Static Magnetic Field Accelerates Diabetic Wound Healing by Facilitating Resolution of Inflammation

Impaired wound healing is commonly encountered in patients with diabetes mellitus, which may lead to severe outcomes such as amputation, if untreated timely. Macrophage plays a critical role in the healing process including the resolution phase. Although magnetic therapy is known to improve microcirculation, its effect on wound healing remains uncertain. In the present study, we found that 0.6 T static magnetic field (SMF) significantly accelerated wound closure and elevated reepithelialization and revascularization in diabetic mice. Notably, SMF promoted the wound healing by skewing the macrophage polarization towards M2 phenotype, thus facilitating the resolution of inflammation. In addition, SMF upregulated anti-inflammatory gene expression via activating STAT6 and suppressing STAT1 in macrophage. Taken together, our results indicate that SMF may be a promising adjuvant therapeutic tool for treating diabetic wounds.


Introduction
The diabetic foot ulcer is one of the most common and severe complications of diabetes mellitus because of impaired wound healing [1,2]. More than one million diabetes patients have to undergo lower limb amputation per year worldwide [3], which makes up approximately 50%-70% of all limb amputations. The standard treatment for diabetic wounds includes debridement of the wound, treatment of any infection, revascularization, and off-loading of the ulcer [4]. Although several strategies, such as the wound healing peptides, have been used with high efficiency [5,6], some refractory wounds and high costs of wound care predispose the patients to delay the treatment. Thus, it is desirable to explore alternative and cost-effective therapies for the patients with severe diabetic wounds.
Static magnetic field (SMF) has been applied in medicine as a tool to increase bone regeneration and promote drug delivery [7,8]. Accumulating evidences have demonstrated multiple beneficial effects of magnetic therapy, such as the recovery of the soft tissue and nerve system injury and insomnia [9][10][11][12][13]. Studies have also shown that SMF may influence the production of inflammatory cytokines released by macrophages and lymphocytes [14]. However, the therapeutic effect of SMF on diabetic wound healing remains to be determined.
During the process of wound healing, macrophages plays a critical role in modulating the inflammation and angiogenesis [15]. Basically, the macrophages are classified into two phenotypes: the "classically" activated macrophage (M1) and "alternatively" activated macrophage (M2) [16]. The M1 macrophage exhibits a proinflammatory function and promotes bacterial clearance and host defense by increasing phagocytosis and the production of inflammatory cytokines, while the M2 macrophage facilitates the resolution of inflammation and angiogenesis and promotes tissue remodeling by releasing anti-inflammatory cytokines and growth cytokines [17][18][19]. In vitro, SMF suppresses the production of inflammatory cytokines released by macrophages and lymphocytes [14]. However, the mechanism underlying SMF-mediated regulation of inflammation reaction is still unclear.
In this study, we observed that SMF significantly accelerated wound closure and revascularization by driving macrophages towards M2 polarization and inflammatory resolution through balancing STAT1/STAT6 signaling. The results suggest that SMF may serve as an effective therapeutic approach for diabetic wound.

Animals.
Male BKS-Lepr em2Cd479 /Nju (db/db) mice at 8-12-week old were used in this study. All mice were purchased from GemPharmatech Co. Ltd. and were maintained in mouse barrier facilities of Tianjin Medical University. All in vivo experiments complied with the Guidelines of the Institutional Animal Care and Use Committee of Tianjin Medical University that approved all protocols.

Wound Healing Model.
Mice were anesthetized by inhalation of isoflurane; the dorsal surface was shaved, washed with povidone iodine solution, and cleaned with an alcohol swab. Two excisional wounds were made on each side of the midline of the shaved dorsum using a sterile 8-mm punch biopsy tool (Miltex, USA). The wounds were covered with self-adhesive dressings (Cofoe). Diabetic mice with excisional wounds were housed on the top of the magnetic or nonmagnetic plate (230 mm × 130 mm × 15 mm) within the cage. Wound sizes were monitored under Leica Microsystems (Leica Microsystems Ltd.) and calculated using ImageJ software (National Institutes of Health). Injured skin tissues were subjected to paraffin embedding, serial sectioning, and subsequent hematoxylin and eosin (H&E) staining. Then, wound healing was assessed by measuring the largest distances between epithelial tips or panniculus carnosus edges in H&E-stained tissue using CaseViewer (3DHISTECH) [20].

Peritoneal Macrophage Isolation and Treatment.
The peritoneal macrophages were induced by intraperitoneal injection of 3% Brewer's thioglycolate as described previously [21]. The macrophages were allowed to adhere at 37°C overnight under 5% CO 2 , and unattached cells were removed by washing with fresh medium before use. Macrophage polarization was induced with 1 μg/ml LPS (Sigma, L2880) or 20 ng/ml IL-4 (Proteintech, 214-14).

Quantitative
Real-Time PCR. Total RNA from peritoneal macrophage and skin tissues were extracted using the TRIzol reagent (Invitrogen), and the cDNAs were synthesized using the Reverse Transcription Reagent Kit (Takara Bio Inc.) according to the manufacturer's instruction. The resulting cDNAs were amplified with 40 cycles by real-time PCR. Each sample was analyzed three times and normalized to a reference RNA using β-actin as the internal control. Sequences of primers used for real-time PCR to analyze the mouse samples are summarized in Table 1. . The cells were then washed twice before analysis. Stained cells were processed on a FACSAria flow cytometer (BD). The final data were analyzed using FlowJo (v.9; Tree Star).

Migration
Assay. Macrophage migration was evaluated using a Transwell system (Corning, 3422, NY, USA). Approximately 5 × 10 4 macrophages were suspended in 100 μl serum-free medium and seeded onto the upper chambers. Then, 500 μl RPMI medium 1640 basic (Gibco) with 30% fetal bovine serum was added to the lower chambers. After incubation for 24 h at 37°C under 5% CO 2 , the medium was removed from the upper chamber and the macrophages on the upper side of the chamber were scraped off with a cotton swab. The cells on the lower side of the upper chamber were fixed, stained with 0.1% crystal violet, photographed, and counted under a microscope (magnification 200x).

Statistical
Analyses. Data were analyzed using unpaired Student's t-test or repeated measures ANOVA, followed by Fisher's least significant difference analysis for multiple comparisons. P < 0:05 was considered statistically significant.

Results
3.1. SMF Accelerates Wound Healing in db/db Mice. To investigate the therapeutic effect of SMF on diabetic injury, the db/db mice were housed in a 230 mm × 130 mm × 15 mm plate with 24 magnetic pieces (0.6 T) embedded (Figure 1(a)). As shown in Figure 1(b), the SMF treatment promoted wound healing by reducing wound sizes at different time points. Moreover, the wound closure rate in db/db mice exposed to SMF was dramatically higher than that in the control group (Figure 1(c)). Histological analyses revealed significantly shorter distances between the epithelial tips of punched wound and distances between the edges of the panniculus carnosus in the SMF group at day 3 and day 7 postoperatively (Figures 1(d)-1(g)), suggesting that reepithelialization and wound contraction were enhanced in db/db mice exposed to SMF. In addition, the number of CD31-positive cells in the regenerative tissue in SMFtreated mice was notably higher than that in the control group (Figure 1(h)), indicating that SMF enhanced the revascularization in injured tissues.

SMF Promotes Wound Healing by Skewing Macrophage
Polarization towards M2 Phenotype. We first examined the effect of SMF on macrophage infiltration in the injured tissue from db/db mice. SMF treatment significantly increased macrophage recruitment (Mac-3 + ) at day 3 (acute inflammatory phase) and day 7 (tissue regeneration phase) (

SMF Accelerates Resolution of Inflammation by
Promoting M2 Macrophage Polarization in Mice. Consistent with microphage recruitment at an acute inflammation stage (day 3), in vitro transwell assay revealed that direct migration of culture macrophages was remarkably enhanced under SMF treatment (Figure 4(a)). We also examined the cytokine expression in wound tissues and found that the expression levels of proinflammatory genes (iNOS, IL-6, IL-Iβ, and CCR7; Figure 4(b)) in injured tissues were elevated at day 3 in SMF-treated mice and paradoxically were downregulated at day 7 and day 14 (Figures 4(c) and 4(d)). Meanwhile, SMF significantly raised the expression levels of reparative genes (CD206, Fizz1, Arg-1, and IL-10) at day 7 (Figure 4(c)). Collectively, these results indicate that SMF promotes inflammation resolution by modulating the expression profile of pro/antiinflammation cytokines in injured tissues.

SMF Promotes the M2 Macrophage Polarization In Vitro.
To examine the impact of SMF on M2 polarization in culture, peritoneal macrophages were isolated from db/db mice and then challenged by LPS or IL-4 to induce M1 or M2 polarization, respectively. After 24 h of treatment with 0.6 T SMF ( Figure 5(a)), the RT-PCR assay revealed that proinflammatory cytokines, such as IL-6, IL-12, and MCP1, were markedly downregulated (Figures 5(b)-5(d)). In contrast, antiinflammatory cytokines, such as YM-1, MRC1, and Arg-1 (Figures 5(e)-5(g)), were significantly upregulated in the SMF group, indicating that SMF suppresses M1 polarization, while promoting M2 polarization. The effects were further confirmed by Western blot assay, which showed a reduction of proinflammatory iNOS expression ( Figure 5(h)) and increased expression of anti-inflammatory Arg-1 ( Figure 5(i)) in SMFtreated macrophages.

SMF Regulates Macrophage
Polarization via STAT1 and STAT6 Activation. Canonically, LPS/TLR signaling skews macrophage towards the M1 phenotype by activation of STAT1 and IL-4 promotes macrophage towards the M2 phenotype via STAT6. As expected, SMF inhibited LPSinduced STAT1 phosphorylation (Figure 6(a)), while augmented IL-4-induced STAT6 phosphorylation was observed in the macrophages (Figure 6(b)). These data suggested that SMF promotes the macrophage polarization by regulation of STAT1/STAT6 activation.

Discussion
Wound healing is frequently impaired in patients with diabetes mellitus, and its treatment is still a big challenge. Here, we show that SMF accelerates wound healing in diabetic mice by promoting macrophage polarization to M2 phenotype. Mechanistically, SMF suppressed STAT1-mediated proinflammatory gene expression and facilitated STAT6mediated anti-inflammatory gene expression in macrophages. Thus, SMF may be a useful therapeutic means for diabetic wound care. SMF has been applied as a noninvasive and effective therapeutic method in various clinical practices. The low-frequency magnetic therapy has been officially approved by the US Food and Drug Administration (FDA) for orthopedic applications in treating pain and edema in superficial soft tissues, because SMF can modulate cell metabolism, proliferation, and apoptosis [9]. Prolonged period of exposure to SMF may assist the control of hypertension [22]. Meanwhile, SMF exerts a positive role in the treatment of osteoarthritis and nonunion fracture [23][24][25]. In this study, we found that     SMF on wound healing in diabetic patients warrants further investigation.
The wound healing process can be divided into three phases: inflammation, proliferation, and remodeling [28]. At the beginning of the inflammation phase, M1-like macrophages, also named proinflammatory macrophages, are recruited and aggregated in a large number to eliminate bacteria, foreign debris, and dead cells [29,30]. As such, massive M1 macrophages infiltrated the wound area by day 3 after injury. SMF increases macrophage infiltration, which aids wound healing at an acute inflammation stage. In some pathological conditions such as diabetes and infection, wounds may fail to achieve sufficient healing due to chronic inflammatory reaction; consequently, the proliferation and remodeling stage of healing would not occur [31]. After SMF treatment, more anti-inflammatory macrophages, referred to as M2 type, were recruited at day 7 and 14 postinjury, which facilitates resolution of inflammation and wound healing. During the process of the wound healing and scar formation at a late stage, vascularization is necessary to provide  sufficient oxygen and nutrition. CD31 expression, a vascularization marker, was enhanced in the wound areas at 7 days after SMF treatment. This finding conforms to the previous report that macrophages play particularly important roles in vascularization [32,33]. The macrophages can be categorized into proinflammatory, prowound healing, and proresolving ones based on their roles in different stages of the wound healing process [34]. The proinflammatory macrophages produce nitric oxide, ROS, IL-1, IL-6, and TNF-α [35], which mediate and facilitate the process of inflammation. The prowound healing macrophages produce elevated levels of growth factors such as PDGF, insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and transforming growth factor β1 (TGF-β1) [35,36], which aid in cellular proliferation, granulation tissue formation, and angiogenesis. The proresolving macrophages suppress inflammation via upregulation of IL-10, arginase 1, and TGF-β1 [35,37]. The expression of inflammatory cytokines, iNOS, MCP-1, IL-6, IL-1β, and CCR7, was increased in the injured tissue at an acute inflammation stage by SMF, probably due to increased recruitment of proinflammatory macrophages. Notably, anti-inflammatory cytokines, CD206, Fizz1, IL-10, and Arg-1, were increased in wound tissues by SMF at both day 3 and day 7, due to increased M2 transdifferentiation induced by SMF. Moreover, SMF can enhance the secretion of IL-10 and inhibit the secretion of proinflammatory cytokines, such as IL-6, IL-8, or TNF-α [14]. IL-10 has been substantiated to promote a regenerative process of wound healing [38], suggesting that SMF can promote resolution of inflammation in the proliferative phase.
Accumulating studies have reported that the alteration in the JAK/STAT pathway may result in impaired wound healing in a diabetes model and promotes alternative activation of macrophage [39][40][41][42][43][44]. Activated STAT3 induces the upregulation of iNOS expression, increases NO production in keratinocytes, and promotes angiogenesis in the wound tissue [45]. Macrophage function is always impaired in patients with diabetes, such as STAT-6-mediated M2 polarization. In addition, diabetic conditions, such as high glucose, activate STAT-1 signaling transduction [46]. We found that SMF induced upregulation of STAT6 phosphorylation and downregulation of STAT1 phosphorylation in macrophage, demonstrating that SMF may modulate the JAK/STAT pathways.
In summary, our study showed for the first time the effect of 0.6 T SMF on wound healing in diabetic mice. These results indicate that SMF accelerates diabetic wound healing by promoting macrophage polarization and resolution of inflammation through modulation of the JAK-STAT pathway. Therefore, focusing on SMF in therapeutic interventions might be useful for treating diabetic wound by renormalizing the healing process.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
Ying Yu is a fellow of the Jiangsu Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.

Authors' Contributions
Wenlong Shang and Guilin Chen contributed equally to this work.