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. 2026 May 30;33:101148. doi: 10.1016/j.reth.2026.101148

Adipose-derived mesenchymal stem cells promote diabetic wound healing through ALDOA secretion

Jingjing Wen a,b, Hui Ye a,⁎
PMCID: PMC13242010  PMID: 42255492

Abstract

Introduction

Diabetic wound healing is impaired by hyperglycemia-induced metabolic dysregulation and chronic inflammation. Adipose-derived mesenchymal stem cell (ADSC)-derived exosomes, noted for rich bioactive molecules and immunomodulation, are promising for wound healing, but their mechanisms in diabetic wounds remain unclear. This study investigated their impact and mechanism.

Methods

HaCaT cells were treated with high glucose to mimic in vitro diabetic conditions. C57BL/6 mice were treated with streptozotocin to construct the diabetic mouse model and induce skin wound. Exosomes were characterized by transmission electron microscopy and nanoparticle tracking analysis. Its role in vitro was evaluated by measuring cell viability, transwell and scratch wound assays. Hematoxylin-eosin and Masson staining were used to evaluate the pathological changes in mouse skin tissues. Microarray analysis, RNA sequencing and KEGG pathway enrichment analysis were performed to investigate the underlying mechanisms.

Results

Results showed that ADSC-derived exosomes enhanced migration and ECAR levels in HG-induced HaCaT cells, and promoted wound healing in diabetic mice. ALODA was enriched in glycolysis pathway, and its expression was reduced in HG-induced HaCaT cells, which was increased following exosomes treatment, even higher than that in the control group. ALDOA knockdown in exosomes inhibited migration and glycolytic activity increased by ADSCs-derived exosomes in HG-induced HaCaT cells. Moreover, ALDOA overexpression promoted migration and glycosis in HG-induced HaCaT cells.

Conclusions

These findings indicate that ADSC-derived exosomes enhance diabetic wound healing by upregulating ALDOA and promoting glycolysis, providing new experimental evidence for developing exosome-based therapies targeting impaired wound healing in diabetic patients.

Keywords: Diabetes, Wound healing, Adipose-derived mesenchymal stem cells, Exosomes, ALDOA

1. Introduction

Diabetes mellitus is a global public health challenge. According to recent statistics, approximately 463 million people worldwide have been diagnosed with diabetes, and this number is projected to reach 700 million by 2045 [1]. Among the different types of diabetes, the incidence of type 2 diabetes mellitus (T2DM) has increased markedly, particularly in low- and middle-income countries [2]. Notably, the incidence of T2DM is also increasing among younger populations, a trend that has raised widespread concern [3].

Patients with diabetes often suffer from impaired wound healing, primarily due to metabolic disturbances and chronic inflammatory responses caused by hyperglycemia [4,5]. Research has shown that the hyperglycemic state in diabetes suppresses the function of fibroblasts and endothelial cells, reducing their proliferation and migration capacities during the wound healing process, thereby delaying tissue repair [6]. Additionally, immune responses are compromised in diabetic patients, making wounds more susceptible to infection and further exacerbating healing difficulties [7]. In the diabetic milieu, all phases of wound healing-including inflammation, proliferation, and remodeling-are adversely affected, ultimately leading to the formation of chronic wounds or ulcers [8]. Therefore, investigating the specific mechanisms underlying impaired wound repair in diabetic patients and developing novel therapeutic strategies to enhance wound healing in this population have become urgent clinical priorities.

Adipose-derived mesenchymal stem cells (ADSCs) are primarily isolated from adipose tissue, especially subcutaneous fat. Studies have shown that ADSCs maintain robust proliferative capacity and multilineage differentiation potential during in vitro culture, making them well-suited for applications in regenerative medicine and tissue engineering [9]. Moreover, ADSCs exhibit potent immunomodulatory properties and can promote tissue repair and regeneration through the secretion of cytokines and growth factors [10]. In the context of wound healing in patients with diabetes, ADSCs have been found to improve the wound healing process by enhancing angiogenesis and cell migration, thereby accelerating the rate of healing [11]. In addition, exosomes derived from ADSCs are increasingly recognized as important mediators in intercellular communication and the regulation of biological functions. These exosomes can carry a variety of bioactive molecules to modulate the behavior of target cells [12]. These characteristics highlight the broad application prospects of ADSCs in diabetic wound healing and other fields of regenerative medicine.

Recently, multiple studies have demonstrated that exosomes derived from ADSCs play a crucial role in the wound healing process. These nanovesicles significantly promote the proliferation and migration of key cellular components in wound repair, particularly fibroblasts and endothelial cells, by delivering bioactive molecules including TGF-β and VEGF that activate critical growth signaling pathways [13,14]. Moreover, the anti-inflammatory effects of ADSC-derived exosomes in wound healing have attracted considerable attention. Research has revealed that ADSC exosomes can effectively modulate the local immune response and inhibit the infiltration of inflammatory cells, thereby reducing inflammatory reactions at the wound site [15,16]. ADSC-derived exosomes facilitate wound healing by promoting macrophage polarization toward the pro-reparative M2 phenotype, which not only reduces inflammatory responses but also actively enhances tissue regeneration [17]. These findings highlight the potential of ADSC-derived exosomes in promoting wound healing. However, their specific therapeutic role in diabetic wound healing remains to be fully elucidated.

In this study, we systematically investigated the effects of ADSC-derived exosomes on the proliferation and migration of high glucose-treated skin cells and diabetic wound healing in mice through comprehensive in vitro and in vivo experiments, while elucidating the underlying mechanisms. These findings provide novel insights into developing exosome-based therapies for impaired wound healing under diabetic conditions.

2. Methods

2.1. Animal study

Specific pathogen-free (SPF)-grade C57BL/6 mice (male-female ratio 1:1, 8-12 weeks old) were acclimatized for one week before the establishment of the diabetic wound model. The mice were randomly divided into three groups: the normal control group, the diabetes mellitus (DM) group, and the DM + exosomes group (n = 6 per group). Diabetes was induced in the DM and DM + exosomes groups by a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg). 72 h after injection, blood glucose levels were measured in a random subset of mice, and all showed levels ≥16.7 mmol/L, confirming successful induction of the diabetic model.

Following diabetic wound model establishment, wound creation was performed according to the method described by Li et al. [18] Mice were anesthetized by inhalation of 3% isoflurane. The dorsal hair of all mice was removed using an electric clipper and depilatory cream. The skin was then cleaned with alcohol, and full-thickness wounds (6 mm in diameter) were created on both sides of the midline using a biopsy punch, extending through the panniculus carnosus.

After wound creation, exosome treatment was performed. Mice in the HG + exosomes group received subcutaneous injections of 100 μg/mL exosome solution around the wound area. All wounds were disinfected daily with povidone-iodine. The treatment lasted for 10 consecutive days, and wound images were captured on days 0, 5, and 10 to calculate the wound healing rate.

At the end of the treatment period, mice were euthanized by cervical dislocation under 3% isoflurane anesthesia. Skin tissues surrounding the wounds were collected and fixed in 4% paraformaldehyde for subsequent histological analysis.

2.2. Histological analysis

Skin tissues fixed in 4% paraformaldehyde was embedded in paraffin and made into 5 μm of paraffin sections. The sections were mounted on slides, deparaffinized in xylene, and rehydrated with descending ethanol concentrations prior to staining. Hematoxylin and eosin (HE) staining was conducted using a HE staining kit (Beyotime). The paraffin sections were stained with hematoxylin for 10 min and counterstained with eosin for 2 min. Afterwards, sections were dehydrated through graded ethanol, cleared in xylene, and mounted with neutral resin. The sections were observed under a microscope.

A Masson staining kit was performed to stain the paraffin sections. Paraffin sections were sequentially stained with hematoxylin (5 min), Ponceau-acid fuchsin (10 min), and light green (1 min), each followed by distilled water rinses. Differentiation steps included acidic solution (30 s) after hematoxylin, phosphomolybdic acid (2 min) after Ponceau-acid fuchsin, and acidic solution (1 min) after light green. Sections were dehydrated through graded ethanol each cleared in xylene, and observed under a microscope.

2.3. Isolation and characterization of ADSCs-derived exosomes

Human ADSCs (SUNNCELL, Wuhan, China) were cultured in the specific ADSCs medium (SUNNCELL) at 37 °C with 5% CO2. ADSCs at 70-80% confluency were collected, washed twice with PBS, and cultured in 10 mL of fresh medium for two days. Subsequently, the culture medium was harvested and sequentially centrifuged at 300×g for 15 min, 3000×g for 15 min, and 20,000×g for 70 min. The supernatant was filtered through a 0.2 μm membrane filter to remove cells and large debris. Exosomes were then pelleted by ultracentrifugation at 120,000×g for 70 min, washed with PBS to remove contaminating proteins, subjected to a second round of ultracentrifugation, and finally resuspended in 1 mL of PBS for subsequent use.

The morphology and size distribution of exosomes derived from ADSCs were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). For TEM analysis, exosomes were placed onto a copper grid for 3-5 min, fixed with 2% (w/v) phosphotungstic acid for 2-3 min, and subsequently examined under a transmission electron microscope (JEOL, Tokyo, Japan). Particle size and concentration of the exosomes were further assessed using a NanoSight NS300 system (Malvern, Worcestershire, UK) in accordance with the manufacturer's protocol.

2.4. Cell culture and treatment

Human epidermal cells HaCaT were provided by SUNNCELL. The cells were cultured in Dulbecco's modified eagle's medium (Gibco, Grand Island, CA, USA) supplemented with 10% fetal bovine serum (Gibco) in a humidified 5% CO2 incubator at 37 °C. HaCaT cells were exposed to 30 mM glucose (high glucose, HG) for 24 h to mimic diabetic skin wound condition in vitro. Following HG treatment, the cells were treated with 100 μg/mL exosomes for 24 h.

2.5. Cell transfection

Short hairpin RNA targeting ALODA (shALDOA), shRNA targeting negative control (shNC), ALDOA overexpression plasmids (pcDNA3.1-ALDOA) and its negative control (pcDNA3.1) were synthesized by GeneScript (Nanjing, China). HaCaT cells seeded in 6-well plates were transfected with pcDNA3.1-ALDOA or empty vector at 70-80% confluence using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The ADSCs-derived exosomes were transfected with shALDOA or shNC using the Exo-Fect Exosome Transfection Kit (Univ-bio, Shanghai, China) according to the manufacturer's protocol.

2.6. Western blot

Total protein extraction was performed using RIPA lysis buffer (Invitrogen) and its contraction was measured using a BCA kit (Beyotime). The protein samples were loaded on 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skimmed milk for 1 h, and incubated overnight at 4 °C with ptimart antibodies, inclusing anti-CD63 (1: 1000, ab134045, Abcam, Cambridge, UK), anti-TSG-101 (1: 1000, ab125011, Abcam), anti-Calnexin (1: 1000, ab22595, Abcam) and anti-GM130 (1: 1000, ab52649, Abcam). Afterwards, the membranes were washed with TBST and incubated with secondary antibodies (1: 10,000, ab6721, Abcam) for 2 h. The blots were visualized using the ECL reagent (Beyotime).

2.7. Cell viability assay

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Yeasen, Shanghai, China). Briefly, HaCaT cells were plated in 96-well plates and subjected to the designated treatments. Subsequently, 10 μL of CCK-8 reagent was introduced into each well, followed by incubation at 37 °C for 1 h. The optical density was recorded at 450 nm, and viability was determined using the specific formula.

2.8. Transwell assay

Cell migration was evaluated using a Transwell® assay (Corning, NY, USA). Briefly, HaCaT cells were seeded into the upper chamber and allowed to migrate for 24 h. Following incubation, non-migratory cells on the upper surface of the membrane were carefully removed by PBS washing. The translocated cells on the lower side were then fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min. Quantification of migration was performed by counting the stained cells under an optical microscope.

2.9. Cell scratch assay

The cell migration ability of HaCaT cells was evaluated through a scratch wound assay. Briefly, cells were plated in 6-well culture plates at an initial density of 2 × 105 cells per well and allowed to adhere until they attained ∼85% monolayer confluency. A standardized linear scratch was then generated in each well using a sterile 200-μL pipette tip. Following two gentle washes with PBS to remove dislodged cells, fresh ECM medium was added to support cell migration. The wound closure dynamics were monitored periodically, and images of the scratch regions were captured at 0 h and 48 h post-scratching using an optical microscope. The migration rates were quantified using ImageJ software.

2.10. Real-time cell metabolism assay

The glycolytic flux of HaCaT cells was evaluated by monitoring the extracellular acidification rate (ECAR) with an XFp Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA). Cells were suspended in sterile XF Base Medium supplemented with 10 mM d-glucose (pH 7.4), equilibrated at 37 °C for 30 min, and subsequently subjected to ECAR quantification. Measurements were recorded after sequential administration of 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose.

2.11. RNA sequencing and bioinformatic analysis

The GSE15932 dataset was downloaded from the Gene Expression Omnibus (GEO) database, which includes peripheral blood samples from patients with type 2 diabetes mellitus (T2DM) and healthy controls. Raw microarray data were normalized using the robust multi-array average (RMA) algorithm to correct background intensity and ensure inter-sample comparability. Batch effects were adjusted using the “ComBat” function in the sva R package. Differentially expressed genes (DEGs) between T2DM and control groups were identified via the limma R package, with significance thresholds set at adjusted p < 0.05 (false discovery rate, FDR) and |log2 (fold change)| ≥ 1.5. Hierarchical clustering and heatmap visualization of DEGs were performed using the pheatmap R package, with Euclidean distance and complete linkage methods applied for both rows (genes) and columns (samples). Gene expression values were standardized by Z-score transformation prior to heatmap generation.

Total RNA was extracted from control and exosome-treated HaCaT cells, and RNA sequencing (RNA-seq) was performed by Novogene (Beijing, China) using the Illumina HiSeq platform. Raw sequencing reads were quality-checked with FastQC and aligned to the human reference genome (GRCh38) using STAR software. Gene expression levels were quantified as transcripts per million (TPM) via the RSEM algorithm. Differential gene expression analysis was conducted using the DESeq2 R package, with significance thresholds set at adjusted p < 0.05 (false discovery rate, FDR) and |log2 (fold change)| ≥ 1.0. DEGs were visualized as a heatmap using the pheatmap R package, with Euclidean distance and complete linkage clustering applied to standardized (Z-score) gene expression values across samples.

DEGs from the GSE15932 dataset and RNA-seq data were intersected using the VennDiagram R package. Overlapping genes were subjected to KEGG pathway enrichment analysis via the clusterProfiler R package, with pathways considered significant at FDR<0.05.

2.12. Quantitative real-time PCR (qPCR)

Total RNA was extracted from cultured HaCaT cells following the TRIzol reagent protocol (Invitrogen). RNA concentration and purity were assessed spectrophotometrically using a NanoDrop instrument (Thermo Scientific, Waltham, MA, USA). Subsequently, first-strand cDNA was synthesized from total RNA using the HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China), following the manufacturer's instructions. qPCR amplification was carried out in triplicate on a StepOnePlus system (Thermo Scientific) with SYBR green master mix (Vazyme). Gene expression levels were normalized to the endogenous control GAPDH, and relative quantification was determined using the comparative 2−ΔΔCt method. The primers of ALDOA were as follows: 5′-ATGCCCTACCAATATCCAGCA-3’ (sense) and 5′-GCTCCCAGTGGACTCATCTG-3’ (antisense).

2.13. Statistical analysis

All data were analyzed using SPSS 22.0 software. Data were presented as the mean ± standard deviation (SD) derived from at least three replicates. Comparisons between two or more groups were analyzed using Student's t-test or one-way analysis of variance (ANOVA). P < 0.05 were recognized as statistically significant.

3. Results

3.1. Characterization of the exosomes

Results of TEM suggested that exosomes isolated from ADSCs exhibited a typical vesicular morphology with a double-membrane structure, which was consistent with the morphological characteristics of exosomes (Fig. 1A). The results of NTA showed that the size distribution of exosomes was mainly concentrated between 100 and 200 nm (with a peak concentration of 5.35 × 106 particles/mL), indicating a relatively uniform particle size (Fig. 1B). Moreover, Western blot revealed that the exosomal marker proteins CD63 and TSG101 were upregulated in exosomes but were not detected in the cell lysate. In contrast, the endoplasmic reticulum marker Calnexin and the Golgi apparatus marker GM130 were highly expressed in the cell lysate but absent in exosomes, confirming the high purity of the isolated exosomes (Fig. 1C). These results indicated that the exosomes isolated from ADSCs meet the core criteria for exosome identification and can be used in subsequent experiments.

Fig. 1.

Fig. 1

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Characterization of the exosomes (A) The characteristic of ADSCs-derived exosomes was identified by TEM. (B) The size of ADSCs-derived exosomes was measured by NTA. (C) The protein levels of CD63, TSG101, Calnexin and GM130 were detected by Western blot.

3.2. ADSCs-derived exosomes promotes wound healing in HG-induced HaCaT cells

We employed HG to induce HaCaT cells in vitro to mimic diabetic skin wound conditions and investigated the therapeutic effects of ADSCs-derived exosomes. Results demonstrated that HG treatment significantly reduced HaCaT cell viability, which was markedly restored following exosome administration (Fig. 2A). Similarly, HG-induced suppression of HaCaT cell migration was significantly alleviated by exosome treatment (Fig. 2B and C). Scratch wound healing assays revealed that the migration capacity of HG-treated HaCaT cells was significantly impaired compared to the control group, whereas exosome intervention prominently enhanced wound closure rates (Fig. 2D). These findings indicate that HG inhibited the wound-healing potential of HaCaT cells, while exosome treatment effectively reversed this impairment (Fig. 2E). Furthermore, the HG-reduced ECAR was partially restored upon exosome treatment (Fig. 2F). Collectively, our data demonstrated that ADSCs-derived exosomes promoted wound healing in HG-induced HaCaT cells.

Fig. 2.

Fig. 2

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ADSCs-derived exosomes promotes wound healing in HG-induced HaCaT cells (A) Cell viability of HaCaT cells was detected using a CCK-8 kit. (B and C) Transwell assay was performed to evaluate cell migration of HaCaT cells. (D and E) A scratch wound assay was performed to assess the migration and healing of HaCaT cells. (F) ECAR was detect to evaluate glycolysis in HaCaT cells.

3.3. ADSCs-derived exosomes promotes wound healing in DM mouse model

Subsequently, we investigated the therapeutic effects of ADSCs-derived exosomes on wound healing in DM mouse model. On day 5, compared to the normal group, DM mice exhibited significantly delayed wound closure, whereas exosome-treated DM mice displayed smaller wound areas and enhanced wound closure. On day 10, DM mice still presented with visible wounds, while exosome-treated DM mice showed near-complete wound closure, comparable to the normal group, indicating that exosomes accelerate wound closure in diabetic mice (Fig. 3A). Histological analysis via HE staining revealed that the normal group exhibited intact epidermis, well-organized dermal cellular architecture, and minimal inflammatory infiltration. In contrast, DM mice showed delayed epidermal repair, extensive inflammatory cell infiltration in the dermis, and disordered tissue structure. Notably, exosome administration significantly increased epidermal thickness, reduced inflammatory responses, and promoted dermal cell proliferation and tissue regeneration in DM mice. Masson staining further demonstrated that the normal group displayed densely packed, well-aligned collagen fibers, whereas DM mice exhibited reduced collagen deposition and disorganized fiber arrangement. Importantly, exosome treatment markedly restored collagen content and improved fiber alignment in DM mice, approaching the structural integrity observed in the normal group (Fig. 3B). These findings collectively demonstrated that ADSCs-derived exosomes enhanced epidermal repair and collagen deposition in diabetic wounds. In summary, our results provided evidence that ADSCs-derived exosomes improved skin wound healing capacity in DM mice.

Fig. 3.

Fig. 3

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ADSCs-derived exosomes promotes wound healing in DM mouse model (A) The wound healing progression in diabetic mouse models was evaluated by measuring wound closure percentages on days 0, 5, and 10 post-wounding. (B) HE and Masson staining were performed to evaluate pathological changes of skin tissues of diabetic mice at day 10 post-wounding.

3.4. ADSCs-derived exosomes increases ALDOA mRNA expression in HaCaT cells

Subsequently, we explored the underlying mechanism by which ADSCs-derived exosomes promote diabetic wound healing. We identified DEGs between peripheral blood samples from patients with T2DM and healthy controls from the GSE15932 dataset (Fig. 4A). Additionally, RNA-seq was performed to profile DEGs in HaCaT cells treated with ADSCs-derived exosomes versus untreated control cells, as shown in Fig. 4B. Intersection analysis of these DEGs revealed 43 commonly regulated genes (Fig. 4C). KEGG pathway enrichment analysis was performed on these genes, and the key top ten signaling pathways were obtained. Among them, the glycolysis pathway was most relevant to diabetic wound healing, and the key gene of this pathway was ALDOA (Fig. 4D). Next, qPCR demonstrated a significant downregulation of ALDOA mRNA expression in HG-induced HaCaT cells relative to controls, whereas exosome treatment notably enhanced its expression, exceeding even that of the control group (Fig. 4E). These findings indicate that ALDOA expression was suppressed in HG-induced HaCaT cells but was rescued by ADSCs-derived exosomes administration. This condition may be mediated through the additional secretion of ALDOA by ADSCs-derived exosomes.

Fig. 4.

Fig. 4

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ADSCs-derived exosomes increases ALDOA mRNA expression in HaCaT cells (A) Microarray analysis was performed to identify the DEGs in the peripheral blood between patients with T2DM and the healthy controls from the GSE15932 dataset. (B) The DEGs between HaCaT cells treated with ADSCs-derived exosomes or not were identified from RNA-seq results. (C) A Venn diagram illustrated the intersection of DEGs identified from RNA-seq results and those from the GSE15932 dataset. (D) These 43 intersecting genes were used for KEGG pathway enrichment analysis. (E) The ALDOA mRNA expression in HaCaT cells was measured by qPCR.

3.5. ALDOA knockdown in ADSCs-derived exosomes impairs their therapeutic efficacy in promoting wound healing in HG-induced HaCaT cells

To validate the hypothesis that ADSCs-derived exosomes promote diabetic wound healing through the secretion of ALDOA, we specifically knocked down ALDOA expression in exosomes (Fig. 5A). Next, we found that the exosome-mediated enhancement of cell viability and migration in HG-induced HaCaT cells was significantly attenuated following ALDOA knockdown (Fig. 5B and C). Scratch wound healing assays further demonstrated that ALDOA knockdown in exosomes suppressed the migratory capacity of HG-treated HaCaT cells, thereby abrogating the exosome-induced recovery of wound healing (Fig. 5D). To elucidate the molecular mechanism by which ALDOA induces cell migration, we further examined the expression of core migration-related proteins. We found that HG treatment downregulated the protein levels of MMP9 and N-cadherin while upregulating the epithelial marker E-cadherin. ADSCs-derived exosomes reversed these HG-induced changes; however, these pro-migratory effects were completely abrogated when ALDOA in the exosomes was knockeddown (Fig. S1A). These findings confirm that ALDOA derived from ADSCs exosomes promotes HaCaT cell migration under high glucose conditions by facilitating EMT (via downregulating E-cadherin and upregulating N-cadherin) and enhancing extracellular matrix degradation (via upregulating MMP9). Additionally, while exosome administration restored the reduced ECAR in HG-induced HaCaT cells, this metabolic rescue effect was diminished by ALDOA knockdown (Fig. 5E). In conclusion, these findings indicate that ALDOA knockdown in ADSCs-derived exosomes compromises their therapeutic efficacy in restoring HG-impaired wound healing in HaCaT cells.

Fig. 5.

Fig. 5

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ALDOA knockdown in ASDC-derived exosomes impairs their therapeutic efficacy in promoting wound healing in HG-induced HaCaT cells (A) ALDOA mRNA expression in HaCaT cells was measured by qPCR. (B) Cell viability of HaCaT cells was detected using a CCK-8 kit. (C) Transwell assay was performed to evaluate cell migration of HaCaT cells. (D) A scratch wound assay was performed to assess the migration and healing of HaCaT cells. (E) ECAR was detect to evaluate glycolysis in HaCaT cells.

3.6. ALDOA overexpression promotes wound healing in HG-induced HaCaT cells

We further directly validated the role of ALDOA in wound healing in vitro. HaCaT cells were transfected with pcDNA3.1-ALDOA, qPCR confirmed a significant upregulation of ALDOA mRNA expression post-transfection (Fig. 6A). Subsequently, we investigated the effects of ALDOA overexpression on cell viability, migration, and glycolysis in HaCaT cells with HG treatment. Results demonstrated that ALDOA overexpression effectively restored the HG-impaired cell viability and migration (Fig. 6B and C). Notably, the wound-healing capacity of HG-treated HaCaT cells, which was compromised by HG treatment, was partially rescued following ALDOA knockdown (Fig. 6D). Furthermore, ALDOA overexpression elevated the ECAR suppressed by HG in HaCaT cells (Fig. 6E). Additionally, we assessed intracellular ATP levels. The results demonstrated that HG treatment significantly decreased intracellular ATP levels in HaCaT cells. ALDOA overexpression markedly restored ATP production; however, this restorative effect was completely abolished by the glycolysis inhibitor 2-DG (Fig. S2A). These findings confirm that the ALDOA-induced increase in intracellular ATP is directly attributable to enhanced glycolysis, and that the elevated ATP levels mediate the pro-healing effects of ALDOA. Collectively, these findings indicated that ALDOA overexpression enhanced wound healing capacity in HG-induced HaCaT cells.

Fig. 6.

Fig. 6

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ALDOA overexpression promotes wound healing in HG-induced HaCaT cells (A) ALDOA mRNA expression in HaCaT cells was measured by qPCR. (B) Cell viability of HaCaT cells was detected using a CCK-8 kit. (C) Transwell assay was performed to evaluate cell migration of HaCaT cells. (D) A scratch wound assay was performed to assess the migration and healing of HaCaT cells. (E) ECAR was detect to evaluate glycolysis in HaCaT cells.

4. Discussion

ADSCs-derived exosomes have been widely applied in wound healing therapy, primarily by delivering bioactive molecules to promote cell proliferation, migration, angiogenesis, and inflammation resolution, thereby accelerating wound closure [19]. Notably, they also exhibit great potential in treating diabetic wound healing. For example, Song et al. [20] demonstrated that ADSC-derived exosomes encapsulated in an ECM hydrogel significantly promotes the proliferation, migration, and tube-forming capacity of HaCaT cells, and accelerated wound healing in diabetic mice by enhancing cell proliferation, stimulating angiogenesis, and reducing inflammation. Moreover, Ren et al. [21] demonstrated that ADSC-derived exosomes activate autophagy, thereby enhancing the proliferation and migration of epidermal cells and promoting cutaneous wound healing in diabetic mice. In the present study, we found that ADSC-derived exosomes enhanced the migratory capacity and ECAR in HG-induced HaCaT cells, and promoted cutaneous wound healing in diabetic mice. These findings provide new experimental evidence supporting the therapeutic potential of ADSC-derived exosomes in the treatment of diabetic wound healing.

Glycolysis is the primary pathway for cells to rapidly generate ATP under conditions of hypoxia or high metabolic demand, and its activation is crucial for restoring the structure and function of wounded tissues [22]. Evidence indicates that glycolysis is impaired in diabetes [23]. Narayanan et al. [24] also observed a reduction in glycolysis levels in a diabetic mouse model with delayed wound healing, which may be associated with the failure of diabetic wound healing. Notably, Ma et al. [25] demonstrated that PDK4 rescues high glucose-induced senescent fibroblasts and promotes diabetic wound healing by enhancing glycolysis. Kim et al. [26] revealed that PKM2 promotes wound healing and angiogenesis during the healing process by enhancing glycolysis and interacting with the Wnt/β-catenin signaling pathway. These results indicate that enhanced glycolysis is beneficial for promoting wound healing in diabetic conditions, providing the necessary energy for the proliferation of various cell types involved in the healing process. Our findings further demonstrate that ADSCs-derived exosomes increase the ECAR in HG-induced HaCaT cells, suggesting that their pro-healing effects are, at least in part, mediated through the activation of glycolytic metabolism.

Additionally, our findings suggest that ADSCs-derived exosomes promoted diabetic wound healing through the secretion of ALDOA. ALDOA, a key glycolytic enzyme, catalyzes the reversible cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate [27]. ALDOA is critically involved in metabolic reprogramming during pathological processes, where its upregulation supports enhanced glycolytic flux to meet the elevated energy demands of proliferating and migrating cells. For instance, Li et al. [28] demonstrated that ALDOA promotes the proliferation and migration of intrahepatic cholangiocarcinoma cells by enhancing their glycolytic activity. Sun et al. [29] found that ALDOA is upregulated in colorectal cancer (CRC) tissues, and its high expression promotes CRC cell proliferation and migration while activating glycolytic activity. Notably, an early study by Tochio et al. [30] demonstrated that ALDOA promotes the migration of HaCaT cells by inducing the formation of lamellipodia. This study was the first to reveal the role of ALDOA in epidermal cell growth. Liu et al. [31] demonstrated that ADSCs-derived exosomes enhance the migration capacity and glycolytic activity of HaCaT cells under high glucose conditions by upregulating key molecules such as ALDOA, thereby promoting the healing of skin injuries in rats. These results demonstrate that ALDOA promotes wound healing by mediating glycolysis. However, whether ALDOA is involved in diabetic wound healing remains unclear. Our study found that ALDOA was upregulated in exosome-treated HaCaT cells and downregulated in HG-induced HaCaT cells, while exosome treatment on the HG group increased the expression level of ALDOA, even higher than that in the control group, suggesting that ADSCs-derived exosomes treatment may secrete ALDOA mRNA. ALDOA knockdown in exosomes reversed the promotion of ADSCs-derived exosomes on the migration and glycolytic activity of HG-induced HaCaT cells, confirming that ADSCs-derived exosomes promoted diabetic wound healing through the secretion of ALDOA.

To sum up, our results demonstrated that ADSCs-derived exosomes enhance the migration and glycolytic activity of HG-induced HaCaT cells through ALDOA secretion, thereby promoting wound healing in diabetic mouse model. The findings of this study provide novel insights into the mechanism by which ADSC-derived exosomes facilitate diabetic wound healing, highlighting the potential of ALDOA as a key mediator in exosome-mediated therapeutic effects. These results may contribute to the development of exosome-based therapies targeting impaired wound healing in diabetic patients.

Consent to participate

Not applicable.

Ethics approval

This study was approved by the Ethics Committee of SHENZHEN PKU-HKUST MEDICAL CENTER (Approval Number: 2026-395).

All animal experiments should comply with the ARRIVE guidelines.

All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. J W drafted the work and revised it critically for important intellectual content; H Y was responsible for the acquisition, analysis and interpretation of data for the work; J W made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

Funding

The study was supported by Fujian Provincial Science and Technology Innovation Joint Fund Project Plan (2024Y9369) and Fujian Provincial Natural Science Foundation (Project No. 2023J01720).

Declaration of competing interest

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

Acknowledgements

Not applicable.

Footnotes

Peer review under responsibility of the Japanese Society for Regenerative Medicine.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2026.101148.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (112KB, docx)

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Associated Data

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Supplementary Materials

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Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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