C1q tumor necrosis factor associated protein 3 protects HaCaT via phosphoinositide 3-kinase/protein kinase B activation by PTEN reduction

Abstract

BACKGROUND

Delayed wound repair is commonly observed among individuals with diabetes.

AIM

To determine the mechanism of C1q tumor necrosis factor associated protein 3 (CTRP3) in regulation of wound healing.

METHODS

Wounds were created in wild-type C57BL/6J and db/db diabetic mice. CTRP3 expression was measured in mouse blood on days 0, 4, 8, 12, 16 and in high glucose-treated HaCaT cells. CTRP3’s role was assessed via cell growth and migration assays. Vascular endothelial growth factor (VEGF) levels were detected. A phospho-kinase array identified phosphorylated kinases. Co-immunoprecipitation verified CTRP3 interaction with phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway proteins.

RESULTS

CTRP3 expression increased significantly with the passage of wound healing time and CTRP3 expression is low in high-glucose-treated HaCaT cells. High glucose treatment significantly suppressed the cell growth and migration of HaCaT cells, while high expression of CTRP3 reversed the effects of high glucose. Meanwhile, the growth factor VEGF down-regulated by high glucose treatment in HaCaT cells significantly elevated in HaCaT cells with highly-expressed CTRP3. CTRP3 promotes wound healing in diabetic mice in vivo. Moreover, CTRP3 activates PI3K/AKT pathway by reducing PTEN protein level.

CONCLUSION

CTRP3 accelerates diabetic wound healing, potentially by protecting keratinocytes through downregulating PTEN and activating the PI3K/AKT pathway, presenting a novel therapeutic target.

Key Words: Diabetes; Wound healing; C1q tumor necrosis factor associated protein 3; Protein kinase B; PTEN

Core Tip: High expression of C1q tumor necrosis factor associated protein 3 (CTRP3) promotes the wound healing of diabetic mice, and this may related to the protective effects on keratinocytes. The underlying mechanism is linked to the activation of phosphoinositide 3-kinase/protein kinase B pathway by reducing the PTEN protein level. Taken together, CTRP3 may be a novel target to accelerate the wound healing in diabetes.

INTRODUCTION

Characterized by chronic hyperglycemia, diabetes confers a serious risk to human health. Disrupted wound healing represents a common and chronic complication in diabetic patients[1]. Some diabetic patients may have foot ulcers, which takes a long time to heal, and has become one of the important reasons for amputation[2,3]. Globally, an estimated 18.6 million people are affected by diabetic foot ulcers, with an infection rate of approximately 50%. Among these infected ulcers, up to 20% necessitate hospitalization, and 15%-20% of the moderate to severe cases ultimately lead to lower-extremity amputation[4]. Individuals with diabetic foot ulcers face a five-year mortality rate of 30%, while the rate jumps to over 70% for patients following an above-foot amputation[5].

Wound healing is a vital physiological process that maintains the integrity of the skin barrier. This process entails the precise and coordinated interaction of numerous cell types and signaling pathways[6]. In individuals with diabetes, the wound healing process is often impeded by a range of underlying pathophysiological factors. These can include an exaggerated immune response, decreased blood vessel formation, peripheral nerve damage, and a buildup of extracellular matrix materials, as well as a disruption in the balance of matrix remodeling[7]. At the molecular level, these macroscopic impairments are driven by more intricate mechanisms. Key factors contributing to delayed wound healing in diabetic contexts encompass epigenetic modifications, aberrant activation of pattern recognition receptors, mitochondrial dysfunction, and disruptions in cellular energy metabolism[8-10]. Understanding the intricate mechanisms behind the challenges of wound healing in diabetes is vital. It provides significant insights that can guide treatments for the condition and inform the development of new clinical therapies and medications.

C1q tumor necrosis factor associated protein 3 (CTRP3) is an adipokine that has been identified in recent years[11]. It possesses anti-inflammatory, antioxidant, and anti-apoptotic characteristics, which suggest it could be significantly protective against cardiovascular and metabolic diseases[12,13]. Patients with diabetes and diabetic nephropathy exhibited decreased serum levels of CTRP3. Moreover, they constitute an independent factor affecting insulin resistance[14,15]. Notably, individuals with diabetes tend to have lower serum concentrations of CTRP3 compared to non-diabetic individuals[14]. The current study explores the potential role of CTRP3 in safeguarding keratinocytes and enhancing the wound healing process. Findings from this research could offer novel perspectives on therapeutic approaches for diabetic wound healing.

The primary objective of this research was to evaluate the therapeutic potential of CTRP3 in diabetic wound repair and to uncover its mechanism of action. To this end, we utilized both in vivo diabetic mouse models and in vitro keratinocyte assays, focusing on elucidating the role of CTRP3 in keratinocyte protection and healing acceleration.

MATERIALS AND METHODS

Animal grouping

The study was approved by the Ethics Committee of Xi’an Jiaotong University School of Medicine (approval No. 2023-65). Male db/db mice (7-9 weeks old, 40-50 g) were purchased from the Model Animal Research Center of Nanjing University. Male control db/m mice (7-9 weeks old, 20-25 g) purchased from Shanghai Laboratory Animal Center were selected as the normal control group (n = 6). Male db/db mice were randomly divided into 3 groups with 6 mice in each group: Diabetes group, diabetes mice with injection of adenovirus (Ad) carrying CTRP3 (diabetes + Ad-CTRP3), and its negative control (diabetes + Ad-NC).

Preparation of skin wound

Anesthesia in mice was induced by inhalation of 3% isoflurane. The dorsal hair was shaved to expose skin prior to excision for wounds. The wound was located 4 cm from the base of the neck and 7 mm from the midline of the back. A 6 mm diameter circular wound was prepared and the skin was completely damaged. Photographs of wound were recorded on the day of surgery (day 0) and every other day post-injury. The transparent film with mesh was affixed to the wound on day 0, 4, 8 12 and 16, and the wound area was analyzed and quantified by scanner and Adobe Photoshop Creative Suite 6 (version 13.0, URL link: https://www.adobe.com/cn/). At the same time, we drew blood samples from the murine tail tip at each time point, and centrifuged them at 4 °C at a rate of approximately 3000 rpm for 10 minutes (Sorvall BP, Thermo Fisher; Waltham, MA, United States) and serum CTRP3 expression was detected. Afterwards, after ensuring deep anesthesia with 5% isoflurane, we humanely euthanized the mice by cervical dislocation.

Histological analysis

Skin samples were fixed in 4% paraformaldehyde and processed for histology through paraffin embedding and sectioning (5 μm). Following deparaffinization and rehydration, sections were stained with hematoxylin and eosin (HE). Microvessel density (MVD) was assessed by first identifying the region of highest vascular density at × 400 magnification, then counting vessels in five random high-power fields within that region. The average of these counts represented the MVD for each sample.

Enzyme-linked immunosorbent assay

Serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions. Briefly, serum samples were thawed on ice and diluted appropriately as required. Aliquots of 50 μL of each standard and sample were added to the pre-coated 96-well plates provided in the kits (TNF-α: Mouse TNF-α ELISA Kit, Invitrogen, BMS607-3TEN; IL-6: Mouse IL-6 ELISA Kit, Invitrogen, BMS603-2; IL-10: Mouse IL-10 ELISA Kit, Invitrogen, BMS614). We then incubated the plates for 2 hours at room temperature. After discarding the liquid and washing the plates four times, we added 100 μL of biotinylated detection antibody per well for a 1-hour incubation. Following another wash, we introduced 100 μL of streptavidin-horseradish peroxidase (HRP) solution, including for 30 minutes in the dark. After a final wash, color was developed by adding 100 μL of 3,3’,5,5’-tetramethylbenzidine substrate for 15 minutes. We terminated the reaction with 50 μL of 2 N sulfuric acid and immediately measured the optical density at 450 nm. Cytokine concentrations were interpolated from the standard curve.

Cell culture and treatment

The human immortalized keratinocytes HaCaT cells were obtained by Procell Life Science and Technology and cultured in a 37 °C incubator containing 5% carbon dioxide. HaCaT cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 (31870074; Giboco; Grand Island, NY, United States), 10% fetal bovine serum (FBS) (10099158; Giboco), 100 U/mL penicillin and 0.1 mg/mL streptomycin (15070063; Giboco). HaCaT cells were treated with 5.6 mmol/L glucose or 30 mmol/L glucose (HG) for 48 hours[16].

The stably overexpressed CTRP3 or PTEN HaCaT cells were then established. Firstly, the CTRP3 and PTEN gene complementary DNA (cDNA) sequence were obtained from NCBI database. The CTRP3/PTEN cDNA was then cloned into the lentiviral vector plasmid Hanbio Lentivirus Vector (pHBLV)-cytomegalovirus (CMV)-multiple cloning site (MCS) (Solarbio; Beijing, China). The empty vector was used as the NC. The CMV promoter is a strong promoter that can drive efficient expression of the gene in various cell types. The MCS consists of multiple restriction enzyme cutting sites for the insertion of the target gene. The constructed lentiviral vector pHBLV-CMV-MCS-CTRP3 (CTRP3) or pHBLV-CMV-MCS-PTEN (PTEN) was transfected with pMD2.G and psPAX2 packaging plasmids (GenScript Probio; Nanjing, Jiangsu Province, China) into HEK293T cells to produce lentiviral particles. The culture supernatant containing the lentiviral particles was collected 48 hours later. For lentiviral transduction, HaCaT cells were incubated with lentivirus in the presence of polybrene (8 μg/mL; Sigma-Aldrich; St. Louis, MO, United States) overnight. The stably expression of the CTRP3 was verified by quantitative polymerase chain reaction (qPCR). CTRP3 was overexpressed in HaCaT cells 48 hours before HG treatment.

Cell growth and migration assay

Cell growth was determined by a cell counting kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay according to the previous studies[17,18]. Cell migration was measured by transwell assay (Millipore; St. Louis, MO, United States). We seeded HaCaT cells in serum-free RPMI 1640 into the upper chamber of a Transwell apparatus, with the lower chamber filled with RPMI 1640 containing 10% FBS. After 36 hours, we fixed the cells that had migrated to the underside of the membrane with methanol and stained them with 0.1% crystal violet (Sigma-Aldrich). To assess cell migration, a uniform scratch was created with a sterile pipette tip in a confluent monolayer of HaCaT cells, which had been cultured in six-well plates (Beyotime, FCP060) for 12 hours. The wound closure was monitored by capturing images at 0 hour and 24 hours after creating the scratch.

Western blot assay

We harvested total proteins using radio immunoprecipitation assay lysis buffer (Beyotime; Shanghai, China) plus a protease inhibitor cocktail (Merck; Billerica, MA, United States). The determination of protein concentration was carried out using a commercial bicinchoninic acid kit (Beyotime) according to the supplied instructions. Equal amounts of protein (30 μg) from different samples were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis via electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore). Afterwards, the polyvinylidene difluoride membranes was blocked in 5% skim milk for 1 hour in tris-buffered saline with Tween-20 (TBST) to prevent non-specific antibody binding. The primary antibodies were added and incubated at 4℃ overnight. The membranes were washed with TBST three times. Then, the blots were incubated with HRP labeled secondary antibody at 37 °C for 2 hours. Afterwards, we thoroughly washed the membrane with TBST to remove unbound secondary antibody. Visualization of the bands was achieved using an electrochemiluminescence detection system (Bio-Rad Laboratories, United States). The information of antibodies used in this study were listed as follows: Anti-CTRP3 (Thermo Fisher, PA5-20149), anti-phosphoinositide 3-kinase (PI3K) (Thermo Fisher, PA5-99518), anti-phospho-PI3K (Thermo Fisher, PA5-121306), anti-protein kinase B (AKT) (Thermo Fisher, MA5-14916), anti-phospho-AKT (Thermo Fisher, PA5-36780).

Quantitative real-time PCR

Following isolation from blood and HaCaT cells using TRIzol reagent (Invitrogen), total RNA was reverse-transcribed into cDNA with a qPCR RT Master Mix (Takara), following the respective manufacturers’ protocols. qPCR analyses were performed using SYBR Green (Takara). Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase. The primer sequences were listed as follows: Mouse CTRP3 (forward: 5’-GCCTTTGCTTTTCCTCCCATT-3’, reverse: 5’-CCTTGGTAACCACGAAATCCA-3’), human CTRP3 (forward: 5’-TCTCCACAAACCGGAGGACTA-3’, reverse: 5’-CCTTGGTAGCCTCGAAAGC-3’), and human VEGF (forward: 5’-GAGGAGCAGTTACGGTCTGTG-3’, reverse: 5’-TCCTTTCCTTAGCTGACACTTGT-3’). The relative expressions of genes were calculated using the 2^-ΔΔCt method.

Phospho-kinase array

Kinase phosphorylation was assessed with a Human Phospho-Kinase Array kit (RD Systems) according to the manufacturer’s protocol[19]. In brief, quantified HaCaT cell lysates were applied to the array for overnight incubation at 4 °C. After a series of incubations with biotinylated antibodies and HRP-streptavidin, the results were visualized by chemiluminescence. A signal was produced at each capture spot corresponding to the amount of a phosphorylated protein bound.

Co-immunoprecipitation

We performed co-immunoprecipitation (Co-IP) to assess potential interactions. First, we constructed and sequenced PcDNA3.1 vectors carrying Flag-CTRP3, hemagglutinin-PI3K, Myc-AKT, and His-PTEN. HEK293T cells, maintained in Dulbecco’s Modified Eagle Medium with 10% FBS and antibiotics under standard conditions, were then transfected with these plasmids (singly or in combination) using a lipofectamine-based method. Following 48 hours of expression, we conducted the Co-IP assay with a Pierce kit. In this procedure, 500 μg of cell lysate was pre-cleared and incubated overnight at 4 °C with antibody-coated beads. After elution, the immunoprecipitated proteins were probed by western blot with anti-His (for PTEN; 51-2400) or anti-Flag (for CTRP3; PA5-20149) antibodies, using total input and immunoglobulin G as controls.

Statistical analysis

Data are expressed as mean ± SD unless otherwise stated. Group differences were evaluated by Student’s t-test (two groups) or one-way analysis of variance with Tukey’s post-hoc test (multiple groups) using SPSS software (v20.0, IBM). A P value < 0.05 was deemed statistically significant.

RESULTS

CTRP3 expression is low in the high-glucose-treated keratinocytes

Considering that CTRP3 plays a regulatory role in metabolic diseases including diabetes, we first detected the expression level of CTRP3 in the blood of normal and diabetic mice during the wound healing process, and the results indicated that the messenger RNA (mRNA) (Figure 1A) and protein (Figure 1B) levels of CTRP3 increased significantly with the passage of wound healing time. At the same time, CTRP3 levels in the diabetic group were prominently lower than those in the normal group from day 8. The CTRP3 expression levels were prominently suppressed in HaCaT cells by high glucose treatment, compared to normal glucose treatment group (Figure 1C and D). Therefore, we speculate that CTRP3 is lowly expressed in the diabetic microenvironment.

Figure 1 C1q tumor necrosis factor associated protein 3 expression is low in the high-glucose-treated HaCaT cells. A: Quantitative polymerase chain reaction (qPCR) analysis of messenger RNA (mRNA) levels of C1q tumor necrosis factor associated protein 3 (CTRP3) in blood samples collected from normal and diabetic mice at days 0, 4, 8, 12, and 16; B: Western blot analysis of protein levels of CTRP3 in blood samples collected from normal and diabetic mice at days 0, 4, 8, 12, and 16; C: QPCR analysis of mRNA levels of CTRP3 in HaCaT cells treated with 5.6 mmol/L glucose or 30 mmol/L glucose 48 hours later; D: Western blot analysis of protein levels of CTRP3 in HaCaT cells treated with 5.6 mmol/L glucose or 30 mmol/L glucose 48 hours later. ^bP < 0.01 vs 5.6 mmol/L glucose group. ^dP < 0.0001 vs normal group. mRNA: Messenger RNA; CTRP3: C1q tumor necrosis factor associated protein 3; NG: 5.6 mmol/L glucose group; HG: 30 mmol/L glucose group; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Overexpression of CTRP3 ameliorates the detrimental effects of high glucose on the function of keratinocytes

Subsequently, to further delineate the influence of CTRP3 on keratinocyte biology, a CTRP3 overexpression system was generated in HaCaT cells. As shown in Figure 2A, the mRNA level of CTRP3 increased significantly after transfection to more than ten times that of control cells (Figure 2A). High glucose treatment significantly suppressed the cell growth of HaCaT cells, but the cell growth capacity of HaCaT cells with highly expressed CTRP3 was enhanced according to the results of CCK-8 assay (Figure 2B) and EdU method (Figure 2C and D). Meanwhile, the images and the corresponding quantitative analysis results of transwell (Figure 2E) and scratch wound healing (Figure 2F) assays suggested that suppressed migrate ability induced by high glucose treatment was also alleviated by over-expression of CTRP3 in HaCaT cells. Besides, the growth factor VEGF down-regulated by high glucose treatment in HaCaT cells significantly elevated in HaCaT cells with highly-expressed CTRP3 (Figure 2G). Taken together, high expression of CTRP3 may help to maintain the cellular function of keratinocytes in vitro.

Figure 2 Overexpression of C1q tumor necrosis factor associated protein 3 ameliorates the detrimental effects of high glucose on the function of keratinocytes. A: Quantitative polymerase chain reaction (qPCR) analysis of messenger RNA (mRNA) levels of C1q tumor necrosis factor associated protein 3 in HaCaT cells before and after transfection; B: Cell viability of HaCaT cells before and after transfection is detected by cell counting kit-8 assay; C and D: Representative images and quantitative analysis of HaCaT cells before and after transfection were measured by 5-ethynyl-2’-deoxyuridine method to obtain the cell growth; E: Cell migration of HaCaT cells before and after transfection was detected by transwell method; F: Representative images and quantitative analysis of HaCaT cells before and after transfection were measured by scratch wound healing method to obtain the cell migration; G: QPCR analysis of mRNA levels of vascular endothelial growth factor in HaCaT cells before and after transfection. ^bP < 0.01 vs vector group. ^cP < 0.001 vs 5.6 mmol/L glucose group. ^dP < 0.0001 vs 5.6 mmol/L glucose group. ^fP < 0.01 vs 30 mmol/L glucose + vector group. ^gP < 0.001 vs 30 mmol/L glucose + vector group. ^hP < 0.0001 vs 30 mmol/L glucose + vector group. mRNA: Messenger RNA; CTRP3: C1q tumor necrosis factor associated protein 3; NG: 5.6 mmol/L glucose group; HG: 30 mmol/L glucose group; DAPI: 4’,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2’-deoxyuridine; VEGF: Vascular endothelial growth factor.

CTRP3 promotes wound healing in diabetic mice

Subsequently, the accelerating role of CTRP3 on wound healing of diabetic mice was investigated in vivo. In the normal group of mice, the wound size was about 10% of the original wound size by day 16. In diabetic mice, the wound size was less than 20% healed by day 16. The wound healing rate of mice injected with CTRP3 over-expression ad was significantly faster than that of diabetic mice in the vector group, and the wound size in diabetes + Ad-CTRP3 group had shrunk to about 40% of the initial wound size by day 16 (Figure 3A and B). Histological examination of wound tissues by HE staining revealed complete epithelialization, mature granulation tissue, diminished inflammatory cell infiltration, and regression of blood vessels with dense collagen deposition in the normal control group. On the contrary, the model group exhibited incomplete epithelialization, with persistent infiltration of inflammatory cells, extravasated red blood cells, and visible hematoma. The Ad-CTRP3 group showed well-defined epithelial layers and a notable reduction in inflammatory cells (Figure 3C). Diabetes was associated with a pro-inflammatory state, characterized by increased TNF-α and IL-6 and decreased IL-10 relative to normal controls. This inflammatory imbalance was effectively mitigated by CTRP3 overexpression, which restored the levels of these cytokines toward normal (Figure 3D-F).

Figure 3 C1q tumor necrosis factor associated protein 3 promotes wound healing in diabetic mice. A: The representative images of wound in mice of each group; B: The quantitative analysis of wound size in mice of each group; C: The hematoxylin and eosin staining images of wound tissues in mice of each group; D-F: Serum levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-10 were measured using commercially available enzyme-linked immunosorbent assay kits. ^dP < 0.0001 vs normal group. ^hP < 0.0001 vs diabetes + adenovirus-negative control group. CTRP3: C1q tumor necrosis factor associated protein 3; Ad: Adenovirus; NC: Negative control; TNF: Tumor necrosis factor; IL: Interleukin.

CTRP3 activates PI3K/AKT pathway by reducing PTEN protein level

CTRP3 was found to specifically promote AKT phosphorylation. This finding was obtained through a phospho-kinase array analysis, which was conducted to elucidate the signaling mechanisms mediated by CTRP3 (Figure 4A and Supplementary Figure 1). Then the STRING analysis also demonstrated that CTRP3 protein is linked to AKT protein (Figure 4B). Therefore, we speculated that CTRP3 may regulate the PI3K/AKT pathway in HaCaT cells. Our data suggested that over-expression of CTRP3 significantly elevated the phosphorylated PI3K and phosphorylated AKT protein levels (Figure 4C). However, the Co-IP results revealed that CTRP3 not only cannot bind to PI3K protein, but also cannot bind to AKT protein (Figure 4D). Therefore, we also detected the binding relationship between the upstream protein of PI3K/AKT pathway PTEN and the CTRP3 protein, and the results demonstrated that PTEN interacts with CTRP3 (Figure 4D and E). Meanwhile, the western blots analysis also suggested that CTRP3 over-expression down-regulated PTEN protein levels (Figure 4F).

Figure 4 C1q tumor necrosis factor associated protein 3 activates phosphoinositide 3-kinase/protein kinase B pathway by reducing phosphatase and tensin homolog deleted on chromosome ten protein level. A: The cell lysates of HaCaT cells before and after transfection were analyzed using the human phospho-kinase array; B: The STRING analysis was performed to verify the interaction between C1q tumor necrosis factor associated protein 3 (CTRP3) and protein kinase B (AKT) proteins; C: Representative images of key proteins of phosphoinositide 3-kinase/AKT pathway in HaCaT cells before and after transfection; D and E: Co-immunoprecipitation analysis demonstrated the interaction between CTRP3 and AKT proteins; F: Representative images of PTEN protein in HaCaT cells transfected with overexpressed CTRP3 vector or control vector. CTRP3: C1q tumor necrosis factor associated protein 3; AKT: Protein kinase B; AMPK: Adenosine 5’-monophosphate-activated protein kinase; c-Jun: C-Jun N-terminal kinase; CREB: Cyclic-adenosine 5’-monophosphate response binding protein; EGFR: Endothelial growth factor receptor; eNOS: Endothelial nitric oxide synthase; ERK: Extracellular signal-regulated kinase; GSK: Glycogen synthase kinase; HSP: Heat shock protein; MSK: Mitogen- and stress-activated protein kinases; PDGFR: Platelet-derived growth factor receptor; STAT: Signal transducer and activator of transcription; PI3K: Phosphoinositide 3-kinase; p-PI3K: Phosphor-phosphoinositide 3-kinase; p-AKT: Phosphor-protein kinase B; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; HA: Hemagglutinin; IgG: Immunoglobulin G.

PTEN overexpression reverses the protective effects of CTRP3 in keratinocytes

PTEN expression was then significantly elevated in HaCaT cells (Figure 5A). The protective effects of over-expression of CTRP3 on HaCaT cells were reversed by PTEN over-expression by promoting cell growth (Figure 5B-D) and migration (Figure 5E-H). Meanwhile, the VEGF mRNA levels elevated by over-expression of CTRP3 were reduced by PTEN over-expression (Figure 5I). Consistent with these observations, the increased phosphorylation levels of PI3K and AKT resulting from CTRP3 overexpression were also suppressed by PTEN overexpression (Figure 5J).

Figure 5 PTEN reversed the protective effects of C1q tumor necrosis factor associated protein 3 on HaCaT cells. A: Quantitative polymerase chain reaction (qPCR) analysis of messenger RNA (mRNA) levels of PTEN in HaCaT cells before and after transfection; B: Cell viability of HaCaT cells before and after transfection is detected by cell counting kit-8 assay; C and D: Representative images and quantitative analysis of HaCaT cells before and after transfection were measured by 5-ethynyl-2’-deoxyuridine method to obtain the cell growth; E and F: Cell migration of HaCaT cells before and after transfection was detected by transwell method; G and H: Representative images and quantitative analysis of HaCaT cells before and after transfection were measured by scratch wound healing method to obtain the cell migration; I: QPCR analysis of mRNA levels of vascular endothelial growth factor in HaCaT cells before and after transfection; J: Representative images of key proteins of phosphoinositide 3-kinase/protein kinase B pathway in HaCaT cells before and after transfection. ^bP < 0.01 vs vector group. ^dP < 0.0001 vs 5.6 mmol/L glucose group. ^hP < 0.0001 vs 30 mmol/L glucose + vector group. ^jP < 0.01 vs 30 mmol/L glucose + C1q tumor necrosis factor associated protein 3 + vector group. ^kP < 0.001 vs 30 mmol/L glucose + C1q tumor necrosis factor associated protein 3 + vector group. ^lP < 0.0001 vs 30 mmol/L glucose + C1q tumor necrosis factor associated protein 3 + vector group. mRNA: Messenger RNA; CTRP3: C1q tumor necrosis factor associated protein 3; NG: 5.6 mmol/L glucose group; HG: 30 mmol/L glucose group; DAPI: 4’,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2’-deoxyuridine; PI3K: Phosphoinositide 3-kinase; p-PI3K: Phosphor-phosphoinositide 3-kinase; p-AKT: Phosphor-protein kinase B; AKT: Protein kinase B; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; VEGF: Vascular endothelial growth factor.

DISCUSSION

In diabetic wounds, the disorders of transcription factors, epigenetic modifications, and non-coding RNAs can lead to a variety of downstream molecular disorders by regulating the transcription and translation of genes related to wound healing[20,21]. Inhibition of inflammation regression, angiogenesis, and re-epithelialization regulated by these downstream molecules ultimately lead to poor wound healing[22-24]. The regulation of the selective expression of these genes is located in the upstream of the signal pathway related to wound healing, and can strongly regulate multiple downstream effector molecules, which has many effects on wound healing. Therefore, we urgently need to invest more research in the development of new drugs that target these molecules to address the conundrum of poor wound healing in diabetes.

CTRP3 may be a biomarker for predicting metabolic diseases, and studies have found that its level is lowered in the presence of various adverse cardiometabolic risk factors[25]. Serum CTRP3 level is negatively correlated with body mass index, triglyceride, fasting blood glucose glycosylated hemoglobin and fasting insulin levels[14]. During adipocyte differentiation, CTRP3 may modulate lipid metabolism by playing an inhibitory role[26]. For example, studies have shown that after CTRP3 gene knockout in mice, transforming growth factor-β1 expression levels in liver, fat, and serum are reduced, and serum IL-6 levels are increased[27]. Moreover, the level of CTRP3 is negatively correlated with the occurrence of diabetes and is an independent factor affecting insulin resistance[28]. Exogenous administration of CTRP3 reduces insulin resistance and increases the expression level of PI3K protein in a dose-dependent manner, indicating that CTRP3 may improve insulin resistance through reducing inflammation and enhancing insulin signaling[29]. In diabetic rat models from insulin resistance to the onset stage of dominant diabetes, the expression of CTRP3 in visceral adipose tissue is gradually reduced[30]. These results suggest that CTRP3 can regulate glucose metabolism in diabetes mellitus. In this study, our data suggested that CTRP3 also lowly expressed in diabetic mice blood, and CTRP3 levels increased with the healing of the wound.

Studies have shown that keratinocytes can produce antibacterial skin and have a scavenging effect on invading pathogens. Keratinocytes can also activate T cells directly through antigen presentation, thereby mediating immune responses[31]. In addition, when the skin is damaged, keratinocytes will quickly enter the repair state and begin to synthesize more keratinocytes to strengthen the protective barrier of the skin and prevent the invasion of pathogenic microorganisms[9,32,33]. A key function of keratinocytes is to orchestrate skin cell renewal and differentiation, processes essential for wound healing and tissue regeneration[34]. High glucose environment may affect the proliferation and migration of keratinocytes. 30 mmol/L glucose inhibits HaCaT cell differentiation by uprating c-Myc and S100A6, which may indirectly affect cell proliferation[35]. Zhang et al[36] observed that exposure to 30 mmol/L glucose promoted the proliferation of HaCaT cells relative to exposure to 9 mmol/L glucose. Secretion of KRT17 from skin cells under high glucose stimulation drives keratinocyte proliferation and migration, which represents a potential mechanism for impaired wound healing in diabetes[37]. Our data, however, suggested that 30 mmol/L glucose treatment inhibited cell proliferation of HaCaT cells. Glucose concentration of 25 mmol/L significantly inhibited the proliferation and migration of human gingival fibroblasts[38]. Therefore, the regulatory effect of high glucose on the wound healing process of these cells is complex and worthy of further investigation. We further explored the regulatory role of CTRP3 in keratinocytes treated with high glucose. The results also suggest that CTRP3 has protective effect on keratinocytes by weakening the effects of high glucose. The PI3K/AKT signaling pathway has been implicated in mediating the quality repair of wounds under diabetic conditions[39-42]. Our study then preliminatively investigated the possible involvement of CTRP3 overexpression in diabetic wound healing by inducing PI3K/AKT signaling pathway activation. Mechanically, CTRP3 binds to PTEN protein, the upstream protein of PI3K/AKT pathway, to plays a protective role in keratinocytes. Due to the lack of specific CTRP3 pharmacological inhibitors and the possibility that knockout of CTRP3 may affect the physiological state of cells and thus affect the activity of the signaling pathway, it is unclear whether inhibiting CTRP3 in keratinocytes will inhibit the activation of PI3K/AKT signaling pathway. This should be further investigated in the future.

In a high-glucose environment, HaCaT cells may exhibit functional impairments such as reduced proliferation and migration, changes that are similar to the cellular behavior observed during diabetic wound healing. This study is the first to explore the role and potential mechanism of CTRP3 in the regulation of diabetic wound healing in vivo and in vitro. Targeting CTRP3 on wound healing treatment is a promising therapeutic method. Supported by the evidence from this study, CTRP3 demonstrates therapeutic potential for diabetic wound repair, meriting future clinical exploration. However, some limitations of this study need to be further discussed and solved in the future. For instance, there are also limitations to using HaCaT cells in diabetic wound healing studies. Although HaCaT cells are a type of epidermal cell, they are immortalized cell lines that may not fully mimic the response of normal human epidermal cells in a diabetic state. In addition, the conditions of cell culture in vitro differ from the in vivo environment, such as oxygen concentration, cell-cell interactions, and the composition of the extracellular matrix, and these differences may affect the clinical relevance of the findings. In vitro studies of wound healing, in addition to HaCaT cells, there are fibroblasts responsible for the synthesis of extracellular matrix and collagen[43], macrophages that promote wound healing[44], endothelial cells that promote new blood vessels[45], and mesenchymal stem cells that can secrete exosomes to promote wound healing[46]. Different cell types play different roles in different stages of wound healing, so the function and interactions of these cells should be considered when designing experiments. Moreover, to avoid differences in sex hormone levels, male mice were selected for in vivo experiments. Follow-up studies need to expand the sample size and include female mice to study wound healing.

CONCLUSION

Our findings demonstrate that CTRP3 promotes wound healing in diabetic mice. This effect appears to be mediated, at least in part, through its protective actions on keratinocytes. Mechanistically, our data suggest that CTRP3 activates the PI3K/AKT pathway, likely via downregulation of PTEN. Taken together, this study highlights CTRP3 as a potential therapeutic target, suggesting a novel strategy for promoting wound healing in diabetic patients. Future studies are warranted to elucidate the precise mechanism of CTRP3-PTEN interaction and to explore the efficacy of CTRP3-based therapeutics in more complex disease models.