSCs Regulate Myocardial Protection through the AHR/NLRP3 Signaling Pathway. Western blot (A) and qPCR (B) analysis of NLRP3, Bax, NF-κB, caspase-1, AHR, CYP1A1, and CYP1A2 protein and mRNA expression in myocardial cells (biological replicates, n = 3). (C) ELISA detection of inflammatory cytokine levels in the culture supernatant (biological replicates, n = 6). Western blot (D) and qPCR (E) analysis of AHR protein and mRNA expression in hUC-MSCs (biological replicates, n = 3). Groups: AC16 (AC16 cells cultured under normal conditions); AC16-H/R (AC16 cells subjected to hypoxia/reoxygenation); hUC-MSCs+AC16-H/R (co-culture of AC16 (lower chamber) and hUC-MSCs (upper chamber)); hUC-MSCs+AC16-H/R (co-culture of AC16-H/R (lower chamber) and hUC-MSCs (upper chamber)). “**” indicates p < 0.01; “***” indicates p < 0.001.

In parallel, AHR expression increased by 1.8-fold after H/R and was further enhanced to about 2.6-fold by hUC-MSCs, accompanied by a 2.1-fold and 2.4-fold rise in its downstream targets CYP1A1 and CYP1A2 (p < 0.001), indicating activation of the AHR pathway.

ELISA results confirmed that H/R markedly elevated IL-1β, TNF-α, and IL-18 secretion (3.2-, 3.9-, and 3.0-fold increases, respectively, vs. control; p < 0.001), whereas hUC-MSCs decreased these cytokine levels by 45–60% (p < 0.001) (Figure 2(C)). Moreover, hUC-MSCs exposed to injured AC16 cells exhibited a ~ 9-fold increase in AHR expression (Figure 2(D,E)), suggesting that hUC-MSCs respond to injury signals by upregulating AHR, which may be transferred to AC16 cells through exosomes.

3.3. AHR knockdown eliminates hUC-MSC-Mediated protection

To confirm the role of AHR in hUC-MSC – mediated cardioprotection, hUC-MSCs with AHR knockdown (KD-AHR) were co-cultured with H/R-treated AC16 cells (Figure 3(A,B)). CCK-8 assay showed that cell viability in the KD-NC group increased to 3.48-fold compared with the H/R group (p < 0.001), whereas KD-AHR hUC-MSCs failed to restore viability (1.03 ± 0.10), showing no significant difference from H/R (0.98 ± 0.07, p > 0.05). Similarly, LDH release and apoptotic rate were significantly reduced in the KD-NC group (LDH: 106.18 ± 6.8 mU/mL; apoptosis: 17.05 ± 1.7%) compared with H/R (LDH: 268.44 ± 8.4 mU/mL; apoptosis: 40.15 ± 0.59%, p < 0.001), but remained elevated in KD-AHR (LDH: 275.07 ± 11.64 mU/mL; apoptosis: 39.52 ± 0.33%, p > 0.05) (Figure 3(C–E)). These findings demonstrate that AHR knockdown abolishes the protective effects of hUC-MSCs against H/R-induced cardiomyocyte injury.

Figure 3.

AHR Knockdown in hUC-MSCs Eliminates Their Cardioprotective Effects. Western blot (A) and RT-qPCR (B) analysis of AHR protein and mRNA expression in hUC-MSCs (biological replicates, n = 3). (C) CCK-8 assay to assess myocardial cell viability (biological replicates, n = 6). (D) LDH release assay to assess myocardial cell damage (biological replicates, n = 6). (E) Flow cytometry analysis of myocardial cell apoptosis rate (biological replicates, n = 3). Groups: AC16-H/R (AC16 cells subjected to hypoxia/reoxygenation); AC16-H/R+hUC-MSCs-KD-NC (co-culture of AC16-H/R (lower chamber) and hUC-MSCs-KD-NC (upper chamber)); AC16-H/R+hUC-MSCs-KD-AHR (co-culture of AC16-H/R (lower chamber) and hUC-MSCs-KD-AHR (upper chamber)). “***” indicates p < 0.001. n = 3.

To further explore the underlying mechanism, qRT-PCR and Western blot analyses revealed that KD-AHR hUC-MSCs had no significant effect on the mRNA or protein levels of NLRP3, AHR, or their downstream targets (p > 0.05), whereas KD-NC hUC-MSCs markedly downregulated NLRP3 by approximately 70% and upregulated AHR by 3-fold compared with the H/R group (p < 0.001) (Figure 4(A,B)). Consistent with these molecular findings, ELISA demonstrated that KD-NC co-culture reduced IL-1β, TNF-α, and IL-18 concentrations by 48.6%, 52.3%, and 55.7%, respectively (p < 0.001), whereas KD-AHR failed to decrease these cytokine levels (p > 0.05) (Figure 4(C)). Together, these results indicate that AHR is indispensable for the anti-inflammatory and cytoprotective effects of hUC-MSCs against H/R-induced injury.

Figure 4.

AHR Knockdown Abolishes the Regulatory Effect on AHR/NLRP3 Signaling and Cytokine Release. Western blot (A) and RT-qPCR (B) analysis of NLRP3 and AHR-related proteins and mRNA expression in myocardial cells (biological replicates, n = 3). (C) ELISA detection of inflammatory cytokine levels in the culture supernatant (biological replicates, n = 6). Groups: AC16-H/R (AC16 cells subjected to hypoxia/reoxygenation); AC16-H/R+hUC-MSCs-KD-NC (co-culture of AC16-H/R (lower chamber) and hUC-MSCs-KD-NC (upper chamber)); AC16-H/R+hUC-MSCs-KD-AHR (co-culture of AC16-H/R (lower chamber) and hUC-MSCs-KD-AHR (upper chamber)). “***” indicates p < 0.001.

3.4. AHR-Enriched exosomes protect AC16 cells from H/R injury

To determine whether hUC-MSC-derived exosomes mediate AHR transfer, AHR-overexpressing hUC-MSCs were generated by plasmid transfection, and their exosomes (AHR-exo) were subsequently isolated and characterized. Transmission electron microscopy (TEM) revealed the typical cup-shaped morphology of exosomes (Figure 5(A)). Western blot analysis confirmed the presence of the exosomal marker CD81 and the absence of the endoplasmic reticulum protein Calnexin (Figure 5(B)), indicating successful isolation and high purity of the exosome preparations. qRT-PCR and Western blot analyses further demonstrated that AHR-exo contained substantially higher levels of AHR mRNA (approximately 8-fold) and protein (approximately 10-fold) than exosomes from control hUC-MSCs (NC-exo, p < 0.01) (Figure 5(C,D)). These findings confirm that AHR was successfully incorporated into hUC-MSC-derived exosomes, supporting their potential role in intercellular AHR transfer.

Figure 5.

Extraction and Identification of AHR overexpressing Exosomes. (A) Exosome morphology observed under transmission electron microscopy. (B) CD81 expression was positive and Calnexin expression was negative in the exosomes from the hUC-MSCs-Control group, confirming successful exosome extraction. Western blot (B) and RT-qPCR (C) analysis of AHR protein and mRNA expression in exosomes. “**” indicates p < 0.01; “***” indicates p < 0.001. biological replicates, n = 6.

Treatment with AHR-exo significantly improved the viability of H/R-injured AC16 cells and reduced cytotoxicity and apoptosis (viability increased ~2.9 ± 0.3-fold; LDH decreased ~55%; apoptosis rate declined from 32.17 ± 1.07% to 25.00 ± 1.61%, p < 0.001), whereas NC-exo showed no significant effect (p > 0.05) (Figure 6(A–C)). Western blot analysis revealed that AHR-exo downregulated NLRP3, Bax, NF-κB, and cleaved caspase-1 expression by 35–60%, while significantly upregulating AHR and its downstream targets CYP1A1 and CYP1A2 (p < 0.05) (Figure 6(D)). ELISA further confirmed a marked reduction in pro-inflammatory cytokines, with IL-1β, TNF-α, and IL-18 levels decreased by 52.4%, 39.7%, and 58.2%, respectively (p < 0.001) (Figure 6(E)). Together, these results demonstrate that AHR-enriched exosomes exert potent cardioprotective effects by attenuating inflammation and apoptosis through activation of the AHR/NLRP3 signaling pathway.

Figure 6.

Protective Effect of AHR-Overexpressing hUC-MSCs Exosomes (hUC-MSCs-AHR-exo) on Myocardial H/R Injury. (A) CCK-8 assay to assess myocardial cell viability (biological replicates, n = 6). (B) LDH assay to assess myocardial cell damage (biological replicates, n = 6). (C) Myocardial cell apoptosis rate was assessed by flow cytometry analysis (biological replicates, n = 3). (D) Analysis of NLRP3 and AHR-related protein expression in heart cells using Western blot (biological replicates, n = 3). (E) ELISA detection of inflammatory cytokine levels in the culture supernatant (biological replicates, n = 6). “*” indicates p < 0.05; “**” indicates p < 0.01; “***” indicates p < 0.001.

3.5. AHR-exosomes modulate immune cell phenotypes toward an anti-inflammatory profile

AHR-enriched exosomes promote anti-inflammatory immune polarization. In THP-1–derived macrophages, AHR-exo treatment markedly enhanced M2 polarization while suppressing the M1 phenotype (M2/M1 ratio increased by ~2.8-fold, p < 0.001). Western blot analysis showed significant reductions in NLRP3, NF-κB, and cleaved caspase-1 expression (decreased by 45–60%), accompanied by upregulation of AHR and its downstream effectors CYP1A1 and CYP1A2 (~2.5–3.0-fold increase, p < 0.001) (Figure 7(A,B)). Consistently, ELISA revealed decreased secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-18 reduced by 50–65%) together with elevated levels of anti-inflammatory cytokines IL-10 and TGF-β (increased by ~2.2- and 2.5-fold, respectively; p < 0.001) (Figure 7(C)). These findings indicate that AHR-exo reprogram macrophages toward an anti-inflammatory phenotype through activation of AHR signaling and suppression of the NLRP3 inflammasome.

Figure 7.

hUC-MSCs-AHR-exo Induce Macrophage Polarization toward the M2 phenotype via the AHR/NLRP3 Axis. (A) Flow cytometry analysis of macrophage phenotype conversion (biological replicates, n = 3). (B) Western blot analysis of NLRP3, caspase-1, NF-κB, and AHR-related protein expression in macrophages (biological replicates, n = 3). (C) ELISA detection of inflammatory cytokine levels in macrophage culture supernatant (biological replicates, n = 6). “**” indicates p < 0.01; “***” indicates p < 0.001. Model: LPS-induced macrophage model.

Similarly, in dendritic cells, AHR-exo inhibited cellular activation and antigen presentation. Treatment significantly downregulated CD40, MHC II, and O×62 (reduced by 45–60%, p < 0.001), while markedly upregulating the inhibitory ligand CD274 (PD-L1; ~30-fold increase, p < 0.001) (Figure 8(A)). In T cells, AHR-exo reduced the proportion of CD3⁺ CD8⁺ cytotoxic T cells by ~80% and increased regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺) by ~2.1-fold (p < 0.001) (Figure 8(B)). Collectively, these data demonstrate that AHR-enriched exosomes orchestrate a broad anti-inflammatory immune remodeling by reprogramming macrophages, dendritic cells, and T cells, thereby promoting an immunosuppressive microenvironment.

Figure 8.

hUC-MSCs-AHR-exo Modulate DC and T Cell Phenotype Through AHR/NLRP3 Axis. (A) Flow cytometry analysis showing the surface marker expression of DCs: CD40, MHCII, O×62 (activation markers), and CD274 (tolerogenic marker) (biological replicates, n = 3). (B) Flow cytometry analysis of T cell subsets, showing changes in CD3⁺ CD8⁺ cytotoxic T cells and regulatory T cells (Tregs) (biological replicates, n = 3). Model: LPS-induced DC and T cell co-culture model. DC cells and T cells were co-cultured in direct contact. “**” indicates p < 0.01; “***” indicates p < 0.001.

Western blot and ELISA analyses further validated the systemic anti-inflammatory effects of AHR-exo. Treatment markedly reduced the expression of inflammation-related proteins – including NLRP3, NF-κB, and cleaved caspase-1—and decreased the secretion of pro-inflammatory cytokines IL-1β, TNF-α, and IL-18 (p < 0.001) (Figure 9(A,B)). Together, these results indicate that AHR-enriched exosomes exert comprehensive myocardial protection by modulating the immune microenvironment, suppressing inflammatory signaling, and restoring immune homeostasis.

Figure 9.

hUC-MSCs-AHR-exo Suppress Inflammatory Signaling in DC – T Cell Co-cultures. (A) Western blot analysis of NLRP3, caspase-1, NF-κB, and AHR-related proteins (AHR, CYP1A1, CYP1A2) in co-cultured DCs and T cells (biological replicates, n = 3). (B) ELISA detection of cytokine levels in the supernatant, showing reduced IL-1β, TNF-α, IL-18 and increased IL-10, TGF-β after AHR-exo treatment. Model: LPS-induced DC and T cell co-culture model. DC cells and T cells were co-cultured in direct contact (biological replicates, n = 6). “**” indicates p < 0.01; “***” indicates p < 0.001.

4. Discussion

Myocardial hypoxia/reoxygenation (H/R) injury remains a major therapeutic challenge in ischemic heart disease, characterized by excessive inflammation, oxidative stress, and cardiomyocyte apoptosis [3,4]. In this study, we demonstrate that exosomes derived from AHR-overexpressing hUC-MSCs effectively mitigate H/R-induced myocardial injury, primarily through modulation of the AHR/NLRP3 signaling axis and immune regulation.

The AHR is a ligand-activated transcription factor capable of sensing both endogenous metabolites (such as kynurenine) and environmental ligands. It acts as a critical regulator of immune metabolism, oxidative stress, and inflammatory signaling. In cardiovascular and immune contexts, AHR activation has been shown to limit cardiomyocyte apoptosis and promote anti-inflammatory immune polarization [25–28]. The NLRP3 inflammasome, a cytoplasmic multiprotein complex, is a key driver of sterile inflammation during myocardial ischemia – reperfusion injury. Its activation promotes caspase-1 cleavage and release of IL-1β and IL-18, which amplify cell death and tissue damage [29–33]. A key finding is that AHR-enriched exosomes suppress NLRP3 inflammasome activation, as reflected by reduced expression of NLRP3, caspase-1, and pro-inflammatory cytokines such as IL-1β and IL-18. These results align with previous studies suggesting that AHR inhibits inflammasome assembly by competing for shared cofactors or suppressing transcriptional activation [36]. Our data extend these findings by suggesting that exosomes act as delivery vehicles for functional AHR protein, thereby enabling paracrine modulation of recipient cells. The loss of protective effects following AHR knockdown in hUC-MSCs further supports AHR’s central role. Moreover, accumulating evidence indicates a reciprocal regulatory relationship between AHR and NLRP3, in which AHR activation constrains inflammasome assembly and cytokine maturation, thereby mitigating excessive inflammatory injury [34–36].

In addition, AHR-overexpressing exosomes exerted systemic immunomodulatory effects, promoting M2 macrophage polarization, dampening dendritic cell maturation, and expanding regulatory T cells. These shifts likely synergize with NLRP3 suppression to establish an anti-inflammatory milieu conducive to tissue repair. While this dual targeting of cardiomyocyte-intrinsic and immune-mediated injury is promising, it remains to be clarified whether the observed changes in immune phenotypes are direct effects of AHR cargo or secondary to improved cardiomyocyte health.

This study also provides several notable strengths and innovations. It is among the first to engineer hUC-MSCs to overexpress AHR and to verify the functional transfer of AHR via exosomes. The dual action of AHR – attenuating intrinsic myocardial damage while reprogramming immune responses – offers a multifaceted therapeutic strategy against ischemic injury. Furthermore, the combined use of biochemical (Western blot, qPCR, and ELISA) and phenotypic (flow cytometry) analyses strengthens the consistency and reliability of the findings. Together, these results build upon existing work on MSC-derived exosomes by highlighting AHR – a relatively underexplored protein cargo – as a functional component mediating both local and systemic repair. While previous research has focused mainly on exosomal miRNAs (e.g. miR-133a, miR-182), the present work underscores the importance of protein effectors such as AHR. Its broad regulatory influence over inflammation, metabolism, and oxidative balance positions AHR as a key integrator of reparative signaling [28,40,41].

Although the present study primarily focused on the inflammatory and immunoregulatory roles of the AHR/NLRP3 axis, it is worth noting that AHR also exerts dual regulatory effects on oxidative stress and mitochondrial homeostasis. Previous studies have shown that AHR activation can either promote reactive oxygen species (ROS) generation [42] or enhance antioxidant defense and mitochondrial biogenesis depending on the cellular context [43,44]. Given this complexity, future investigations integrating redox and mitochondrial signaling analyses – such as assessing ROS levels, antioxidant enzyme activity, and mitochondrial biogenesis markers (e.g. PGC-1α, TFAM) – will be valuable for clarifying how AHR contributes to the maintenance of cellular homeostasis during myocardial injury [45,46].

Despite the robustness of our findings, several limitations should be acknowledged. First, although AHR activation consistently suppressed NLRP3 signaling, the precise molecular mechanism – whether through direct transcriptional regulation or intermediate pathways – remains to be clarified. Second, the present study was conducted primarily in vitro, and the simplified co-culture systems cannot fully recapitulate the hemodynamic, neurohormonal, and multicellular complexity of the in vivo environment. Third, while we focused on macrophages, dendritic cells, and T cells, additional immune and nonimmune cell types may also contribute to the overall inflammatory remodeling. Finally, exosomes carry a heterogeneous cargo of proteins, RNAs, and lipids, and the possibility that other bioactive molecules cooperated with AHR cannot be excluded. Future in vivo and multi-omics studies are therefore warranted to validate the specificity and translational relevance of these findings. Nevertheless, these limitations do not undermine the central conclusion that AHR-enriched hUC-MSC – derived exosomes exert potent cardioprotective and immunoregulatory effects through modulation of the AHR/NLRP3 axis.

From a translational perspective, the findings support the therapeutic potential of engineered exosomes as a cell-free, customizable platform for myocardial repair. Compared to MSC transplantation, exosomes offer advantages including enhanced safety, reduced immunogenicity, and greater scalability. AHR-enriched exosomes may be particularly well-suited for adjunctive use in reperfusion therapies, where they could reduce injury severity and promote immune tolerance. However, careful optimization of exosome dosing, timing, and delivery route is needed prior to clinical translation.

5. Conclusion

This study provides novel insights into how AHR-enriched exosomes derived from hUC-MSCs mitigate H/R-induced myocardial injury by concurrently suppressing NLRP3-driven inflammation and reprogramming immune responses. While the findings advance the mechanistic understanding of exosomal therapy, future work should focus on in vivo validation, comprehensive molecular characterization, and addressing potential sources of bias to better inform clinical translation.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Abbreviations

H/R

myocardial hypoxia-reoxygenation

hUC-MSCs

human umbilical cord mesenchymal stem cells

DALYs

disability-adjusted life years

PCI

percutaneous coronary intervention

AHR

aryl hydrocarbon receptor

LPS

lipopolysaccharide

TEM

transmission electron microscopy

Authors’ contributions

Ying Qin and Dongyan Shen-Experiment, data collection and analysis, and writing the draft manuscript; Kequan Guo, Kequan Guo, Kequan Guo, and Yongchao Li -Investigation; Yixin Jia-Conception, supervision, funding, and revision of the manuscript. All authors approved the final of the version to be published and agreed to be accountable for all aspects of the work.

Availability of data and materials

All data in this study are available from the corresponding author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2025.2609649