Introduction

Wound healing in individuals with type 2 diabetes mellitus (T2DM) presents a significant clinical challenge due to impaired healing mechanisms [1,2]. Approximately 15% of diabetic patients develop foot ulcers during their lifetime, with these ulcers being progressed to lower limb amputation. Estimates suggest that 15–25% of patients with diabetic foot ulcers ultimately require amputation [3,4]. Despite advancements in wound dressings and diabetic foot ulcer management, the persistently high incidence of diabetic foot complications implies the limitations of current treatments. This ongoing clinical burden calls for the development of more effective therapeutic approaches, including advanced wound dressings and regenerative medicine strategies, to improve healing outcomes.

Diabetic foot ulcer is multifactorial and closely associated with the systemic dysregulation seen in T2DM. The delayed healing in diabetic wounds is frequently attributed to chronic inflammation, poor vascularization, slow re-epithelialization, deficient extracellular matrix components, and severe peripheral neuropathy [1]. Compromised immune cell function and impaired leukocyte recruitment delay microbial clearance and promote chronic inflammation. This persistent inflammatory state creates a hostile microenvironment that delays the progression to the proliferative phase of healing [5]. Simultaneously, keratinocyte migration and proliferation―critical processes for re-epithelialization―are significantly reduced in diabetic wounds [6]. These interlinked dysfunctions culminate in prolonged wound persistence and a high risk of infection and amputation, underscoring the need for molecular-level interventions targeting the wound microenvironment.

Zinc plays a pivotal role in physiological wound healing due to its involvement in cell proliferation and migration, tissue regeneration, and immune modulation [7]. In the context of diabetes, where inflammation is often prolonged, zinc contributes to the resolution of sustained inflammatory responses by supporting the downregulation of pro-inflammatory signaling pathways and aiding in immune homeostasis. Additionally, zinc promotes re-epithelialization by enhancing keratinocyte proliferation, migration, and differentiation. It supports cytoskeletal organization and growth factor responsiveness, enabling efficient closure and restoration of the epithelial barrier. Given its essential role in wound repair and tissue regeneration, optimal zinc homeostasis is crucial for effective healing, particularly in patients with chronic wounds such as diabetic foot ulcers.

While zinc is essential for orchestrating key phases of wound repair, excessive accumulation in the wound microenvironment can exert detrimental effects, particularly in diabetic conditions. Aberrant zinc levels may lead to cellular toxicity, impair wound healing, and disturb intracellular signaling pathways essential for orderly healing [8]. In particular, excessive intracellular zinc accumulation disrupts mitochondrial function and triggers apoptosis through stress-induced zinc release and signaling pathways [9]. Furthermore, excessive zinc has been shown to inhibit nicotinamide adenine dinucleotide phosphate oxidase activity, thereby compromising the elimination of invading pathogens during the inflammatory phase of wound healing [10]. Elevated zinc levels can suppress T-cell function, further disrupting the immune coordination required for efficient tissue repair [11]. These maladaptive responses suggest that while zinc is beneficial in appropriate concentrations, its dysregulation under diabetic conditions may aggravate, rather than alleviate, delayed wound healing.

This study hypothesizes that co-treatment with a zinc receptor agonist can mitigate the adverse effects of excessive zinc exposure while preserving its therapeutic benefits in diabetic wound healing. Rather than relying on high levels of exogenous zinc, which may lead to cytotoxicity and impaired epithelial regeneration, the use of a zinc receptor agonist aims to activate downstream zinc-mediated signaling pathways in a more controlled manner. By mimicking the beneficial effects of zinc on inflammation resolution and re-epithelialization, this approach may reduce the required zinc dosage while minimizing its accumulation-related toxicity. This strategy offers a targeted means to enhance wound repair under diabetic conditions.

In the present study, we explored the collective effects of zinc and its receptor agonists, particularly GPR39 agonist, on inflammation and cell migration in vitro, as well as their impact on wound healing in vivo using a type 2 diabetic mouse model. Zinc’s cytotoxicity has already been observed, particularly with high concentrations of zinc chloride demonstrating significant cytotoxic effects on human keratinocytes [12,13]. Furthermore, the cytotoxicity of zinc nanoparticles has been identified, leading to extensive research efforts aimed at mitigating its toxic effects [14]. Despite the multitude of beneficial roles zinc plays in human physiology, certain limitations persist. Our hypothesis postulates a synergistic effect with zinc and GPR39 agonists in augmenting the wound recovery process by modulating macrophage activity, thereby maximizing positive outcomes while minimizing cytotoxicity.

Materials and Methods

1. Reagents

Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, and fetal bovine serum (FBS) were purchased from Cytiva. Easy-BLUE^TM and PRO-PREP^TM were obtained from iNtRON Biotechnology. For the first strand cDNA Synthesis, AccuPower RT PreMix was purchased from Bioneer and TB Green Premix Ex Taq was obtained from Takara Bio. The polymerase chain reaction (PCR) primers were synthesized from Cosmogenetech. Antibodies for extracellular signal-regulated kinase (ERK), and phospho-ERK were purchased from Cell Signaling Technology. EZ-CYTOX for cell viability was purchased from DoGenBio. Streptozotocin (STZ) was obtained from Sigma- Aldrich. A high-fat diet was purchased from Research Diets, and isoflurane was also obtained from Hana Pharm.

2. Cell culture

RAW264.7 cells were maintained in RPMI-1640 supplemented with 10 % heat-inactivated FBS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37℃ in 5% CO₂ incubator. RAW264.7 cells were exposed to lipopolysaccharide (LPS), zinc chloride or TC-G-1008 for the indicated time.

HaCaT cells were cultured in high-glucose DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 U/ mL penicillin and 100 µg/mL streptomycin) at 37℃ in 5% CO₂ incubator. HaCaT cells were exposed to LPS, zinc chloride or TC-G-1008 for the indicated time.

3. Cell viability assessment

RAW264.7 cells or HaCaT cells were seeded onto 96-well plates at a density of 10⁴ cells/well and cultured until the confluency of each well reached 80%. The cells were incubated with the indicated concentrations of LPS, zinc chloride, or TCG- 1008 for the specified time. Subsequently, 10 µL of EZCYTOX was added into each well and incubated at 37℃ in 5% CO₂ incubator for 2 hours. The absorbance was measured at 450 nm using a GloMax Explorer Multimode Microplate Reader (Promega).

4. In vitro scratch test

HaCaT cells were cultured in four-well plates (Thermo Fisher Scientific) and grown to the confluence at 37℃ in 5% CO₂ incubator. A scratch was created by manually scraping the cell monolayer with a 200 µL pipette tip. Subsequently, cells were incubated with the indicated concentrations of LPS, zinc chloride, or TC-G-1008 for 48 hours. The wound area was measured using DP72 (Olympus) to compare the remaining wound area at 0 hour and 48 hours after scratch.

5. Transwell migration assay

HaCaT cells were incubated in serum-free conditions overnight. After that, cells were detached and seeded into the upper inserts (8.0 µm pore size; Corning Inc.) at a density 1 × 105 cells/well in 100 µL of DMEM in Transwell Migration Assay plates (Corning Inc.). The lower chamber was added with 600 µL DMEM containing 1% FBS with LPS, zinc chloride, or TCG- 1008 or the conditioned media.

The conditioned media were prepared by treating bone marrow-derived macrophages (BMDMs) with pretreatment of LPS (100 ng/mL) for 24 hours, followed by co-treatment of zinc chloride (30 µM) or TC-G-1008 (1 µM) for an additional 24 hours. The final conditioned medium was obtained by mixing the treated medium with fresh medium at a 1:1 ratio. After 24 hours, cells that had migrated to the lower surface of the insert membrane were fixed, stained with DAPI, and imaged at 200 × magnification. The number of migrated cells was quantified using ImageJ software.

6. Quantitative real-time PCR

Total RNA was isolated from cells using easy-BLUE^TM RNA extraction reagent and complementary DNA was synthesized from 2 µg of total RNA using AccuPower RT PreMix. Quantitative PCR was conducted using TB Green Premix Ex Taq in 7500 Real-Time PCR System (Applied Biosystems). Expression levels of the gene were compared after normalization to internal control, human glyceraldehyde-3-phosphate dehydrogenase (gapdh). The human primer sequences used for quantitative real-time PCR (qRT-PCR) were as follows: gapdh, forward 5’-CCA TCT TCC AGG AGC GAG ATC-3’ and reverse 5’-GCC TTC TCC ATG GTG GTG AA-3’; il-1β, forward 5’-CTC CAG GGA CAG GAT ATG GA-3’ and reverse 5’-TCT TTC AAC ACG CAG GAC AG-3’; il-6, forward 5’-ACT CAC CTC TTC AGA ACG AAT TG-3’ and reverse 5’-CCA TCT TTG GAA GGT TCA GGT TG-3’; Tnf-α, forward 5’-GAG GCC AAG CCC TGG TAT G-3’ and reverse 5’-CGG GCC GAT TGA TCT CAG C-3’; nos2, forward 5’-AAC AAC GTG GAG AAA ACC CCA-3’ and reverse 5’-GGG TCG ATG GAG TCA CAT GC-3’.

7. Animal study

All the procedures adhered to the guidelines outlined in the IACUC Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care (SNU- 230601-7-2). Male C57BL/6 mice, aged two weeks, were obtained from Raon Bio.

8. T2DM induction

Following a two-week quarantine period, T2DM mice were generated by administering a high-fat diet for eight weeks, followed by intraperitoneal injection of STZ (40 mg/kg body weight). Two weeks post-injection, blood samples were collected and hemoglobin A1c (HbA1c) levels were measured using a Mouse HbA1c Assay Kit (ELK Biotechnology).

9. Hydrogel preparation

The 0.04 g of zinc oxide (ZnO) was dispersed in 100 mL of distilled water at room temperature using an ultrasonic homogenizer for 30 minutes. Subsequently, 1.5 g of sodium alginate was dissolved in the ZnO suspension, followed by the addition of 6 mL of glycerol. The mixture was stirred continuously for 16 hours to ensure homogeneity. After stirring, either 10% dimethyl sulfoxide (DMSO) or 1.3 mg of TC-G-1008 dissolved in 10% DMSO was added to the solution. The resulting mixture was degassed for 24 hours to eliminate entrapped air bubbles. Following degassing, a mixed solvent system comprising 40% polyethylene glycol 300, 5% Tween-80, and 45% physiological saline was added to complete the formulation. The final solution was then dried in a conventional oven at 37℃ for 24 hours. To induce cross-linking, an aqueous solution containing 2.5% calcium chloride (CaCl₂) and 2.5% zinc chloride (ZnCl₂) was prepared. This cross-linking solution was poured over the dried hydrogel formulation and allowed to react for 10 minutes. After cross-linking, the hydrogel was thoroughly washed with Milli- Q water to remove residual ions, and excess water was allowed to drain. Finally, the hydrogel was punched into uniform, application-ready sizes using a biopsy punch.

10. In vivo wound healing assay

Normal diet mice [15] and type 2 diabetic mice (DM) were anesthetized with isoflurane. Their dorsal hairs were shaved, and two 8 mm diameter, full-thickness wounds were created with a biopsy punch at either side of the dorsal central line in experimental animals (normal diet mice, n = 10; DM, n = 10). Each group further divided into two subgroups: sodium alginate hydrogel group (SA, n = 5) and ZnO nanoparticle–TC-G- 1008 conjugated sodium alginate hydrogel group (ZT, n = 5). In all groups, the wounds were wrapped with Tegaderm and Coban tape (3M Health Care). Wounds were photographed on days 0, 2, 4, 6, and 8 post-surgery using a camera. The surface wound area was quantified at each time point using the ImageJ software. The rate of wound closure was determined by calculating the wound area (wound closure rate = [wound area at day 0 – wound area at the indicated day] / wound area at day 0).

11. Statistical analysis

All the quantitative data are presented as the mean ± standard deviation. Statistical significance was analyzed by Student’ s t-test or one-way ANOVA with Tukey post hoc analysis by Prism 5. p-value of ＜ 0.05 was considered statistically significant. To analyze the interaction effects of diabetes status and treatment (zinc and TC-G-1008) on wound healing, two-way ANOVA was performed followed by Tukey’s honestly significant difference test. Statistical significance was set at p ＜ 0.05.

Results

1. Non-cytotoxic dose optimization of zinc and TC-G-1008

To explore the cytotoxic effects of zinc and TC-G-1008 on macrophages, the cell viability of RAW264.7 cells was evaluated using a WST assay. Zinc displayed a progressive increase in cell proliferation compared to the control group; nevertheless, cell viability declined post-treatment with 20 µM zinc chloride (Fig. 1A). A notable 70% reduction in cell viability was observed in the group treated with 50 µM zinc (Fig. 1A). The impact of TC-G-1008 was also investigated, revealing a significant drop in cell viability at a concentration of 10 µg/mL, with a declining trend noticeable from 1 µg/mL onwards (Fig. 1B). After analyzing the mRNA levels of proinflammatory cytokines as illustrated in Fig. 2A, the concentrations of 1 µM TC-G-1008 and 30 µM zinc chloride were chosen as the final concentrations. Following stimulation with 100 ng/mL LPS, the cell viability of RAW264.7 cells was evaluated post-co-treatment with zinc chloride and TC-G-1008. While LPS led to a marked reduction in cell viability, the co-treatment reinstated the viability of RAW264.7 cells (Fig. 1C).

2. Suppression of LPS-induced inflammation by zinc and TC-G-1008

Macrophages play a pivotal role in the inflammatory phase of wound healing. Nevertheless, the presence of proinflammatory cytokines poses a significant obstacle to the effective healing of wounds. The current research examines the activation of M1 polarization through the application of 100 ng/mL of LPS, alongside an evaluation of the dose-dependent impact of zinc on anti-inflammatory responses. Remarkably, zinc chloride proved effective in diminishing the mRNA expression of il-1β, il-6, Tnf-α, and nos2 in a dose-dependent manner (Fig. 2A). Moreover, the administration of 30 and 40 µg/mL of zinc chloride led to a notable reduction in the mRNA levels of il-1β, il-6, and Tnf-α (Fig. 2A). To optimize the anti-inflammatory efficacy, 30 µg/mL of zinc chloride was identified as the optimal concentration. Preceding the co-administration of zinc chloride and TC-G-1008, cells were exposed to 100 ng/mL of LPS for 1 hour in advance. Subsequently, a quantification of the mRNA levels of il-1β , il-6 , Tnf-α , and nos2 was conducted. Sole treatment of TC-G-1008 exhibited a reduction in the LPSinduced upregulation of il-1β, il-6 , and Tnf-α, whereas zinc chloride alone not only reduced the expression of il-1β, Tnf-α, and il-6 but also suppressed nos2 levels (Fig. 2B). The combined treatment yielded a further decline in il-6 and tnf- α (Fig. 2B).

3. Promotion of keratinocyte migration

Prior to evaluating the migratory impact of the co-treatment, a WST assay was executed to assess its cytotoxicity. Zinc chloride demonstrated a trend of a dose-dependent increase in cell viability after 24 hours. However, after 72 hours, a significant increase in cell viability was observed only at 70 and 100 µM, while other concentrations did not show statistically significant differences compared to the 0 µM group (Fig. 3A). The assessment of cytotoxicity involved a combination of 1 µM of TC-G-1008 and 30 µM of zinc chloride, revealing no statistically significant variances among the three groups (Fig. 3B).

Subsequent to this, an in vitro scratch assay was carried out to examine the migratory influence of the combination on cells. While LPS impeded keratinocyte migration, the separate application of zinc chloride and TC-G-1008 reinstated migration to basal levels after 48 hours (Fig. 3C). Additionally, the co-treatment notably boosted migration compared to the untreated group (Fig. 3C). To validate these findings, a transwell migration assay was performed. It was observed that LPS did not hinder migration, suggesting that inflammation positively influences cell migration early point. Conversely, zinc chloride and TC-G-1008 promoted HaCaT cell migration compared to LPS treated group, with the combined treatment displaying the most significant impact (Fig. 3D).

4. Enhanced diabetic wound healing with zinc/TC-G-1008 hydrogel

A high-fat diet in conjunction with a solitary administration of low-dose STZ effectively triggered the development of T2DM. The levels of HbA1c were validated in both cohorts (Fig. 4A). After the induction of diabetes for eight weeks, a biopsy punch was utilized to generate lesions on the dorsal skin of the mouse, with the dimensions of the wounds being assessed every 48 hours (Fig. 4B). Delayed wound healing was observed in T2DM groups, and the hydrogel containing both zinc nanoparticles and TC-G-1008 exhibited a capacity to promote wound repair in T2DM groups (Fig. 4C). Strikingly, the rate of wound closure manifested at an earlier time frame in the control group, while in the T2DM group, this phenomenon was observed at a later juncture (Fig. 4D).

5. Inflammation reduction and skin repair by therapeutic hydrogel

To assess the therapeutic potential of zinc in combination with TC-G-1008 under diabetic wound conditions, we performed quantitative PCR using mouse skin tissue samples. Expression levels of the pro-inflammatory cytokines il-1β and Tnf-α were markedly elevated in the DM in DM-SA group. Notably, treatment with the combination of zinc and TC-G-1008 (DM-ZTSA group) led to a significant reduction in il-1β expression, suggesting a potential anti-inflammatory effect (Fig. 5A).

To investigate the paracrine effects of macrophage-mediated inflammation on keratinocyte migration, a transwell assay was performed using HaCaT cells seeded in the upper chamber and conditioned media in the lower chamber. After 24 hours of incubation in transwell, migrated HaCaT cells were quantified to assess cell motility. In the normal diet [15] group, treatment with TC-G-1008 or with both TC-G-1008 and zinc chloride significantly increased migration relative to both the control and LPS (L) groups (Fig. 5B). These findings indicate a robust pro-migratory effect of TC-G-1008 in a non-diabetic inflammatory context. Under diabetic conditions, a significant improvement was observed only with the LPS and TC-G-1008 (L+T) treatment group when compared to LPS alone. The addition of zinc chloride in the LPS, TC-G-1008, and zinc chloride (L+T+Z) treated DM group did not further enhance migration to a statistically significant extent. These results suggest that TC-G-1008 alone is sufficient to partially rescue keratinocyte migration under diabetic inflammatory conditions, potentially by modulating macrophage-derived paracrine signals. These findings collectively suggest that TC-G-1008, particularly in the presence of zinc nanoparticles, can attenuate inflammation and improve cellular responses critically for wound healing in normal groups.

Discussion

This study elucidates the therapeutic potential of a combinational approach using zinc and the GPR39 agonist TC-G-1008 in promoting wound healing, particularly under diabetic conditions. Our findings highlight the dual capacity of this treatment strategy to attenuate inflammation and enhance keratinocyte migration, two critical components frequently impaired in chronic wounds associated with T2DM.

In vivo experiments using a type 2 diabetic mouse model confirmed that the hydrogel formulation containing both ZnO nanoparticles and TC-G-1008 significantly accelerated wound closure. Although delayed healing was evident in diabetic groups compared to controls, the co-treated DM exhibited improved wound contraction rates, particularly at later time points (Fig. 4C). These results highlight the potential of this therapeutic strategy in overcoming the chronic inflammation and impaired cellular responses that hinder wound repair in diabetic conditions.

Mechanistically, the activation of ERK signaling is a key driver of keratinocyte migration [16]. Western blot analysis demonstrated that TC-G-1008 substantially increased ERK phosphorylation and co-treatment with zinc chloride further augmented this effect (Supplementary Fig. 1). This implies a synergistic activation of the ERK pathway, which may contribute to the enhanced migratory behavior of keratinocytes observed in vitro.

Importantly, the anti-inflammatory effects of the combined treatment were evident at the molecular level. qRT-PCR analysis of diabetic mouse skin revealed a marked reduction in the expression of il-1β following the hydrogel conjugated with ZnO nanoparticles and TC-G-1008, suggesting sufficient suppression of pro-inflammatory signaling (Fig. 5A). Furthermore, a BMDM-keratinocyte Transwell migration demonstrated that TC-G-1008, particularly under non-diabetic inflammatory conditions, robustly enhanced keratinocyte migration. Under diabetic conditions, TC-G-1008 alone significantly improved migration, while the addition of zinc did not further enhance this effect, indicating that TC-G-1008 may serve as the primary modulator of paracrine signaling in this context.

The role of TC-G-1008 in regulating keratinocyte behavior has been previously suggested, with one study reporting its ability to promote keratinocyte proliferation via ERK pathway activation [17]. Building upon this finding, our study provides further evidence supporting the contribution of TC-G-1008 to wound healing, particularly by enhancing keratinocyte migration with zinc application. These results suggest that TCG- 1008 has multifaceted effects on keratinocyte function, acting through ERK signaling to modulate both proliferation and migration. This positions TC-G-1008 as a promising pharmacological agent capable of addressing multiple deficiencies in diabetic wound repair, with the potential for even greater synergistic effects when combined with zinc.

In complex clinical contexts where topical zinc use is limited ―such as in Wilson’s disease or certain dermatological conditions―a personalized therapeutic approach may involve zinc dose reduction combined with TC-G-1008 [18]. This strategy could help mitigate potential adverse effects while maintaining therapeutic efficacy. In cases where the optimal concentration of zinc varies among patients, personalized zinc dosing could be implemented. With the aid of three-dimensional printing technologies, patient-specific hydrogel patches could be readily fabricated and applied, offering a customizable and scalable solution for individualized wound care. Moreover, if the therapeutic efficacy of the zinc and TC-G-1008 combination demonstrated in this study is validated in clinical settings with optimal concentrations, it could serve as a promising alternative to existing effector molecules incorporated in commercial wound dressings. This approach may facilitate direct integration into current bandage formulations without requiring substantial changes in manufacturing infrastructure, thus enabling efficient scale-up and commercialization.

The combination of zinc and TC-G-1008 exhibited a synergistic effect, significantly enhancing keratinocyte migration in vitro and promoting wound healing in vivo under diabetic conditions. However, it remains unclear whether this synergy arises specifically from zinc’s amplification of GPR39 signaling or parallel, independent actions of zinc on cellular behavior. Zinc is known to influence numerous signaling cascades, and its interaction with GPR39 may not be limited to receptor agonism. Future studies aimed at dissecting the mechanistic basis of this synergy are essential to fully exploit its therapeutic potential in chronic wound management. Additionally, investigating the effects of this combination therapy on other critical cell types involved in wound healing, such as fibroblasts and endothelial cells, will help establish a comprehensive understanding of its therapeutic scope. Elucidating the downstream targets of GPR39 activation and zinc-mediated signaling will also provide valuable insights into the mechanism of the synergy of zinc and TC-G-1008.

In conclusion, the combined application of zinc and TC-G- 1008 represents a promising strategy for enhancing wound healing in T2DM by concurrently mitigating inflammation and stimulating keratinocyte migration. This dual-action approach may offer significant advantages in the clinical management of chronic wounds, particularly in diabetic patients.