Omentin-1 promotes wound healing in diabetic mice by improving vascular endothelial cell function

Abstract

Diabetes is a metabolic disease that affects global human health, with 25% of diabetic patients suffering from chronic non-healing ulcers. Omentin-1, also referred to as intelectin-1 and encoded by the itln1 gene, is a recently discovered adipokine that modulates inflammation, cellular activities, and vascular tone. Research indicates that omentin-1 provides protective benefits in several conditions, including atherosclerosis and diabetic retinopathy. Nevertheless, the therapeutic potential of omentin-1 in promoting diabetic wound healing is still not well understood. Our results revealed a significant decrease in ITLN1 levels in the cutaneous tissues of diabetic mice. Subcutaneous injection of omentin-1 reduced endothelial cell apoptosis, enhanced angiogenesis, and accelerated wound repair in diabetic mice. In vitro studies demonstrated that omentin-1 upregulated VEGF expression in endothelial cells, improved endothelial function under high-glucose conditions, reduced high glucose-induced endothelial apoptosis, and promoted endothelial tube formation. Further mechanistic studies revealed that omentin-1 improved endothelial function and promoted angiogenesis under high-glucose conditions by mediating the PI3K/AKT/FOXO1 pathway. Our results suggest that omentin-1 is a potential adjunct or therapeutic agent for treating chronic non-healing diabetic wounds by enhancing endothelial cell function and promoting vascularization.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-24682-5.

Introduction

Diabetes mellitus constitutes a chronic metabolic disease of worldwide significance, characterized by persistent elevation of blood glucose levels¹. Chronic, refractory cutaneous lesions constitute a prevalent diabetic complication, particularly affecting lower limb tissue². Approximately 15–20% of diabetic foot patients ultimately require amputation, with a post-amputation five-year mortality rate as high as 50–70%³. The global annual healthcare expenditure for diabetic foot treatment amounts to tens of billions of dollars⁴. Diabetic wounds not only impose severe physical and psychological distress on patients and their families but also place considerable economic burdens on healthcare infrastructure. Therefore, identifying promising new therapeutic strategies to accelerate diabetic wound healing is a top priority in clinical practice.

Cutaneous wound repair constitutes a precisely coordinated biological cascade involving multiple cellular participants including inflammatory cells, vascular endothelial cells, and dermal fibroblasts. Any impairment in this process may result in prolonged wound healing⁵. Endothelial dysfunction is a major contributor to impaired wound healing in diabetes. Persistent hyperglycemia damages endothelial cells, leading to reduced cell migration and proliferation, along with increased apoptosis⁶. Endothelial cells play a crucial role in angiogenesis during wound healing, and their dysfunction impairs the formation of local microvascular networks, resulting in ischemia and hypoxia at the wound site. Moreover, apoptotic endothelial cells release pro-inflammatory mediators that further destabilize vascular structures, exacerbating wound healing impairment^7,8. Thus, developing effective approaches to restore endothelial function is crucial for improving diabetic wound treatment.

Omentin-1 (ITLN1), a novel adipocytokine discovered in recent years, shows predominant expression in the omentum and perivascular adipose tissue⁹. Earlier research has demonstrated that omentin-1 enhances endothelium-dependent vasodilation by promoting the production of nitric oxide (NO) in endothelial cells¹⁰. Additionally, omentin-1 inhibits endoplasmic reticulum stress and oxidative stress in mouse aortic endothelial cells induced by high glucose, reducing the accumulation of reactive oxygen species (ROS)¹¹. Moreover, omentin-1 has been implicated in regulating apoptosis in various disease models, including ischemic stroke and intervertebral disc degeneration^12,13. Although omentin-1’s endothelial protective properties are well-established, its role in diabetic wound repair remains poorly understood, and the precise molecular pathways mediating its endothelial regulatory effects require further elucidation.

Our experimental approach involved: (1) creating full-thickness skin wounds in diabetic (STZ-treated) mice to test omentin-1’s wound repair efficacy, (2) analyzing its vascular effects, and (3) conducting mechanistic studies in cultured endothelial cells.

Materials and methods

Antibodies and reagents

Streptozotocin (STZ, 18883-66-4), and D-Glucose (D9434) were obtained from Sigma-Aldrich (USA). Omentin-1(00020-06-50) was provided by Aviscera Bioscience (USA). The annexin V-FITC/PI Apoptosis Detection Kit was obtained by Yeasen Biotechnology (China). β-tubulin (AC030), ITLN1 (A7234), β-actin (AC026) and GAPDH (A7234) were provided by Abclonal (China). FOXO1 (18592-1-AP) was provided by proteintech (China). p-FOXO1 was provided by Cell Signal Technology (USA). VEGFA (ER30607), AKT1/2/3 (ET1609-51), and p-AKT (ET1607-73) were provided by HUABIO (China).

Animal experiments

The animal experiment was approved by the Experimental Animal Ethics Committee of Nanjing Hospital, Nanjing Medical University (Approval No.: DWSY-23125429). We confirm that all animal experiments were conducted in accordance with the relevant guidelines and regulations. Male C57/B6 mice (6–8 weeks old) were obtained from Beijing SiPeiFu Biotechnology Co., Ltd. and maintained at the Laboratory Animal Center of Nanjing Hospital, Nanjing Medical University. Using the random number table method, mice were randomly allocated into three groups using the random number table method: the healthy control (NC) group, the diabetes mellitus (DM) group, and the diabetes mellitus with omentin-1 treatment (DM + omentin-1) group. A diabetic mouse model was established by intraperitoneal injection of streptozotocin (STZ, 80 mg/kg/day) for three consecutive days. Fasting blood glucose levels were assessed in the fourth week post-injection, and mice with levels exceeding 16.7 mmol/L were deemed to have successfully established the diabetic model. Next, a 1 cm circular full-thickness excisional wound was created on the dorsum of each mouse, including both healthy and diabetic groups. To prevent skin contraction from interfering with the evaluation of wound healing, a circular silicone splint (thickness: 1 mm, inner diameter: 13 mm, outer diameter: 21 mm) was fixed around the wound edge with 5-0 nylon sutures. NC and DM mice were administered 100 μL solvent subcutaneously each day, while the DM + omentin-1 group was treated with 100 μL omentin-1 (300 ng/mL) via daily subcutaneous injection¹⁴.

Cell culture

HUVECs were obtained from the Chinese Academy of Sciences Cell Bank. HUVECs were grown in DMEM containing 1% penicillin–streptomycin and 10% FBS. Control cells were kept in 5 mM glucose medium, whereas high-glucose-treated cells were kept in 33 mM glucose medium.

Western blot

Protein samples (equal quantities) were separated by SDS-PAGE and transferred to PVDF membrane (Sigma-Aldrich, Germany). Subsequently, rapid closure buffer (Epizyme Biotech, China) was used for 10 min (min) and specific primary and secondary antibodies were incubated successively. Images were obtained by chemiluminescence imaging.

Transwell migration assay

DMEM resuspension HUVECs without FBS were inoculated with an 8 μm aperture Transwell inserter (upper chamber) (Corning, USA), with DMEM containing 10% FBS added to the lower chamber. After incubation for 8 h, the upper compartment cells were fixed (using 4%PFA), stained with 0.1% crystal violet, and subsequently counted under a microscope.

Scratch assay (wound healing assay)

Upon reaching full confluence in 6-well plates, HUVECs were subjected to a scratch along the central axis using a 200 μL pipette tip of each well. Images were then taken at different points in time.

Tube formation assay

HUVECs (2 × 10^5 cells/Wells) were inoculated in 24-well plates pre-coated with Matrigel for 8 h and recorded using Olympus microscope.

In vitro apoptosis assessment

Cell suspensions from all experimental groups were pelleted in microcentrifuge tubes and washed with PBS. Following PBS aspiration, cells were stained for 15 min with Annexin V-FITC (5 μL) and propidium iodide (10 μL) solution. After staining, cells were washed and reconstituted in fresh Binding Buffer. Subsequently, apoptosis was assessed using flow cytometry.

Colony formation assay

HUVECs were inoculated in a 6-well culture plate (1 × 10³ cells/well). After the cells had adhered and stabilized, omentin-1 (200 ng/mL) or its solvent was administered to each well for treatment, followed by incubation for 12–14 days. After medium removal, the cells were washed twice, fixed with PFA, and then stained with crystal violet (15 min). Following a gentle rinse with ddH2O, images were acquired using a camera.

Histological staining, masson staining, immunohistochemical staining, and tissue immunofluorescence staining

Tissues from each group of mice were harvested and treated with 4% paraformaldehyde, followed by embedding and sectioning. Tissue sections underwent xylene deparaffinization and graded ethanol rehydration. Subsequent staining employed either H&E or Masson’s trichrome protocols. For immunostaining, antigen retrieval was performed using citrate buffer prior to immunohistochemistry or immunofluorescence.

In immunohistochemical analysis, following antigen retrieval, endogenous peroxidase activity was inhibited using hydrogen peroxide. The tissue sections were subsequently incubated with the appropriate primary antibody and secondary antibody (Proteintech, China). Lastly, the sections were treated with DAB substrate solution for staining and mounted using neutral resin.

Immunofluorescence was performed by blocking with 10% BSA, then incubating with primary antibodies and corresponding secondary antibodies. After PBS washes, nuclei were counterstained with DAPI (15 min), washed again, and mounted using antifade medium.

Statistics

All statistical analyses were conducted in GraphPad Prism (v10.1.2). Between-group differences were assessed by student’s t-tests or one-way ANOVA. ns denotes no significant difference. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. The results indicated the mean ± SD.

Result

ITLN1 expression is downregulated in the skin tissue of diabetic mice and in HUVECs exposed to high-glucose conditions

To assess ITLN1 levels in cutaneous tissues of diabetic and healthy mice, dorsal skin samples were obtained from each group. Immunohistochemical staining revealed a reduction in ITLN1 expression in the cutaneous tissue of diabetic mice (Fig. 1A, B). Consistently, Western blotting showed diminished ITLN1 protein levels in diabetic murine skin compared to controls (Fig. 1D, F). In addition, we performed immunofluorescence staining to assess ITLN1 expression in endothelial cells of the dorsal skin in mice, using Platelet Endothelial Cell Adhesion Molecule-1 (CD31) as a marker. The results showed that ITLN1 expression in skin endothelial cells was significantly reduced in diabetic mice compared with healthy controls (Supplementary Fig. 1A, B). RT-qPCR analysis revealed a significant decrease in itln1 transcript levels in the skin tissue of diabetic mice relative to healthy controls (Fig. 1C). Our Western blot data indicated that there was no significant difference in ITLN1 expression between HUVECs cultured at 25 mM glucose and those maintained at normal glucose levels (5 mM) (Supplementary Fig. 2A, B). Furthermore, 33 mM glucose is commonly used to represent a severe hyperglycemic condition, and previous studies have demonstrated that this concentration can induce significant stress responses in endothelial cells^15,16. For these reasons, we selected 33 mM to model hyperglycemia and cultured HUVECs in media containing normal (5 mM) glucose and high (33 mM) glucose. Western blotting showed a marked decrease in ITLN1 levels in HUVECs cultured under 33 mM compared to 5 mM glucose conditions (Fig. 1E, G). Subsequently, we treated HUVECs cultured in 5 mM glucose medium with mannitol (28 mM) and found that mannitol did not cause a reduction in ITLN1 expression. This result demonstrates that the decrease in ITLN1 protein expression in HUVECs is specifically induced by high glucose rather than hyperosmolarity (Supplementary Fig. 3A, B). Together, these data indicate that ITLN1 expression is downregulated in the skin of diabetic mice and in HUVECs exposed to high-glucose conditions.

Fig. 1.

ITLN1 expression is downregulated in the skin tissue of diabetic mice and in HUVECs exposed to high-glucose conditions. (A, B) Representative immunohistochemical staining images showing ITLN1 expression in the dorsal skin tissue of healthy and diabetic mice, accompanied by quantitative analysis (n = 3). Scale bars = 100 μm. (C) Relative mRNA levels of itln1 in the skin tissue of healthy and diabetic mice were measured using RT-PCR (n = 5). (D, F) Representative Western blot images of ITLN1 expression in the dorsal skin tissue from healthy and diabetic mice, along with quantitative analysis (n = 5). (E, G) Representative Western blot images showing ITLN1 expression in HUVECs cultured in 5 mM and 33 mM glucose for 20 days, with quantitative analysis (n = 3).

Omentin-1 enhances wound healing in diabetic mice

To assess the therapeutic potential of omentin-1 in diabetic wound repair, standardized 10-mm full-thickness excisional wounds were created on the dorsal skin of mice in all experimental groups. Wound photographs revealed markedly delayed wound closure in diabetic mice compared to healthy controls, while omentin-1 administration significantly improved the healing process (Fig. 2A, B). Additionally, we collected wound bed skin tissue from each group on day 14 and performed hematoxylin and eosin (HE) staining. The results demonstrated that, in the healthy group, wound re-epithelialization was almost complete, and the granulation tissue progressively thinned, closely resembling normal skin tissue. Compared to the healthy group, diabetic mice exhibited significantly reduced re-epithelialization, less granulation tissue formation, and a visibly larger wound gap. However, following omentin-1 treatment, the wound gap was reduced, re-epithelialization was enhanced, granulation tissue formation increased, and hair follicle density was enhanced (Fig. 2C–E). Subsequently, to further evaluate collagen deposition in the wounds of each group, we performed Masson’s trichrome staining. The results indicated that, in comparison to the healthy group, diabetic mice displayed reduced collagen deposition and a more disorganized arrangement of collagen fibers. However, after omentin-1 treatment, collagen deposition increased (Fig. 2F, G). Furthermore, we evaluated the expression of collagen deposition markers, type I collagen (Collagen Type I), and α-smooth muscle actin (α-SMA), through Western blot analysis. The results indicated that, in comparison to the healthy group, diabetic mice displayed decreased expression of α-smooth muscle actin (α-SMA) and type I collagen in wound tissue. However, omentin-1 treatment significantly increased the expression of these markers (Fig. 2H, I). These findings indicate that omentin-1 promotes wound healing in diabetic mice.

Fig. 2.

Omentin-1 promotes dorsal wound healing in diabetic mice. (A) Photographic documentation of dorsal wound healing across experimental groups. Scale bars = 4 mm. (B) Quantitative analysis of the skin wound area during the healing process for each group of mice (n = 4). (C) Representative images of H&E staining of the wound bed on day 14 post-wound formation. The black dashed lines indicate the boundaries of the wound gap, while the red arrows denote the thickness of the granulation tissue. Scale bars = 1 mm. (D, E) Quantification of wound gap and granulation tissue thickness in the wound bed on day 14 post-wound formation (n = 3–4). (F) Representative image of the wound section with Masson’s trichromatic staining on the 14th day after injury. Scale bar = 50 μm. (G) Quantitative assessment of collagen deposition in the wound bed on day 14 post-wound formation (n = 4). (H, I) Representative Western blot images of type I collagen and α-SMA expression in skin tissue at the wound edges on day 14 following wound formation, accompanied by quantitative analysis (n = 3–4).

Omentin-1 mitigates endothelial dysfunction induced by high glucose and enhances endothelial tube formation

To investigate the effects of elevated glucose concentrations on HUVEC function, cells were cultured for a long time in a medium containing either 5 mM or 33 mM glucose. Subsequently, we performed a scratch assay, which showed that 33 mM glucose significantly reduced HUVEC migration compared to the 5 mM glucose group (Fig. 3A, B). Similarly, Transwell migration assays confirmed this reduction in migratory capacity (Fig. 3C, D). To further assess HUVEC proliferation and tube formation under different glucose concentrations, we conducted colony formation and tube formation assays. The results demonstrated that both proliferation (Fig. 3E, F) and tube formation ability (Fig. 3G–I) were markedly inhibited under 33 mM glucose conditions. These findings indicate that HUVEC function is impaired in a high-glucose environment. Therefore, we further examined the influence of omentin-1 on HUVEC function under high-glucose conditions.

Fig. 3.

High glucose impairs HUVEC migration, proliferation, and tube formation in vitro. (A) Representative micrographs of the scratch assay assessing HUVEC migration ability in 5 mM and 33 mM glucose-containing media. Scale bars = 100 μm. (B) The scratch wound assay was quantitatively evaluated (n = 3). (C) Representative images of the Transwell assay used to assess HUVEC migration ability. Scale bars = 100 μm. (D) Quantitative analysis of the Transwell assay was conducted (n = 3). (E) Representative images of the colony formation assay used to assess HUVEC proliferation ability. (F) Quantitative analysis of the colony formation assay (n = 3). (G) Representative images of the tube formation assay. Scale bars = 100 μm. (H, I) Quantitative assessment of the tube formation assay (n = 3).

First, we used CCK-8 experiments to examine the effects of different concentrations of omentin-1 on HUVECs under high glucose conditions. The results demonstrated that omentin-1 at concentrations of 100, 200, 300, and 600 ng/mL did not exhibit cytotoxicity toward HUVECs, with cell viability significantly enhanced at 200 ng/mL (Fig. 4A). A scratch wound healing assay was performed to evaluate omentin-1’s effects on HUVEC migratory capacity in hyperglycemic conditions. The results demonstrated that omentin-1 at 100 ng/mL and 200 ng/mL significantly promoted HUVEC migration relative to the control group (Fig. 4D, E). Additionally, a Transwell migration assay was performed, revealing that omentin-1 significantly enhanced HUVEC migration without a concentration-dependent effect (Fig. 4F, G). Notably, the colony formation assay also demonstrated that omentin-1 enhanced HUVEC proliferation (Fig. 4H, J). Next, we examined omentin-1’s angiogenic potential. The Matrigel-based tube formation assay demonstrated that omentin-1 promoted angiogenesis (Fig. 4I, K, L). vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine and a major regulator of vascular formation¹⁷. Western blot analysis showed that omentin-1 significantly increased VEGFA expression in HUVECs, with the greatest effect observed at 200 ng/mL (Fig. 4B, C). Collectively, these findings demonstrate that omentin-1 mitigates endothelial dysfunction and enhances angiogenesis under high-glucose conditions.

Fig. 4.

Omentin-1 promotes migratory, proliferative, and angiogenic capacities of HUVECs. (A) Assessment of the effects of varying concentrations of omentin-1 on HUVEC viability measured by the CCK-8 assay (n = 3). (B, C) Representative Western blot images and corresponding quantitative analysis of VEGFA expression in HUVECs treated with different concentrations of omentin-1 (n = 4). (D) Representative images of the scratch assay used to assess HUVEC migration ability. Scale bars = 100 μm. (E) Corresponding quantitative analysis of the scratch assay was performed (n = 3). (F) Representative images of the Transwell assay used to assess HUVEC migration ability. Scale bars = 100 μm. (G) Corresponding quantitative analysis of the Transwell assay was conducted (n = 4). (H, J) Representative images of the colony formation assay used to evaluate HUVEC proliferative capacity, accompanied by quantitative analysis (n = 3). (I) Representative images of the tube formation assay are shown. Scale bars = 100 μm. (K, L) Corresponding quantitative analysis of the tube formation assay was performed (n = 3).

Omentin-1 alleviates high glucose-induced endothelial cell apoptosis

To investigate endothelial cell apoptosis under varying glucose concentrations, we cultured HUVECs in media containing 5, 25, and 33 mM glucose for an extended duration. Subsequently, we assessed cell apoptosis using flow cytometry. The findings demonstrated that apoptosis levels did not differ significantly between HUVECs maintained in 5 and 25 mM glucose conditions; however, a marked increase in apoptosis was observed in HUVECs exposed to 33 mM glucose (Fig. 5A, B). Next, we analyzed the expression levels of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in HUVECs following omentin-1 treatment. Western blot analysis showed that Bcl-2 expression was significantly increased and Bax expression was decreased after omentin-1 treatment compared with the control group (Fig. 5C–F). This suggests the anti-apoptotic effect of omentin-1. Further flow cytometry analysis demonstrated that omentin-1 improved high glucose-induced apoptosis in HUVECs (Fig. 5G, H).

Fig. 5.

Omentin-1 reduces endothelial cell apoptosis induced by high glucose levels in vitro. (A, B) Following prolonged culture of HUVECs in media with 5 mM, 25 mM, and 33 mM glucose, apoptosis was evaluated via flow cytometry combined with quantitative analysis (n = 3). (C–F) Representative immunoblots illustrating Bcl-2 and Bax expression in HUVECs treated with omentin-1 or its control vehicle, accompanied by quantitative analysis (n = 3). (G, H) After treating HUVECs cultured in 33 mM glucose with omentin-1 or its vehicle for an extended period, apoptosis was assessed using flow cytometry, along with quantitative analysis (n = 3).

Omentin-1 facilitates angiogenesis and attenuates endothelial cell apoptosis in the wound bed of diabetic mice

To further confirm the pro-angiogenic effect of omentin-1 in diabetic mice, wound bed skin tissue was harvested from each group on day 14 after wound induction, and key angiogenic markers, particularly VEGFA expression, were analyzed. The findings revealed that VEGFA levels were lower in diabetic wound tissue than in healthy controls. However, VEGFA levels increased following omentin-1 treatment (Fig. 6A, B). Prior in vitro studies have shown that omentin-1 suppresses endothelial cell apoptosis. To investigate its effect on endothelial apoptosis during wound repair in mice, immunofluorescence staining was conducted to detect Platelet Endothelial Cell Adhesion Molecule-1 (CD31), a crucial angiogenic marker, and cleaved caspase-3, a key apoptosis marker, in skin tissues from each group. Double-positive staining for CD31 and cleaved caspase 3 indicates apoptotic endothelial cells. The findings indicated that the apoptosis rate of endothelial cells was significantly higher in diabetic mice compared to the healthy group; however, omentin-1 treatment improved this condition (Fig. 6C, D). Furthermore, in diabetic mice, CD31 expression within the wound bed was markedly lower than in healthy mice but showed a slight increase after omentin-1 administration (Fig. 6C, E). Furthermore, in diabetic mice, Bcl-2 expression was downregulated, whereas Bax expression was upregulated in wound skin tissue compared to healthy controls. However, both markers were reversed following omentin-1 treatment (Fig. 6F, G). These findings demonstrate that omentin-1 promotes angiogenesis and reduces endothelial cell apoptosis in the wound bed of diabetic mice.

Fig. 6.

Omentin-1 promotes angiogenesis and suppresses endothelial cell apoptosis in vivo. (A, B) Representative images of immunofluorescence staining showing VEGFA expression in the dorsal skin tissue of each mouse group, accompanied by quantitative analysis (n = 4). Scale bars = 100 μm. (C–E) Representative images of immunofluorescence staining illustrating CD31 and cleaved caspase-3 expression in the dorsal skin tissue of each mouse group, accompanied by quantitative analysis (n = 3). Scale bars = 100 μm. (F, G) Representative images of Western blot analysis showing Bcl-2 and Bax expression in the dorsal skin tissue across different mouse groups.

Omentin-1 improves high glucose-induced endothelial cell dysfunction by mediating the PI3K/AKT/FOXO1 pathway

As a central signaling hub, the PI3K/AKT pathway orchestrates fundamental cellular processes including cell growth, metabolism, and survival. FOXO1, a downstream target of the PI3K/AKT signaling pathway, is considered a transcription factor for pro-apoptotic genes. Its function is inhibited upon phosphorylation, thereby suppressing apoptosis¹⁸. To further investigate the mechanism by which omentin-1 inhibits endothelial cell apoptosis, HUVECs were pretreated with LY294002, a PI3K inhibitor. Western blot analysis revealed that omentin-1 activated the phosphorylation of AKT and FOXO1, which was subsequently inhibited by LY294002 (Fig. 7A). Subsequently, flow cytometry was performed to assess apoptosis in each group of cells. The results showed that LY294002 abolished the protective effect of omentin-1 against high glucose-induced apoptosis in HUVECs (Fig. 7B, C). Additionally, the scratch assay and Transwell migration assays demonstrated that LY294002 abolished the promoting effect of omentin-1 on HUVEC migration (Fig. 7D–G). Following this, a colony formation assay was performed to evaluate the impact of LY294002 on the proliferation of HUVECs stimulated by omentin-1. The findings demonstrated that LY294002 markedly suppressed the proliferation of HUVECs stimulated by omentin-1 (Fig. 7H, I). Notably, the tube formation assay revealed that the enhancing effect of omentin-1 on HUVEC tube formation was also inhibited by LY294002 (Fig. 7J–L). These findings suggest that omentin-1 mitigates high glucose-induced endothelial cell dysfunction via the modulation of the PI3K/AKT/FOXO1 pathway.

Fig. 7.

Omentin-1 improves high glucose-induced endothelial cell dysfunction through the modulation of the PI3K/AKT/FOXO1 pathway. (A) Representative Western blot images depicted the expression levels of p-AKT, AKT, p-FOXO1, and FOXO1 in HUVECs. (B, C) Flow cytometry was employed to assess apoptosis in HUVECs and conduct quantitative analysis (n = 3). (D) Representative images of the scratch assay used to assess HUVEC migration ability. Scale bars = 100 μm. (E) Quantitative assessment of the scratch assay (n = 3). (F) Representative images of the Transwell assay used to assess HUVEC migration ability. Scale bars = 100 μm. (G) Quantitative assessment of the Transwell assay (n = 3). (H, I) Representative images of the colony formation assay used to evaluate HUVEC proliferative capacity, accompanied by quantitative analysis (n = 3). (J) Representative images from the tube formation assay. Scale bars = 100 μm. (K, L) Quantitative assessment of the tube formation assay (n = 3).

Discussion

Diabetes is a non-communicable disease that poses a growing global health challenge, with an increasing incidence rate each year. Many patients suffer from chronic wounds, and approximately 20% of those with diabetic foot ultimately require amputation³. The wound healing cascade involves four precisely coordinated phases: hemostasis, inflammation, proliferation, and remodeling¹⁹. Upon skin injury, hemostasis and inflammation occur almost simultaneously. Platelets and fibrin rapidly form a hemostatic plug to prevent blood loss, while neutrophils are the first to be recruited to the injury site to clear pathogens, followed by macrophage infiltration. During the proliferative phase, endothelial cells, fibroblasts, and keratinocytes play key roles in angiogenesis, extracellular matrix reconstruction, and re-epithelialization. Finally, the tissue remodeling phase completes the wound healing process. These phases are interconnected, and any disruption at any stage can impair wound healing^20–23. Studies suggest that persistent hyperglycemia leads to thickening of the microvascular basement membrane, impaired vasodilation, endothelial cell damage, increased apoptosis, reduced neovascularization, and ischemic hypoxia at the wound site^24–26. Additionally, apoptotic endothelial cells release large amounts of inflammatory factors, exacerbating wound inflammation and further aggravating endothelial cell damage^27,28. These factors collectively impair vascularization and granulation tissue formation in diabetic wounds, thereby hindering the healing process. In this study, we found that diabetic mice exhibited impaired angiogenesis, increased endothelial cell apoptosis, and a reduced wound healing rate, consistent with previous findings.

This study primarily investigated the interplay between omentin-1, angiogenesis, and endothelial cell function. Omentin-1 is a recently identified adipokine possessing anti-inflammatory and anti-apoptotic properties²⁹. Studies have demonstrated that circulating omentin-1 levels exhibit a negative correlation with diabetes and its complications, including diabetic vascular disease and retinopathy^30–33. Our findings indicated that ITLN1 expression in the skin tissue of diabetic mice was significantly reduced compared to that in healthy mice. Similarly, in vitro experiments demonstrated that ITLN1 expression was lower in HUVECs cultured in the high-glucose medium compared to those in low-glucose conditions. Furthermore, subcutaneous injection of omentin-1 significantly improved wound healing in diabetic mice. Notably, omentin-1 inhibited endothelial cell apoptosis in the wound bed while promoting angiogenesis. Consistent with these findings, in vitro experiments confirmed that omentin-1 suppressed high glucose-induced endothelial apoptosis, enhanced endothelial cell proliferation and migration, and facilitated tube formation. VEGF signaling in diabetes represents a complex network. Previous studies have shown that VEGF not only stimulates vascular growth but also indirectly promotes the resolution of inflammation by improving the microenvironment. High-glucose conditions significantly suppress the secretion and expression of VEGFA in endothelial cells³⁴. VEGF does not act in isolation; VEGFA is thought to exert synergistic effects with other pro-angiogenic factors. Therefore, enhancing VEGF secretion in diabetic wounds is of great importance for wound healing. Our results demonstrate that omentin-1 can enhance VEGFA expression in endothelial cells in vitro and promote tube formation^35,36. Collectively, our study demonstrates that omentin-1 significantly promotes angiogenesis and accelerates wound healing by improving endothelial cell function.

Numerous studies have shown that the PI3K/AKT pathway plays a crucial role in regulating cell survival, proliferation, and metabolism³⁷. FOXO1, a downstream target of PI3K/AKT, is considered to be a key transcription factor involved in apoptosis regulation^38,39. To investigate the mechanism underlying omentin-1’s effects, we treated endothelial cells with the PI3K inhibitor LY294002. Our results indicated that omentin-1 significantly enhanced the phosphorylation of AKT and FOXO1, but this effect was suppressed in the presence of LY294002. Furthermore, PI3K inhibition completely negated the omentin-1-induced proliferation and migration of endothelial cells, along with its protective effect against apoptosis. Notably, omentin-1-mediated tube formation was also suppressed by LY294002. These findings suggest that omentin-1 regulates endothelial cell function and facilitates angiogenesis via the PI3K/AKT/FOXO1 pathway.

However, our study has certain limitations. Due to the complexity of the diabetic wound environment, our in vitro experiments only simulated a high-glucose condition and could not fully replicate the intricate physiological and pathological microenvironment in vivo. In addition, the in vivo experiments in this study were primarily conducted using an STZ-induced type 1 diabetes (T1D) model, which mimics the pathological features of T1D, namely absolute insulin deficiency caused by β-cell destruction. It should be noted that the pathogenesis of type 2 diabetes (T2D) is characterized predominantly by insulin resistance and relative insulin deficiency, and is more common in the population. Therefore, this study may not fully recapitulate the insulin-resistant microenvironment of T2D. Considering the mechanistic differences between T1D and T2D, further validation in T2D models will be required in the future to enhance the translational relevance of our findings. In addition, we did not further investigate the specific receptor of omentin-1, nor did we explore the roles of other VEGF family members (such as VEGF-B, VEGF-C, or VEGF-D), which play distinct roles in angiogenesis. Elucidating these mechanisms is important for a more comprehensive understanding of VEGF-related signaling pathways and will help us gain deeper insights into the complexity of diabetic wounds and the therapeutic role of omentin-1. In conclusion, our study demonstrates that omentin-1 alleviates diabetes-induced endothelial cell dysfunction and enhances angiogenesis, thereby facilitating wound healing in diabetic mice, primarily via the PI3K/AKT/FOXO1 signaling pathway.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

XY, YH, and XD jointly conceived and designed the experiments. YH wrote the manuscript. YH and YD conducted the experiments. Statistical analyses were performed by YH, ZM, and PY. Animal studies, including sample collection, were undertaken by YH, YD, and ZM. XY oversaw the overall research, secured funding, and interpreted the results. All authors critically reviewed and approved the final version of the manuscript.

Funding

This work was financially supported by funding from Project of Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (ZDXM-2020-25), Key Project supported by Medical Science and technology development Foundation, Nanjing Department of Health (YKK24110), and Jiangsu Province Graduate Research and Practice Innovation Program Project (KYCX25_4283).

Data availability

The data used to support the findings of this study are available by request to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.