GRAPHICAL ABSTRACT

Highlights

• PKCδ is upregulated in vascular endothelial cells under diabetic conditions.

• PKCδ inhibits GAD1 expression and downregulates GABA synthesis.

• Reduced GABA impairs vascular endothelial cell functions.

• Reduced GABA leads to delayed diabetic wound healing.

INTRODUCTION

Diabetes mellitus stands as a prevalent and detrimental chronic metabolic disorder characterized by elevated blood glucose levels stemming from insulin insufficiency or resistance. The global burden of diabetes is escalating, with projections estimating an increase in prevalence from 10.5% (536.6 million) in 2021 to 12.2% (783.2 million) in 2045 among individuals aged 20 to 79 years [1]. Chronic complications of diabetes mellitus, also known as vascular complications, are the most serious complications in diabetic patients. Diabetic vascular complications can be categorized into macrovascular and microvascular complications [2]. Macrovascular complications encompass ischemic heart disease, stroke, and peripheral artery disease induced by atherosclerosis, while microvascular complications include diabetic retinopathy, nephropathy, neuropathy, and emerging insights into microvascular abnormalities in diabetic cardiomyopathy [3,4].

Among the array of complications, diabetic foot ulcer (DFU) emerges as a multifaceted consequence affecting approximately 18.6 million individuals globally each year, predisposing patients to heightened amputation risks and mortality rates [5]. Despite its clinical significance, DFU poses challenges in treatment efficacy and recurrence prevention [6]. The intricate process of wound healing unfolds through sequential phases of coagulation, inflammation, migration-proliferation, and remodeling, orchestrated by various cellular components such as platelets, inflammatory cells, keratinocytes, fibroblasts, and endothelial cells [7]. Of particular importance in wound healing is angiogenesis, a fundamental process where endothelial cells play a pivotal role. However, in the context of diabetes mellitus, angiogenesis is impaired, characterized by diminished proliferation and migration alongside increased apoptosis of endothelial cells [8].

The pathogenesis of diabetic vascular complications implicates the activation of diverse pathways including the polyol pathway, nonenzymatic glycation pathway, advanced glycation end products pathway, reactive oxygen species pathway, and diacylglycerol (DAG)-protein kinase C (PKC) pathway [9]. PKC is a group of serine/threonine protein kinases that govern multiple signal transduction cascades. They can be divided into three subgroups including conventional PKCs (PKCα, PKCβ, and PKCγ), novel PKCs (PKCδ, PKCε, PKCθ, and PKCη), and atypical PKCs (PKCζ and PKCι/λ). Several PKC isoforms including PKCα, PKCβ, PKCδ, PKCε, and PKCζ have been reported to be activated in vascular cells and tissues, and mediate the progression of diabetic complications. PKCδ, the first identified novel PKC isoform, can be activated independently of calcium by DAG or phorbol esters. Previous investigations have highlighted the involvement of PKCδ in diabetic wound healing. Khamaisi et al. [10] found that PKCδ expression was elevated in fibroblasts from diabetic patients and that PKCδ inhibition in fibroblasts restored their wound repair function. Lizotte et al. [11] also reported that PKCδ inhibition restored pro-angiogenic factors expression and collateral vessel formation. Our prior research delineated the role of PKCδ in mediating high glucose (HG)-induced endothelial apoptosis by upregulating cholesterol synthesis enzymes, thereby impeding wound healing progress. Nevertheless, the comprehensive role of PKCδ in diabetic wounds necessitates further exploration.

Gamma-aminobutyric acid (GABA), a pivotal neurotransmitter in the central nervous system, exhibits the intriguing capacity for synthesis and release by endothelial cells [12]. The enzyme glutamate decarboxylase (GAD) governs GABA synthesis, comprising two isoforms, GAD1 and GAD2, both expressed in endothelial cells. Furthermore, endothelial cells express GABA receptors, namely GABAA and GABAB receptors, underscoring the regulatory role of endothelial-derived GABA in diverse cellular functions, encompassing adenosine triphosphate synthesis, fatty acid metabolism, and protection against oxidative stress [13]. While GABA has been implicated in modulating cell behaviors such as proliferation, migration, and apoptosis, its impact on endothelial functions, including proliferation, migration, apoptosis, tube formation, and diabetic wound healing, remains unexplored [14-16]. This study elucidates that the suppression of the GAD1-GABA pathway by PKCδ leads to diminished endothelial proliferation, migration, tube formation, and enhanced apoptosis, culminating in delayed diabetic wound healing. Topical application of GABA expedites diabetic wound recovery, offering a novel therapeutic avenue for managing diabetic wounds.

METHODS

Chemicals and antibodies

The chemicals utilized in the study included: γ-aminobutyric acid (#HY-N0067, MedChemExpress, Monmouth Junction, NJ, USA), 3-mercaptopropionic acid (3-MPA; #M5801, Sigma, St. Louis, MO, USA), streptozocin (#HY-13753, MedChemExpress). The antibodies employed in the study included: GAD67/GAD1 Rabbit mAb (#A1475, ABclonal Technology, Woburn, MA, USA), caspase 3/p17/p19 polyclonal antibody (#19677-1-AP, Proteintech, Rosemont, IL, USA), β-actin monoclonal antibody (#66009-1-Ig, Proteintech), horseradish peroxidase (HRP)-conjugated affinipure goat anti-rabbit immunoglobulin G (IgG)(H+L) (#SA00001-2, Proteintech), HRP-conjugated affinipure goat anti-mouse IgG(H+L) (#SA00001-1, Proteintech), CoraLite488-conjugated goat anti-rabbit IgG(H+L) (#SA00013-2, Proteintech).

Cell culture and treatment

Human umbilical vein endothelial cells (HUVECs) were acquired from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured at 37°C in a 95% air-5% CO2 atmosphere. Normal glucose (NG; 5.5 mM glucose)-Dulbecco’s Modified Eagle Medium (DMEM; #PM150220, Procell Life Science&Technology, Seoul, Korea), supplemented with 10% fetal bovine serum (FBS; #164210, Procell Life Science & Technology) and 1% antibiotics was employed for conventional cell culture. HG (22.5 mM glucose)-DMEM (#PM150210, Procell Life Science&Technology), supplemented with 10% FBS and 1% antibiotics, was used to mimic a diabetic condition.

Animal and experimental design

Specific pathogen-free C57BL/6 mice (male, 4 weeks old) and db/db mice (male, 8 weeks old) were acquired from SHULAIBAO Biotech (Hubei, China) and bred in a temperature- and humidity-controlled environment with a 12-hour light/dark cycle. Animal care and experimental procedures were approved by the Principles of Animal Use Committee (NIH Publications No. 8023, revised 1978). To establish a mouse model of streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM), mice were fed a high-fat diet for a month. After an overnight fast, one-time intraperitoneal (i.p.) STZ injection (100 mg/kg in 0.1 mol/L citrate buffer, pH 4.5) was done. After 3 days, non-fasted glucose was measured from tail blood, and successful induction of diabetes was confirmed if blood glucose was over 16.67 mM. Control mice were treated in the same way except for i.p. injection of vehicle (citrate buffer). Two weeks after diabetes model was established, serum and dorsal skin tissues were collected to detect GAD1 expression and GABA concentration.

Full-thickness skin wound model was used to monitor the rate of wound healing under different treatments. After anesthesia with sodium pentobarbital (50 mg/kg i.p.), mice were shaved on the back and further depilated with depilatory creams. Circular full-thickness wounds of 6 mm in diameter were made on the back of the mice. To figure out the effect of GABA on wound healing, 100 μL GABA (5 μM) or 100 μL vehicle (phosphate buffered saline [PBS]) was given topically onto each wound daily. To investigate the effects of PKCδ inhibition and GAD1 inhibition on wound healing, mice were divided into three groups: scramble siRNA (si-Scr) group, siRNA targeting the human PKCδ gene (si-PKCδ) group, and si-PKCδ+3-MPA group. For localized knockdown of PKCδ, 8 μL lipo8000 and 0.2 nmol si-PKCδ were diluted in 100 μL PBS and injected surrounding the wounds at 0, 2, 4, 6, 8 days. si-Scr were injected in the same way as control. A 50 μL 3-MPA (1 mM) or 50 μL PBS was given topically onto each wound at 1, 3, 5, 7, 9 days to inhibit GAD1. Photos were taken at 0, 2, 4, 6, 8, 10 days to monitor the wound healing process, and skin around the wounds was collected at 10 days for further staining.

siRNA and plasmid transfection

si-PKCδ and si-Scr were designed and synthesized by GenePharma (Shanghai, China) with specific sequence: si-PKCδ, -GCU GCC AUC CAC AAG AAA UTT- (sense) and -AUU UCU UGU GGA UGG CAG CTT- (antisense); si-Scr, -UUC UCC GAA CGU GUC ACG UTT- (sense) and -ACG UGA CAC GUU CGG AGA ATT- (antisense). The overexpression plasmid of human GAD1 was constructed on the basis of pCDNA3.1. The full-length cDNA of GAD1 was directly synthesized according to its gene sequence and inserted into the XbaI and BamHI sites of the pCDNA3.1 vector (Generalbiol, Anhui, China). Transient transfection was performed using Lipo8000 (#C0533, Beyotime, Shanghai, China) according to the manufacturer’s instructions. For a well of a 6-well plate, 4 μL lipo8000 and 2.5 μg DNA or 200 pmol siRNA were mixed into 125 μL serum-free DMEM medium and incubated for 20 minutes at room temperature. The mixture was then added into a well containing 2 mL serum-free DMEM and cultivated for 6 hours before being replaced with fresh medium. Fresh medium with 1% FBS was used for siRNA transfection and 10% FBS for plasmid transfection.

Cell counting kit 8 assay

Cell counting kit 8 (CCK8) assay (#BL1055D, Biosharp, Hefei, China) was used to examine cell viability according to the manufacturer’s protocol. Approximately 1,500 cells per well were seeded in 96-well plates with five replicates per group. Cell viability was detected every 24 hours post-transfection or drug treatment. CCK8 solution was diluted to one-tenth of the original concentration and 100 μL diluted CCK8 solution was added to a well. The absorbance was detected at 450 nm after incubating for 1.5 hours at 37°C.

Cell cycle assay

Cell cycle assay was performed with a commercial kit (#C1052, Beyotime). HUVECs were detached with 0.25% trypsin and washed once with PBS, followed by 70% ethanol fixation overnight at 4°C. After washing once with PBS, cells were incubated in staining buffer for 30 minutes at 37°C, which contained propidium iodide (PI) and RNase. Flow cytometry was used to detect fluorescence, and FlowJo v10.6.2 (Tree Star Incorporation, Ashland, OR, USA) was used to analyze cell cycle.

EdU assay

5-Ethynyl-2´-deoxyuridine (EdU) assay was performed according to the instructions of a commercial kit (#C0071S, Beyotime). Following treatment, EdU was added to the cells at a final concentration of 10 μM and incubated for another 2 hours. Next, cells were fixed with 4% paraformaldehyde for 15 minutes and permeated with 0.1% Triton X-100 for 10 minutes at room temperature. Then, reaction buffer containing Azide 488 was added and incubated in dark for 30 minutes at room temperature. Finally, nuclei were stained with 4´,6-diamidino-2-phenylindole (DAPI) and the fluorescence was visualized with a fluorescence microscope. ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the ratio of EdU-positive cells.

Scratch wound healing assay

HUVECs were seeded in 6-well plates and incubated at 37°C. Once the cells attached and formed a monolayer, vertical scratches were made using a sterile 1 mL pipette tip. Cells were then rinsed with PBS and replaced with fresh serum-free medium. Images were taken at 0, 24, 48, and 72 hours postscratch. Wound area quantification was performed with ImageJ software. Migrated cells were normalized as (initial wound area–remaining wound area)/(initial wound area)×100%.

Tube formation assay

A 50 μL matrigel (#356234, Corning Inc., Corning, NY, USA) per well was added to a 96-well plate and polymerized for 30 minutes at 37°C. Thirty thousand HUVECs resuspended in 100 μL serum-free medium were added to each well. Cells were then incubated at 37°C for 6 hours before visualization with a microscope. The images were analyzed with ImageJ software.

Metabolomics analysis

Six replicates per group were set and 1×10⁷ cells per sample were collected. The metabolites were extracted and analyzed by BGI company (Shenzhen, China). The extraction process involved mixing the sample with extraction reagent, grinding with magnetic beads, and subsequent centrifugation. The resulting supernatant was analyzed using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS), which was performed by waters 2777c (waters, Milford, MA, USA) in series with Q Exactive HF high resolution mass spectrometer (Thermo Fisher Scientific). Then, the off-line data of mass spectrometry was imported into compound discoverer 3.3 (Thermo Fisher Scientific) software and analyzed in combination with bmdb (BGI metabolome database), mzcloud database and chemspider online database. A data matrix containing information such as metabolite peak area and identification results was obtained. The subsequent analysis was performed on the BGI platform.

Quantitative real-time polymerase chain reaction

Quantitative polymerase chain reaction (qPCR) was implemented to detect the mRNA expression of GAD1. Total RNA was extracted using TRIZOL (#R401-01, Vazyme Biotech, Nanjing, China), followed by reverse-transcription utilizing HiScript III RT SuperMix for qPCR (#R323, Vazyme Biotech) as per the manufacturer’s instructions. Subsequently, qPCR was implemented with ChamQ SYBR qPCR Master Mix (#Q311-02, Vazyme Biotech,). The primers used were as follows: human GAD1, -GCG GAC CCC AAT ACC ACT AAC- (forward) and -CAC AAG GCG ACT CTT CTC TTC- (reverse); mouse GAD1, -AAC GTA TGA TAC TTG GTG TGG C- (forward) and -CCA GGC TAT TGG TCC TTT GTA AG- (reverse). β-Actin was used as a normalization control. All primers were obtained from Sangon Biotech (Shanghai, China).

Western blot

Cell and skin tissue lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (#P0013K, Beyotime) supplemented with 1% protease inhibitors (#HY-K0010, Med-ChemExpress) and 1 mM phenylmethylsulfonyl fluoride (PMSF; #G2008-1ML, Servicebio, Wuhan, China). The supernatant was collected after centrifugation and protein levels were quantified with a bicinchoninic acid (BCA) kit (#P0010, Beyotime). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking in 5% skim milk for 1 hour at room temperature, the membrane was probed with primary antibodies overnight at 4°C, then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Protein bands were visualized with an enhanced chemiluminescence (ECL) reagent (#BL520A, Biosharp), and densitometry analysis was conducted using ImageJ software.

Immunofluorescence

Cells were cultured on coverslips in 12-well plates. After treatment, cells were rinsed with PBS. Next, cells were fixed with 4% formaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, followed by blocking with 1% bovine serum albumin (BSA) for 30 minutes. Then, primary antibody of GAD1 in a 1:100 blocking solution was added to each coverslip and incubated overnight at 4°C. After that, cells were incubated in CoraLite488-conjugated goat anti-rabbit antibody (1:200) for 1 hour at room temperature in the dark. Finally, nucleus was stained by DAPI for 5 minutes and the fluorescence was visualized with a fluorescence microscope. ImageJ software was employed for fluorescence intensity quantification.

Flow cytometry apoptosis assay

Cell apoptosis was examined with a commercial kit (#C1062L, Beyotime) following the prescribed protocol. HUVECs were detached with trypsin solution devoid of ethylenediaminetetraacetic acid (EDTA; 0.25% trypsin) and washed once with PBS. Cells were then resuspended in 100 μL staining buffer containing 5 μL Annexin V-fluorescein isothiocyanate (FITC) and 5 μL PI. After incubating for 20 minutes in the dark at room temperature, fluorescence signals were detected with a flow cytometry. FlowJo v10.6.2 was used to analyze the results.

GABA concentration assay

To detect the GABA concentration in cell culture medium, HUVECs were seeded in a 12-well plate with 1 mL medium per well. A commercial kit (#JL-T0731, Jonln, Jonlnbio, Shanghai, China) was used following the instructions. The detection mixture was boiled for 10 minutes and cooled down to room temperature in ice bath. The absorbance at 645 nm was measured for GABA concentration quantification. GABA concentrations in mouse serum and skin tissue were detect with an enzyme-linked immunosorbent assay (ELISA) kit (#RE10172, ReedBiotech, Wuhan, China), which is more sensitive. Skin tissues (30 mg with 300 μL PBS) were ground by a tissue grinder and supernatant was isolated by centrifugation. Both supernatant and serum were diluted ten times for detection. A 50 μL diluted sample and 50 μL biotinylated detection antibody were added to a well and incubated for 30 minutes at 37°C. Next, 100 μL HRP-conjugated working solution was added and incubated for another 30 minutes at 37°C. Then, 100 μL substrate reagent was added and incubated for 15 minutes at 37°C. Finally, 50 μL stop solution was added and the absorbance at 450 nm was detected. Standard curve was also made to calculate GABA concentrations of samples.

Histological staining

The skin specimens surrounding the wound were harvested at 10 days. Then, they were fixed with 4% paraformaldehyde, dehydrated by gradient ethanol, embedded in paraffin, and cut into 5 μm thick sections. For immunohistochemical staining of CD31, paraffin sections were deparaffinized and rehydrated in gradient ethanol. Next, sections were immersed in sodium citrate (10 mM, PH 6.0) and heated in a microwave for 15 minutes to retrieve the antigen. A 1% H₂O₂ was used to block endogenous peroxidase. Then, the sections were blocked in 1% BSA for 30 minutes at room temperature, followed by CD31 antibody (1:1,000) incubation overnight at 4°C. Subsequently, sections were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. A 3,3ʹ-diaminobenzidine (DAB) kit (#P0202, Beyotime) was used to show the positive signal. Finally, nuclei were stained with hematoxylin before mounting. All stained slices were visualized with a microscope and analyzed with ImageJ software.

Statistical analysis

Data are presented as the mean±standard error of the mean from at least three independent experiments. Unpaired Student’s t-test was used to test the significance of difference between two groups. A P<0.05 was considered statistically significant. Statistical analysis was performed by GraphPad Prism version 8.00 (GraphPad Software Incorporation, San Diego, CA, USA).

Ethics approval and consent to participate

The study was performed following the guidelines provided by the Institutional Animal Care and Use Committee (IACUC) and approved by the Animal Ethics Committee of Huazhong University of Science and Technology ([2022] IACUC Number: 3732).

RESULTS

PKCδ knockdown promoted proliferation, migration, and tube formation of HUVECs under HG conditions

HG conditions are known to impede proliferation, migration, and tube formation while promoting apoptosis of human dermal vascular endothelial cells and HUVECs [17-19]. In our prior research, we confirmed the upregulation of PKCδ in HG-treated HUVECs as well as in the skin of diabetic mice compared with their NG controls, and we demonstrated the role of PKCδ in mediating HG-induced apoptosis in HUVECs. However, the specific impact of PKCδ on proliferation, migration, and tube formation of HUVECs necessitates further investigation.

Utilizing CCK8 assay, cell cycle analysis, and EdU assay, we evaluated the proliferation of HUVECs under HG conditions following PKCδ knockdown. Our results revealed a significant increase in cell viability, as indicated by higher absorbance at 450 nm in the si-PKCδ group compared to controls at 24 and 48 hours post-transfection (Fig. 1A). EdU assay confirmed a higher ratio of EdU-positive cells in PKCδ-knockdown HUVECs 48 hours post-transfection, indicative of enhanced proliferation (Fig. 1B and C). Cell cycle assay further supported these findings, showing a notable increase in the percentage of proliferative cells (G1+S%) in the si-PKCδ group (20.1%) compared to the si-Scr group (15.8%) 48 hours post-transfection (Fig. 1D and E). To assess migration capacity, scratch wound healing assays were conducted, demonstrating a heightened migration rate in HUVECs following PKCδ knockdown. The PKCδ-knockdown group exhibited significantly increased migration rates at 24, 48, and 72 hours compared to control HUVECs (Fig. 1F and G). Additionally, PKCδ knockdown promoted tube formation in HUVECs, characterized by increased junctions, meshes, and nodes at 48 hours post-transfection (Fig. 1H and I). These results revealed that PKCδ knockdown promoted proliferation, migration, and tube formation of HUVECs under HG conditions

Metabolomics analysis and experimental validation reveal decreased GABA synthesis in PKCδ-knockdown HUVECs under HG conditions

Combined with our previous research, we have proved that PKCδ regulated the proliferation, migration, tube formation, and apoptosis of HUVECs under HG conditions. Then, metabolomics analysis was performed to identify potential downstream metabolites affected by PKCδ knockdown. HUVECs were culture in HG-medium and transfected with si-PKCδ or si-Scr. Seventy-two hours after transfection, metabolites were extracted and detected with UPLC-MS. The subsequent analysis was performed on the BGI Dr. Tom platform (https://www.bgi.com/global/service/dr-tom), which identified 20 differentially expressed metabolites (DEMs) between the two groups. Compared with control HUVECs, 16 metabolites were upregulated and four metabolites were downregulated in si-PKCδ-knockdown HUVECs (Fig. 2A and B). The correlation between DEMs as well as their levels were shown in heat maps (Fig. 2C and D). GABA, in addition to being a well-known neurotransmitter, also plays a role in enhancing the protective functions of the skin and promoting wound healing [20-23]. Therefore, we further focused on GABA and its metabolic pathways in this context. Since GAD catalyzes the formation of GABA from glutamate, which is the main pathway for GABA synthesis. We further analyzed data from previous transcriptome sequencing (CNP0004333) and found an upregulation of GAD1 transcription following PKCδ knockdown, indicating a potential inhibitory effect of PKCδ on GAD1 expression and GABA production in HG-treated HUVECs (Fig. 2E). Thus, we may infer that PKCδ inhibits the expression of GAD1 and the production of GABA in HG-treated HUVECs. This comprehensive analysis shed light on the intricate interplay between PKCδ, GABA metabolism, and endothelial cell responses in the diabetic context. To validate the metabolomics findings, GABA levels and GAD1 expression were examined in HUVECs following PKCδ knockdown under HG conditions. Measurement of GABA concentration in the culture medium revealed a 1.2-fold increase in PKCδ-knockdown HUVECs compared to control HUVECs 72 hours after transfection, corroborating the metabolomics data (Fig. 2F). Subsequent assessments of GAD1 mRNA and protein levels 48 hours posttransfection demonstrated a significant upregulation in the si-PKCδ group, with approximately 1.5-fold higher expression compared to controls (Fig. 2G-I). Immunofluorescence analysis further supported these findings, showing a higher fluorescence intensity of GAD1 in PKCδ-knockdown HUVECs (Fig. 2J and K). These results collectively indicated that PKCδ knockdown promoted GABA production and GAD1 expression in HUVECs under HG conditions, reinforcing the regulatory role of PKCδ in GABA metabolism.

Decreased GABA levels and GAD1 expression in HG-treated HUVECs and skin tissues of diabetic mice

Since PKCδ was upregulated in HG-treated HUVECs and in the skin of diabetic mice and inhibited GAD1 expression and GABA synthesis, we examined GAD1 expression and GABA levels under diabetic conditions. HUVECs were cultivated in NG and HG media, respectively. Seventy-two hours posttreatment, the GABA concentration in the medium was examined, revealing an 11.2% decrease in GABA levels in HG-treated HUVECs compared to those in NG-treated HUVECs (Fig. 3A). The mRNA level of GAD1 was assessed 48 hours posttreatment, showing an 84.3% reduction in the HG group (Fig. 3B). Furthermore, the protein level of GAD1 was also decreased by about 40% in HG-treated HUVECs 72 hours posttreatment, as evidenced by Western blot and immunofluorescence (Fig. 3C-F).

Furthermore, the GABA concentration in the serum and skin tissue of STZ-induced T2DM mice was determined. Two weeks after establishing the diabetic mouse model, serum and dorsal skin tissues were collected. Mice injected with vehicle were used as control. ELISA showed decreased concentrations of GABA in serum and skin tissues of diabetic mice compared to control mice (Fig. 3G and H). The mRNA and protein levels of GAD1 in skin tissues were also notably lower in diabetic mice (Fig. 3I and J). In addition, we examined the expression of GAD1 in the skin of db/db mice as well as the concentration of GABA in serum and skin tissues, and the results also showed that GAD1 expression and GABA concentration were lower in db/db mice compared with normal mice (Supplementary Fig. 1A-C). These findings collectively indicate a reduction in GABA production and GAD1 expression in HG-treated HUVECs and skin tissues of diabetic mice.

Inhibition of GAD1 by 3-MPA suppressed proliferation, migration, and tube formation and promoted apoptosis of HUVECs under NG conditions

Building upon previous findings, it can be inferred that PKCδ mediates the inhibition of HUVECs’ proliferation, migration, tube formation, and promotion of apoptosis under HG conditions. PKCδ is also implicated in the downregulation of GAD1 expression and GABA production induced by HG. To investigate the involvement of the PKCδ-GAD1-GABA pathway in the effects triggered by HG, GAD1 expression and GABA levels were modulated, and cellular phenotypes were evaluated.

HUVECs were cultivated in NG and the phenotypes were detected after treatment with 3-MPA (1 mM), an effective inhibitor of GAD1. Forty-eight hours after treatment, the 3-MPA group exhibited significantly decreased cell viability compared to the control group (Fig. 4A). The proportion of EdU-positive cells was also diminished after 3-MPA treatment for 48 hours (Fig. 4B and C). Cell cycle analysis indicated a notable decrease in the percentage of proliferative HUVECs from 55.6% to 49.2% after 48 hours of treatment (Fig. 4D and E). Apoptosis assays conducted 48 hours post-treatment revealed higher expression of cleaved caspase 3 and an increased percentage of apoptotic cells in 3-MPA-treated HUVECs compared to controls (14.9% vs. 6.0%) (Fig. 4F-I). Furthermore, 3-MPA significantly impeded HUVECs’ migration after treatment for 48 and 72 hours by 17.0% and 24.4%, respectively (Fig. 4J and K). Tube formation assay also demonstrated impaired function of HUVECs following 3-MPA exposure (Fig. 4L and M). To conclude a relationship between the GAD1-GABA pathway and PKCδ in diabetic wound healing process, the proliferation and migration of PKCδ-downregulated HUVECs treated with 3-MPA were examined under high-glucose conditions. The results showed that the effect of PKCδ downregulation on cell proliferation and migration in HG-treated HUVECs are nullified by GAD1 inhibition (Supplementary Fig. 2). Moreover, in vivo experiments also illustrated that PKCδ inhibition promoted diabetic wound healing, which was abrogated by GAD1 inhibition (Supplementary Fig. 3A and B). These results demonstrated that inhibition of GAD1 by 3-MPA inhibited proliferation, migration, and tube formation and promoted apoptosis of HUVECs under NG conditions.

Overexpression of GAD1 promoted proliferation, migration, and tube formation and inhibited apoptosis of HUVECs under HG conditions

In this section, the impact of GAD1 overexpression on the phenotypes of HUVECs cultured in HG medium was investigated. The efficacy of GAD1 overexpression plasmid (oe-GAD1) was confirmed by qPCR and Western blot 48 hours post-transfection. The mRNA level of GAD1 exhibited an approximately 1,000-fold increase, while the protein level showed nearly a 4-fold increase, validating the effectiveness of the GAD1 overexpression plasmid (Supplementary Fig. 4A-C).

Subsequent cellular assays revealed that 48 hours post-oe-GAD1 transfection, there was a notable enhancement in cell viability compared to the control overexpression plasmid (oe-Ctr) as indicated by the CCK8 assay (Supplementary Fig. 4D). The percentage of cells in proliferative phase was also upregulated after oe-GAD1 transfection for 48 hours, corroborated by both EdU and cell cycle assays (Supplementary Fig. 4E-H). Additionally, the expression of cleaved caspase 3 decreased after oe-GAD1 transfection for 48 hours, leading to a reduction in the ratio of apoptotic cells from 13.6% to 7.3% as determined by flow cytometry (Supplementary Fig. 4I-L). The scratch wound healing assay demonstrated a higher migration rate of HUVECs overexpressing GAD1 (Supplementary Fig. 4M and N). Furthermore, enhanced tube formation capacity was observed in HUVECs following oe-GAD1 transfection for 48 hours (Supplementary Fig. 4O and P). These findings collectively underscored the promotion of proliferation, migration, and tube formation, along with the inhibition of apoptosis in HUVECs under HG conditions upon GAD1 overexpression.

Exogenous addition of GABA promoted proliferation, migration, and tube formation and inhibited apoptosis of HUVECs under HG conditions

The role of GAD1-GABA pathway in regulating HUVECs’ proliferation, migration, tube formation, and apoptosis was further explored through the examination of the effects of exogenous addition of GABA. HUVECs were cultured in HG medium and treated with either GABA (5 μM) or an equal amount of vehicle (PBS). The CCK8 assay revealed a significant improvement in cell viability after 48 hours of GABA treatment (Fig. 5A). The proportion of cells in the proliferative stage was elevated in GABA-treated HUVECs compared to controls, as evidenced by EdU assay and cell cycle analysis (Fig. 5B-E). A decrease in cleaved caspase 3 expression and a reduction in the percentage of apoptotic cells from 15.1% to 7.5% were observed after 48 hours of GABA treatment (Fig. 5F-I). Furthermore, GABA accelerated the migration of HUVECs by approximately 10% (Fig. 5J and K). The tube formation assay conducted 48 hours post-treatment exhibited an increase in the number of junctions, meshes, and nodes in GABA-treated HUVECs (Fig. 5L and M). These results demonstrated that the exogenous addition of GABA promoted proliferation, migration, tube formation, and inhibited apoptosis in HUVECs under HG conditions.

Topical use of GABA accelerated diabetic wound healing

Given the positive outcomes observed in vitro, the effects of GABA were further evaluated in an in vivo setting to ascertain its potential in promoting diabetic wound healing. A T2DM mouse model induced by STZ was utilized, with wounds created on the back of the mice. Topical application of 100 μL GABA (5 μM) or vehicle (PBS) per wound was performed daily, and images were captured every 2 days until day 10. The results revealed that wounds treated with GABA exhibited significantly accelerated healing compared to those treated with PBS (Fig. 6A and B). Skin tissues surrounding the wounds were collected on day 10, and CD31 and Ki67 staining were conducted. Compared to vehicle-treated wounds, GABA-treated wounds displayed enhanced angiogenesis and proliferation (Fig. 6C-G). These findings highlighted the potential of topical GABA application in promoting wound angiogenesis and expediting wound healing in diabetic mice.

DISCUSSION

DFU represents a prevalent and severe complication of diabetes, affecting 15% to 25% of individuals with diabetes [24]. Understanding the molecular mechanisms underlying DFU is crucial for addressing this complication effectively. Previous studies have highlighted the involvement of PKCδ in delayed diabetic wound healing [10,11]. However, the evidence was limited and the findings were difficult to apply clinically. In our prior investigation, we unveiled a novel pathway through which PKCδ mediates HG-induced apoptosis in HUVECs, contributing to impaired diabetic wound healing. Specifically, PKCδ upregulated the expression of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), leading to cholesterol overload in endothelial cells. We proposed that inhibitors of HMGCR, such as statins, could hold promise as potential therapeutic agents for diabetic wounds. Building upon these insights, our current study further elucidated a novel signaling cascade downstream of PKCδ. Here, we demonstrated that PKCδ downregulates GAD1 expression and GABA production, resulting in the suppression of proliferation, migration, tube formation, and an increase in apoptosis of endothelial cells. Notably, topical application of GABA accelerated the rate of wound healing in diabetic wounds, underscoring the potential of GABA as a therapeutic agent for promoting diabetic wound healing.

The PKC family comprises a group of serine/threonine protein kinases encompassing multiple isoforms. These isoforms are single polypeptides characterized by an N-terminal regulatory region and a C-terminal catalytic region, comprising four conserved structural domains (C1–C4) and five variable regions (V1–V5) [25]. Distinct activation modes exist among conventional, novel, and atypical PKCs due to slight structural variations. Conventional PKCs are activated by phosphatidylserine, calcium, and DAG or phorbol esters, while novel PKCs are activated by phosphatidylserine, DAG or phorbol esters but not by calcium. Atypical PKCs do not respond to DAG or phorbol esters, calcium. Our investigation primarily focused on the role of PKCδ in diabetic wound healing. In addition to PKCδ, PKCβ has also been studied in the context of diabetic wound healing. For instance, Das et al. [26] reported that inhibition of PKCβ II reversed the dysfunction of endothelial progenitor cells and prevented excessive NETosis in diabetic mice. Similarly, Liu et al. [27] identified the activation of the PKCβ-p66shc pathway as contributing to vascular endothelial injury in DFUs. Conversely, Yan et al.’s study [28] demonstrated that a PKC inhibitor (GF 109203X) hindered the pro-healing effect of acellular dermal matrix scaffolds coated with connective tissue growth factor, suggesting divergent roles of different PKC isoforms in regulating diabetic wound healing.

While the exploration of PKC isoforms in diabetic wound healing remains limited, multiple studies have investigated their functions in non-diabetic wound healing contexts. For example, Tsai et al. [29] found that topical administration of N-acetylcysteine accelerated the healing of burn wounds via the PKC/signal transducer and activator of transcription 3 (Stat3) pathway. Moses et al. [30] demonstrated that novel epoxy-tiglianes facilitated skin keratinocyte wound healing response and re-epithelialization through PKC activation. Conversely, Jozic et al. [31] reported that the activation of the phospholipase C (PLC)/protein kinase C (PKC)/glycogen synthase kinase-3 beta (GSK-3β)/β-catenin pathway inhibited wound closure under stress signals. Therefore, the distinct roles of various PKC isoforms in wound healing necessitate separate investigations. Re-epithelialization, a critical aspect of wound repair, has been shown to be influenced by PKC isoforms. For instance, Thomason et al. [32] observed delayed re-epithelialization in PKCα−/− mice and accelerated re-epithelialization in PKCα-overexpressed mice, indicating the importance of PKCα in this process. They further found PKCα−/− mice were unable to upregulate collagen and other extracellular matrix components in response to injury, which resulted in delayed granulation tissue deposition [33]. Additionally, Koppel et al. [34] highlighted the role of PKCδ in mediating proline-rich protein tyrosine kinase 2 (Pyk2)-promoted wound re-epithelialization. Furthermore, inflammation is an important mechanism that delays wound healing. PKCζ has been implicated in tumor necrosis factor α-induced skin inflammatory injury [35]. Given the upregulation of several PKC isoforms in diabetes mellitus, further studies investigating their functions under diabetic conditions are warranted.

GABA serves as the primary inhibitory neurotransmitter in the brain and has been implicated in various neuropsychiatric disorders, such as epilepsy, insomnia, eating disorders, autism, bipolar disorders, anxiety, depression, and alcohol addiction [36,37]. Interestingly, GABA and its synthesizing enzyme, GAD, are also expressed in skin tissues and play roles in regulating skin functions. Ito et al. [20] documented the expression of GAD1 in mouse skin and human dermal fibroblasts (HDFs), with lower GAD1 expression observed in aged mice compared to younger ones, suggesting a link between GABA and aging. GABA benefits skin health in several aspects. It enhances the skin barrier and improves skin moisturization by upregulating the synthesis of hyaluronic acid, filaggrin, and aquaporin 3 and promoting HDFs survival when exposed to H₂O₂ [20-22]. GABA also enhances skin elasticity by stimulating type I collagen expression, inhibiting matrix metalloproteinase 1 expression, and facilitating elastin synthesis and elastic fiber formation [22,38,39]. Besides, GABA application accelerates wound healing by suppressing inflammation and promoting the proliferation and migration of fibroblasts [22,23]. The same results were obtained in our study: GABA promoted the proliferation and migration of HDFs, whereas inhibition of GAD1 by 3-MPA inhibited the proliferation and migration of HDFs (Supplementary Fig. 5). Notably, the GABA signaling pathway supports the survival of skin flaps used for closing skin deficits [40,41]. Furthermore, GABA is implicated in dermatoses such as psoriasis, hyperpigmentation, and melanoma, with reports suggesting its involvement in modulating skin sensitization, melanogenesis, and melanoma initiation [42-45]. Our study delineated the mechanism of GABA signaling inhibition in diabetic wounds and demonstrated the beneficial effects of GABA on wound healing, offering a potential therapeutic approach for DFU management.

GABA is also prominently expressed within the pancreatic islets and plays a crucial role in modulating the activity of endocrine cells, mainly α-cells and β-cells. β-Cells are the major cells expressing GABA in the islets. Among these cells, β-cells are the primary source of GABA production within the islets, exerting both paracrine effects on α-cells and autocrine effects on themselves [46]. Notably, GABA has been reported to suppress glucagon secretion from α-cells through the GABAA receptor rather than the GABAB receptor [46,47]. Intriguingly, GABA and the GABAA receptor have been shown to facilitate the transdifferentiation process from α-cells to β-cells [48-50]. For β-cells, GABA promotes their proliferation, growth, and regeneration, protects them against apoptosis, and facilitates insulin secretion [14,51,52]. Moreover, GABA exhibits anti-inflammatory properties by suppressing systemic inflammation and reducing the production of inflammatory cytokines, thereby mitigating inflammatory damage to pancreatic islets in diabetes mellitus [53-55]. Clinical trials have explored the use of GABA as a treatment for type 1 diabetes mellitus (T1DM), demonstrating favorable safety and tolerability profiles [56,57]. Importantly, GABA selectively activates peripheral GABA receptors without affecting central GABA receptors. While the precise role of GABA in regulating blood glucose levels and the function of α- and β-cells remains debatable, studies have shown that GABA reduces glycated albumin levels and enhances the response of various hormones to hypoglycemia [56-58]. The therapeutic potential of GABA in T1DM suggests an indirect beneficial impact on DFU.

There are also several limitations in our investigation. In our study, we used mice for in vivo studies of diabetic wound healing. However, an important difference in wound healing between mice and humans is that the wound contracts in mice, which confounds quantitative and qualitative evaluation of experimental wound repair [59]. Our findings revealed that PKCδ inhibits GABA production and GAD1 expression through metabolomics and transcriptomics analyses. Further research is warranted to elucidate the mechanistic underpinnings of how PKCδ regulates GAD1. According to data from the Human Protein Atlas, both GABAA and GABAB receptors are expressed in endothelial cells and diabetic skin tissues, whereas GABAB receptors are much more abundantly expressed than GABAA receptors. GABAA receptor agonists were reported to accelerate cutaneous barrier recovery and prevent epidermal hyperplasia induced by barrier disruption [60]. It is very interesting to figure out the role of GABA receptors in diabetic wound healing. Given the clinical utilization of GABA and certain GABA receptor agonists like baclofen and muscimol, additional investigations are needed to evaluate their efficacy in DFU treatment [61,62].

In summary, our study unveils a novel pathway whereby PKCδ regulates diabetic wound healing. PKCδ downregulation of GAD1 production and GABA expression inhibits vascular endothelial cell functions, impairing proliferation and migration while promoting apoptosis, consequently impeding diabetic wound healing. Furthermore, topical application of GABA expedites the healing of diabetic wounds, offering a promising therapeutic approach for managing DFU.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: Y.L., Q.L.

Acquisition, analysis, or interpretation of data: B.L., R.G., B.Z.

Drafting the work or revising: P.Q., P.Z., Y.H., Y.L., Q.L.

Final approval of the manuscript: all authors.

FUNDING

This work was supported financially by grants from the National Natural Science Foundation of China (No. 82270520), and the Fundamental Research Funds for the Central Universities (No. YCJJ20230687).

ACKNOWLEDGMENTS

The raw data of metabolomics has been deposited in the CNGB Nucleotide Sequence Archive (CNSA) repository (CNP000-5512). The original data of the study are available from the corresponding author upon reasonable request.

Fig. 1.

Protein kinase C delta (PKCδ) knockdown promoted proliferation, migration, and tube formation of human umbilical vein endothelial cells under high glucose (HG) conditions. (A) Cell counting kit 8 assay after PKCδ knockdown for 24 and 48 hours. (B, C) 5-Ethynyl-2´-deoxyuridine (EdU) assay and quantification of EdU-positive cells after PKCδ knockdown for 48 hours. The scale bar is 100 μm. (D, E) Cell cycle assay and quantification of the total proportion of G1 and S phase cells after PKCδ knockdown for 48 hours. (F, G) Scratch wound healing assay and quantification of migrated cells after PKCδ knockdown for 24, 48, and 72 hours. The scale bar is 200 μm. (H, I) Tube formation assay and quantification of the number of junctions, meshes, and nodes after PKCδ knockdown for 48 hours. The scale bar is 100 μm. Each experiment was replicated for thrice and representative images were shown. Quantification was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean± standard error of the mean. si-Scr, scramble siRNA; DAPI, 4´,6-diamidino-2-phenylindole; PE-A, phycoerythrin-area. ^aP<0.01, ^bP<0.001, ^cP<0.0001 vs. controls.

Fig. 2.

Metabolomics analysis and experimental validation reveal decreased gamma-aminobutyric acid (GABA) synthesis in protein kinase C delta (PKCδ)-knockdown human umbilical vein endothelial cells under high glucose (HG) conditions. (A, B) The bar chart and volcano plot of differentially expressed metabolites (DEMs). (C) The heat map showing correlation of DEMs. Green *means significant. (D) The heat map showing the levels of DEMs. (E) GABA synthesis pathway and the expression of glutamate decarboxylase 1 (GAD1) mRNA and GABA detected by transcriptomics and metabolomics. (F) Measurement of GABA concentration in culture medium 72 hours after PKCδ knockdown. (G) Quantitative polymerase chain reaction assay of GAD1 mRNA level 48 hours after PKCδ knockdown. (H, I) Western blot assay and quantification of GAD1 protein level 48 hours after PKCδ knockdown. (J, K) Immunofluorescence assay of and quantification of GAD1 protein level 48 hours after PKCδ knockdown. The scale bar is 20 μm. Six replicates per group were used for metabolomics analysis and three replicates per group were used for transcriptomics and experimental validation. Representative images were shown. Quantification in (A-D) was done with the BGI Dr. Tom platform and quantification in (E-K) was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean±standard error of the mean. si-Scr, scramble siRNA; 7-Me-THIQ, (7s,13as)-7-methyl-5,8,13,13a-tetrahydro-6hisoquinolino[ 3,2-a]isoquinolinium; ISET, (5z,8z,11z,14z)-n-[2-(4-sulfamoylphenyl)ethyl]-5,8,11,14-icosatetraenamide; 2-LG, 2-linoleoyl glycerol; OLDA, N-oleoyl dopamine; UDPGA, uridine 5ʹ-diphosphoglucuronic acid; G-GB, G-guanidinobutyrate; DHT, dihydrothymine; PLB, plumbagin; HMBA, hexamethylene bisacetamide; C14-HSL, N-tetradecanoyl-l-homoserine lactone; MISO, misoprostol; SA-Glu, stearoyl glutamic acid; 16-FOPA, 16-feruloyloxypalmitic acid; DiHET, (+/−)11,12-dihydroxy- 5Z,8Z,14Z-eicosatrienoic acid; NOLS, N-oleoyl-l-serine; BPN, butopyronoxyl; 9Z-HODE-Gly, N-[(9z)-3-hydroxy-9-octadecenoyl] glycine; PRM, pramiracetam; LC, lumichrome. ^aP<0.05, ^bP<0.01 vs. controls.

Fig. 3.

Decreased gamma-aminobutyric acid (GABA) levels and glutamate decarboxylase 1 (GAD1) expression in high glucose (HG)-treated human umbilical vein endothelial cells and skin tissues of diabetic mice. (A) Measurement of GABA concentration in culture medium 72 hours after HG treatment. (B) Quantitative polymerase chain reaction assay of GAD1 mRNA level 72 hours after HG treatment. (C, D) Western blot assay and quantification of GAD1 protein level 72 hours after HG treatment. (E, F) Immunofluorescence assay of and quantification of GAD1 protein level 72 hours after HG treatment. The scale bar is 20 μm. (G) Measurement of GABA concentration in the serum of streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) mice. (H) Measurement of GABA concentration in the skin of STZ-induced T2DM mice. (I, J) mRNA and protein level of GAD1 in the skin of STZ-induced T2DM mice. Each experiment was replicated for thrice and representative images were shown. Quantification was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean±standard error of the mean. NG, normal glucose; DAPI, 4ʹ,6-diamidino-2-phenylindole; Veh, vehicle. ^aP<0.01, ^bP<0.001 vs. controls.

Fig. 4.

Inhibition of glutamate decarboxylase 1 (GAD1) by 3-mercaptopropionic acid (3-MPA) suppressed proliferation, migration, and tube formation and promoted apoptosis of human umbilical vein endothelial cells under normal glucose (NG) conditions. (A) Cell counting kit 8 assay after 3-MPA treatment for 24 and 48 hours. (B, C) 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay and quantification of EdU-positive cells after 3-MPA treatment for 48 hours. The scale bar is 100 μm. (D, E) Cell cycle assay and quantification of the total proportion of G1 and S phase cells after 3-MPA treatment for 48 hours. (F, G) Western blot assay and quantification of cleaved-caspase 3 protein level after 3-MPA treatment for 48 hours. (H, I) Flow cytometry assay and quantification of the percentage of apoptotic cells after 3-MPA treatment for 48 hours. (J, K) Scratch wound healing assay and quantification of migrated cells after 3-MPA treatment for 24, 48, and 72 hours. The scale bar is 200 μm. (L, M) Tube formation assay and quantification of the number of junctions, meshes, and nodes after 3-MPA treatment for 48 hours. The scale bar is 100 μm. Each experiment was replicated for thrice and representative images were shown. Quantification was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean±standard error of the mean. DAPI, 4ʹ,6-diamidino-2-phenylindole; Veh, vehicle; PE-A, phycoerythrin-area; FITC-A, fluorescein isothiocyanate-area. ^aP<0.05, ^bP<0.01, ^cP<0.0001 vs. controls.

Fig. 5.

Exogenous addition of gamma-aminobutyric acid (GABA) promoted proliferation, migration, and tube formation and inhibited apoptosis of human umbilical vein endothelial cells under high glucose (HG) conditions. (A) Cell counting kit 8 assay after GABA treatment for 24 and 48 hours. (B, C) 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay and quantification of EdU-positive cells after GABA treatment for 48 hours. The scale bar is 100 μm. (D, E) Cell cycle assay and quantification of the total proportion of G1 and S phase cells after GABA treatment for 48 hours. (F, G) Western blot assay and quantification of cleaved-caspase 3 protein level after GABA treatment for 48 hours. (H, I) Flow cytometry assay and quantification of the percentage of apoptotic cells after GABA treatment for 48 hours. (J, K) Scratch wound healing assay and quantification of migrated cells after GABA treatment for 24, 48, and 72 hours. The scale bar is 200 μm. (L, M) Tube formation assay and quantification of the number of junctions, meshes, and nodes after GABA treatment for 48 hours. The scale bar is 100 μm. Each experiment was replicated for thrice and representative images were shown. Quantification was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean±standard error of the mean. Veh, vehicle; DAPI, 4ʹ,6-diamidino-2-phenylindole; PE-A, phycoerythrin-area; FITC-A, fluorescein isothiocyanate-area. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001 vs. controls.

Fig. 6.

Topical use of gamma-aminobutyric acid (GABA) accelerated diabetic wound healing. (A, B) Images and wound area quantification of GABA- and vehicle (Veh)-treated diabetic wounds. (C-E) CD31 staining and quantification of GABA- and Vehtreated skin tissues surrounding diabetic wounds. The scale bars of the original and enlarged images are 100 and 50 μm, respectively. (F, G) Ki67 staining and quantification of GABA- and Veh-treated skin tissues surrounding diabetic wounds. The scale bars of the original and enlarged images are 100 and 50 μm, respectively. Each experiment was replicated for at least thrice and representative images were shown. Quantification was done using a two-tailed unpaired Student’s t-test. The results are presented as the mean±standard error of the mean. ^aP<0.05, ^bP<0.01, ^cP<0.001, ^dP<0.0001 vs. controls.

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Qin P, Zhou P, Huang Y, Long B, Gao R, Zhu B, Li Y, Li Q. Abnormally Elevated PKCδ Delays Diabetic Wound Healing by Inhibiting the GAD1-GABA Pathway. Diabetes Metab J. 2025 Sep 8. doi: 10.4093/dmj.2024.0450. Epub ahead of print.

Received: Aug 04, 2024; Accepted: May 29, 2025

DOI: https://doi.org/10.4093/dmj.2024.0450.

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