Valproic acid accelerates skin wound healing in mice via its anti-inflammatory and apoptotic cell clearance-promoting effects

Abstract

Objective

To investigate the effects of valproic acid (VPA) on skin wound healing in mice.

Methods

Full-thickness wounds were created in mice, and then VPA was applied. The wound areas were quantified daily. In the wounds, granulation tissue growth, epithelialization, collagen deposition, and the mRNA levels of inflammatory cytokines were measured; furthermore, apoptotic cells were labeled. In vitro, VPA was added to RAW 264.7 cells (macrophages) stimulated with lipopolysaccharide, and apoptotic Jurkat cells were cocultured with the VPA-pretreated macrophages. Then, phagocytosis was analyzed, and the mRNA levels of phagocytosis-associated molecules and inflammatory cytokines were measured in the macrophages.

Results

VPA application significantly accelerated wound closure, granulation tissue growth, collagen deposition, and epithelialization. In wounds, the levels of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β were decreased by VPA, whereas those of IL-10 and transforming growth factor-β1 were increased. Additionally, VPA reduced the number of apoptotic cells. In vitro, VPA inhibited the inflammatory activation of macrophages and promoted the phagocytosis of apoptotic cells by macrophages.

Conclusion

VPA accelerated skin wound healing, which could be partly attributable to its anti-inflammatory and apoptotic cell clearance-promoting effects, indicating that VPA could be a promising candidate for enhancing skin wound healing.

Introduction

Acute and chronic skin wounds represent a global health problem associated with a substantial social and economic burden. ¹ Clinically, it is necessary to find new effective candidate agents for treating skin wounds. Skin wound healing is a complex and dynamic process that includes four coordinated and overlapping phases: hemostasis, inflammation, proliferation, and remodeling. ² Macrophages play a vital role in skin wound healing, and the absence of macrophages in wounds delay the healing process. ³ Macrophages in wounds can be divided into two phenotypes: pro-inflammatory M1 macrophages and anti-inflammatory/reparative M2 macrophages. In the inflammatory phase, M1 macrophages promote inflammation and remove wound debris, pathogens, and apoptotic cells. Then, in the proliferative phase, M2 macrophages resolve inflammation and secrete cytokines that interact with keratinocytes, fibroblasts, and endothelial cells, leading to epithelialization, collagen deposition, angiogenesis, granulation tissue growth, and wound closure.^4,5 After the phagocytosis of apoptotic cells, M1 macrophages switch to the M2 phenotype, ensuring the switch from the inflammatory phase to the proliferative phase and promoting efficient repair. The phagocytosis of apoptotic cells, known as efferocytosis, is a critical process that switches M1 macrophages to the M2 phenotype. ⁶ Efferocytosis deficiency causes M1 macrophage and apoptotic cell accumulation in wounds, resulting in the secretion of a large amount of pro-inflammatory cytokines and prolonged inflammation. ⁷ Excessive inflammation and deficiency of macrophage efferocytosis are important factors delaying skin wound healing, whereas dampening inflammation and promoting the phagocytosis of apoptotic cells by macrophages can accelerate the healing both in normal and chronic wounds.^8,9

Valproic acid (VPA) is a short-chain 2-n-propyl-pentanoic fatty acid and a histone deacetylase inhibitor. ¹⁰ VPA is widely used to control epilepsy because of its ability to inhibit histone deacetylase, which affects the cell cycle, cell differentiation, and apoptosis. VPA is also used to treat other neurological disorders and cancers. ¹¹ As early as 2012, VPA was demonstrated to promote skin wound healing by enhancing keratinocyte motility in a murine model. ¹² However, the therapeutic effects of VPA on skin wound healing are not fully known. Recently, VPA was revealed to regulate the functions of innate and adaptive immune cells. ¹³ According to previous reports, VPA can inhibit macrophage inflammatory activation and promote the phagocytosis of apoptotic cells by macrophages, suggesting the VPA can accelerate skin wound healing by acting on macrophages. ¹⁴ In this study, we sought to elucidate the therapeutic effects of VPA on skin wound healing in mice.

Materials and Methods

Ethics approval and animal welfare

Ethical approval for this study was obtained from the Animal Ethics Committee of Army Medical University, Chongqing, China (No. SYXK(yu)2017‐0002, approval date: 16 September 2021). The study followed international and national guidelines for humane animal treatment and complied with relevant legislation. We ensured good welfare and humane treatment for experimental animals, reduced the number of experimental animals, and minimized their suffering. The reporting of this study conforms to the ARRIVE 2.0 guidelines. ¹⁵

Animals

Wild-type C57BL/6 male mice aged 8 to 12 weeks old were purchased from Vital River Laboratories (Beijing, China). All mice were housed and bred in the animal facility at Army Medical University under pathogen-free conditions. Rodent laboratory chow and water were provided, and the animals were maintained under controlled conditions with a 12-hour/12-hour light/dark cycle and a temperature of 24 ± 2°C.

Wound healing model and assay

Pentobarbital (50 mg/kg; Boster, Wuhan, China) was intraperitoneally injected to anesthetize the mice. Then, 8% Na₂S was used to depilate the dorsal skin, povidone iodine and 75% ethanol were used to sterilize the skin, and wounds (6 mm diameter) were generated on the backs of the mice. According to the random number table method, 18 mice were divided into the phosphate-buffered saline (PBS) group, low-dose (30 mg/kg) VPA (l-VPA) group, and high-dose (60 mg/kg) VPA (h-VPA) group (six mice/group). The mice in the l-VPA and h-VPA groups received intraperitoneal injections of VPA (Sigma-Aldrich, St. Louis, MO, USA) once daily, and the mice in the PBS group were administered the same volume of PBS. ¹⁶ The wounds were photographed daily, and the wound areas were quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). The degree of wound healing was calculated as follows: wound closure (%) = (wound healing area)/(initial wound area) × 100%.

Histological assessment

For the assessment of skin wound histology, the mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneal injection) and sacrificed by cervical dislocation. Wound tissues were harvested, postfixed in 4% paraformaldehyde at 4°C overnight, and then embedded in paraffin. Paraffin blocks were sectioned (4 μm). After dewaxing in xylene and rehydration in an ethanol gradient, the wound tissue sections were stained with hematoxylin–eosin (Sigma-Aldrich), Masson solution (Beyotime Biotechnology, Shanghai, China), or TUNEL (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Following dehydration and vitrification, the sections were viewed under a light or fluorescence microscope (Olympus, Tokyo, Japan).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

RNA was isolated from wound tissues and cultured cells using Tripure Isolation Reagent (Roche, Basel, Switzerland) and reverse-transcribed into cDNA using a Quantscript RT Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR Green qPCR Master Mix (MedChemExpress, Monmouth Junction, NJ, USA) and the CFX96 detection system (Bio-Rad Laboratories, Hercules, CA, USA). Gene expression for each sample was normalized to β-actin expression, which was used as the mouse reference gene, and differences in gene expression were determined using the 2^−ΔΔCT calculation. The sequences of the primers (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) were as follows: β-actin (forward, 5′-TGGAATCCTGTGGCATCCATGAAA-3′; reverse, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′), collagen type I (forward, 5′-ATCACTGCAAGAACAGCGTA-3′; reverse, 5′-TGTTTTCCAAAGTCCATGTG-3′), tumor necrosis factor (TNF)-α (forward, 5′-AACTAGTGGTGCCAGCCGAT-3′; reverse, 5′-CTTCACAGAGCAATGACTCC-3′), interleukin (IL)-6 (forward, 5′-TGATGGATGCTACCAAACTGG-3′; reverse, 5′-TGGTCTTGGTCCTTAGCCACT-3′), IL-1β (forward, 5′-GAAATGCCACCTTTTGACAGTG-3′; reverse, 5′-TGGATGCTCTCATCAGGACAG-3′), IL-10 (forward, 5′-GCTGGACAACATACTGCTAACC-3′; reverse, 5′-ATTTCCGATAAGGCTTGGCAA-3′), TGF-β1 (forward, 5′-TGGTGGACCGCAACAAC-3′; reverse, 5′-AGCCACTCAGGCGTATCAG-3′), Cd36 (forward, 5′-TCGGAACTGTGGGCTCATTG-3′; reverse, 5′-CCTCGGGGTCCTGAGTTATATTTTC-3′), Mertk (forward, 5′-GTGGCAGTGAAGACCATGAAGTTG-3′; reverse, 5′-GAACTCCGGGATAGGGAGTCAT-3′), Mfge8 (forward, 5′-GGACATCTTCACCGAATACATCTGC-3′; reverse, 5′-TGATACCCGCATCTTCCGCAG-3′), Gas-6 (forward, 5′-TCTTCTCACACTGTGCTGTTGCG-3′; 5′-GGTCAGGCAAGTTCTGAACACAT-3′), C1qa (forward, 5′-AAAGGCAATCCAGGCAATATCA-3′; reverse, 5′-TGGTTCTGGTATGGACTCTCC-3′), C1qb (forward, 5′-AACGCGAACGAGAACTATGA-3′; reverse, 5′-ACGAGATTCACACACACAGGTTG-3′), and C1qc (forward, 5′-CAACGCCCTCGTCAGGTT-3′; reverse, 5′-ACAACCCAAGCACAGGGAAGT-3′).

RAW 264.7 cell culture and intervention

RAW 264.7 cells were grown in complete Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific), supplemented with penicillin (100 U/mL, Beyotime Biotechnology, Shanghai, China), streptomycin (100 U/mL, Beyotime Biotechnology), and 10% fetal calf serum (FCS, Gibco) at 37°C in 5% CO₂. In total, 1 × 10⁶ cells were seeded into six-well cell culture plates and cultured overnight. Then, the cells were washed twice with PBS, and VPA (50, 100, or 150 mM) was added to the culture with or without 1 mg/mL lipopolysaccharide (LPS, Sigma-Aldrich) overnight. Then, the expression of inflammatory cytokines was measured by RT-qPCR.

Apoptotic cell generation and in vitro phagocytosis assays

Jurkat cells were cultured in RPMI-1640 medium (RPMI, Gibco) without FCS, and apoptosis was induced by incubation with staurosporine (0.5 μg/mL, Sigma-Aldrich) for 3 hours. Then, the cells were washed three times with PBS and resuspended in RPMI supplemented with 10% FCS. Staurosporine treated yielded a population with 90% apoptotic cells. Before coculture with macrophages, apoptotic cells were labeled with pHrodo™ Green (Molecular Probes, Thermo Fisher Scientific), a pH-sensitive phagocytosis dependent indicator, according to the manufacturer’s protocol. Macrophages and pHrodo-labeled apoptotic cells were cocultured at a ratio of 1:5 (macrophages:apoptotic cells) at 37°C in 5% CO₂ for 1 hour in RPMI supplemented with 10% FCS. Macrophages were pretreated with VPA (150 mM) before coincubation with pHrodo-labeled apoptotic cells. After co-incubation, the cells were resuspended and stained with fluorescently labeled anti-F4/80 (BM8; Sungene Biotech, Tianjin, China). Furthermore, the cells were analyzed by flow cytometry, and the proportion of macrophages that had ingested apoptotic cells was determined.

Flow cytometry

Single-cell suspensions were washed twice, resuspended in staining buffer, and incubated with antibodies against CD16 and CD32 (Sungene Biotech) for 15 minutes to block Fc receptors (0.5 μg/million cells). Then, the cell surface markers were stained by the corresponding labeled antibodies diluted in staining buffer (0.2 µg/million cells in a volume of 100 µL) for 20 minutes at 4°C. After staining, cells were washed and suspended in PBS and then analyzed immediately on a CANTO II (Becton Dickinson, Franklin Lakes, NJ, USA). Flow cytometry data were collected using CellQuest software (Becton Dickinson) and analyzed using FlowJo software (Becton Dickinson).

Statistical analysis

The data were statistically analyzed using SPSS 25.0 (IBM Corp., Armonk, NY, USA). The data were normally distributed, and they were presented as the mean ± standard deviation. Significance was calculated using a two-tailed unpaired Student’s t-test. For all statistical analyses, statistical significance was indicated by P < 0.05.

Results

VPA treatment accelerated skin wound healing

We first investigated whether VPA could accelerate skin wound healing in mice. The healing process includes wound closure, granulation tissue growth, collagen deposition, and epithelialization. ¹⁷ Wound closure was faster in the h-VPA group than in the l-VPA and PBS groups (both P < 0.05, Figure 1a and b). Then, we compared granulation tissue growth, collagen deposition, and epithelialization between the h-VPA and PBS groups. In 5-day-old wounds, the areas of granulation tissue were greater in the h-VPA group than in the PBS group (P < 0.05, Figure 1c). Collagen deposition was greater in the h-VPA group than in the PBS group (P < 0.05), and the mRNA expression of collagen type I was increased after VPA treatment (P < 0.05, Figure 1c). In 3- and 7-day-old wounds, the epidermal distances were smaller in the h-VPA group than in the PBS group (both P < 0.05, Figure 1d). Collectively, these data illustrated that VPA treatment accelerated skin wound healing.

Figure 1.

VPA treatment accelerated skin wound healing. (a) Representative photographs of 0-, 3-, 5-, 9-, and 12-day-old wounds. (b) Wound closure was expressed as the percentage of the wound healing area to initial wound area (n = 6). (c) In 5-day-old wounds, HE staining (black hatch line outlines granulation tissue; original magnification, ×200) was performed to calculate the area of granulation tissue (n = 3). In 5-day-old wounds, Masson trichrome staining was performed to identify collagen deposition (the black hatched line outlines the wound margin, black arrows denote old collagen, and red arrows denote newly formed collagen; original magnification, ×200), and the mRNA levels of collagen type 1 in wounds were quantified (n = 3). (d) In 3- and 7-day-old wounds, HE staining was performed to identify epithelization (black arrows mark the wound margin; original magnification, ×100), and the epidermal distances were calculated (D denotes distance; n = 3). *P < 0.05.

VPA treatment suppressed inflammation and reduced apoptotic cell accumulation in wounds

Inhibiting the expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β and boosting that of the anti-inflammatory cytokines IL-10 and TGF-β1 in wounds promote the healing process. ¹⁸ We measured the mRNA levels of TNF-α, IL-6, IL-1β, IL-10, and TGF-β1 in 5-day-old wounds. The results illustrated that TNF-α, IL-6, and IL-1β levels were lower in the h-VPA group than in the PBS group (all P < 0.05), whereas those of IL-10 and TGF-β1 were higher in the h-VPA group (both P < 0.05, Figure 2a). In addition, increased numbers of apoptotic cells in wounds impair healing. ¹⁹ We analyzed apoptotic cell accumulation in 5-day-old wounds, and the results revealed that the number of apoptotic cells was lower in the h-VPA group than in the PBS group (P < 0.05, Figure 2b). Collectively, these data revealed that VPA treatment suppressed inflammation and reduced apoptotic cell accumulation in wounds.

Figure 2.

VPA treatment suppressed inflammation, reduced apoptotic cell accumulation in wounds, and inhibited the macrophage inflammatory response but promoted macrophage phagocytic activity. (a) The mRNA levels of TNF-α, IL-6, IL-1β, IL-10, and TGF-β1 in 5-day-old wounds (n = 3). (b) In 5-day-old wounds, TUNEL staining was used to identify apoptotic cells, and the numbers of TUNEL-positive cells were counted in 3–5 high-power fields (original magnification, ×400) per mouse (n = 3). (c) The mRNA levels of TNF-α, IL-6, and IL-1β in macrophages (n = 3). (d) Phagocytosis of apoptotic cells by macrophages in vitro was analyzed by flow cytometry (n = 3). (e) The mRNA levels of Cd36, Mertk, Mfge8, Gas-6, C1qa, C1qb, and C1qc in macrophages (n = 3) and (f) The mRNA levels of IL-10 and TGF-β1 in macrophages (n = 3). *P < 0.05.

VPA inhibited the macrophage inflammatory response but promoted its macrophage phagocytic activity

Accumulating evidence suggests a vital role of macrophages in mediating tissue inflammation and apoptotic cell clearance during skin wound healing. ²⁰ Thus, we studied the effects of VPA on macrophages. First, we investigated the effects of VPA on inflammatory macrophage activation. As presented in Figure 2c, after LPS stimulation, increased TNF-α, IL-6, and IL-1β levels were observed, indicating that macrophages underwent inflammatory activation. VPA suppressed the LPS-induced upregulation of TNF-α, IL-6, and IL-1β in a concentration-dependent manner (all P < 0.05), but it had no effect on their levels before LPS stimulation (Figure 2c). Furthermore, we analyzed the effects of VPA on apoptotic cell clearance by macrophages in vitro. As illustrated in Figure 2d, VPA promoted the phagocytosis of apoptotic cells by macrophages (P < 0.05). Because the phagocytic activity of macrophages is mediated by phagocytosis-associated receptors and opsonins, ²¹ we next determined the effect of VPA on molecules associated with macrophage phagocytosis. As presented in Figure 2e, VPA increased the levels of phagocytosis-associated molecules such as Cd36, Mertk, Mfge8, Gas-6, and C1qa (all P < 0.05). Efferocytosis mediates the switch of M1 macrophages to M2 macrophages, which secrete anti-inflammatory cytokines and growth factors to resolve wound inflammation and promote tissue repair. ²¹ We determined the effects of VPA on the macrophage secretion of IL-10 and TGF-β1. As presented in Figure 2f, VPA increased the levels of IL-10 and TGF-β1 after the phagocytosis of apoptotic cells by macrophages (both P < 0.05), but there was no effect before phagocytosis. Together, these data indicated that VPA inhibited the macrophage inflammatory response but promoted the phagocytosis of apoptotic cells by macrophages, resulting in the resolution of wound inflammation and promotion of tissue repair.

Discussion

In this study, we explored the therapeutic effects of VPA on skin wound healing in mice. We found that the VPA administration in mice significantly accelerated the healing process. Moreover, the levels of pro-inflammatory cytokines in wounds, such as TNF-α, IL-6, and IL-1β, were reduced by VPA administration, whereas the levels of the anti-inflammatory cytokines IL-10 and TGF-β1 were increased. In addition, VPA decreased the number of apoptotic cells in wounds. Finally, in vitro experiments revealed that VPA inhibited the macrophage inflammatory response but promoted the phagocytosis of apoptotic cells by macrophages. These data demonstrated that VPA accelerated skin wound healing partly through its anti-inflammatory and apoptotic cell clearance-promoting effects, suggesting that VPA is a promising candidate agent for promoting skin wound healing.

The accumulated evidence suggests a close link between pro-inflammatory cytokines and skin wound healing. Specifically, upregulated pro-inflammatory cytokines impaired the healing process. ²² TNF-α, IL-6, and IL-1β are crucial pro-inflammatory cytokines that perpetuate the positive feedback loop, sustaining inflammation in wounds and leading to reduced proliferation and migration of keratinocytes, fibroblasts, and endothelial cells, which are necessary for tissue repair. ¹⁹ Wound macrophages are the main origins of these inflammatory cytokines. Our in vivo and in vitro experiments illustrated that VPA ameliorated the inflammatory response in wounds and notably inhibited the expression of TNF-α, IL-6, and IL-1β in macrophages, suggesting that VPA accelerates skin wound healing by regulating the macrophage inflammatory response. According to previous reports, the mechanism might be that VPA inhibits the PI₃K/Akt/MDM2 signaling pathway in macrophages to prevent NF-κB activation and drastically reduce the levels of TNF-α, IL-6, and IL-1β.^23,24

Our study also provided evidence that VPA significantly promoted the phagocytic ability of macrophages in vitro and in vivo. Delayed skin wound healing is associated with the accumulation of apoptotic cells in wounds. ⁶ Defects in apoptotic cell clearance can result in secondary necrosis, leading to increase levels of pro-inflammatory cytokines in wounds. ²⁵ Research has indicated that macrophages are the main phagocytic cells. ²⁶ A previous study revealed that VPA can promote the phagocytosis of apoptotic cells by macrophages to treat systemic lupus erythematosus. ¹⁴ In this study, we found that VPA increased the phagocytosis of apoptotic cells by macrophages in vitro. Furthermore, apoptotic cell accumulation in wounds was decreased after VPA treatment. The mechanism might be that VPA increased the expression of phagocytosis-associated molecules in macrophages, leading to increased phagocytic activity. Therefore, the therapeutic effects of VPA on skin wound healing could be partly attributable to its activity to promote apoptotic cell clearance.

This study had some limitations. The potential mechanism by which VPA stimulates macrophages to promote skin wound healing was not investigated, and the sample size in the animal experiments was small. Further studies are warranted.

Conclusion

Our investigation demonstrated that VPA accelerated skin wound healing, which could be partly attributable to its anti-inflammatory and apoptotic cell clearance-promoting effects, suggesting that VPA is a promising candidate agent for enhancing skin wound healing.