Inhibition of skin glucocorticoid synthesis by topical metyrapone administration supports wound healing in diabetic mice

Inhibition of skin glucocorticoid synthesis by topical metyrapone administration supports wound healing in diabetic mice | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 March 2026 V1 Latest version Share on Inhibition of skin glucocorticoid synthesis by topical metyrapone administration supports wound healing in diabetic mice Authors : Vanderlei Fraga-Junior 0000-0003-3155-2388 , Ingrid Waclawiak , Willian Rodrigues Ribeiro , Edson José de Oliveira-Junior , Jeferson Kelvin Alves de Oliveira Silva , Matheus Palazzo , Evelyn Mendes do Nascimento , … Show All … , Matheus Azevedo de Pinho , Ana Paula Lima , Renato Sampaio Carvalho , Anna Cristina Neves Borges , Claudia Mermelstein , Fábio Barrozo do Canto , Thomas Brunner , and Claudia Farias Benjamim [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.177314987.71327230/v1 254 views 81 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Chronic wounds are a major complication of diabetes mellitus (DM), posing a significant burden on healthcare systems worldwide. Despite advances in the field over the past two decades, the incidence of lower-limb amputations due to diabetes remains high. Persistent hyperglycemia sustains a chronic low-grade inflammation, disrupts hormonal homeostasis, and elevates glucocorticoid (GC) levels both systemically and locally, all of which impair wound healing. The enzymes 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and steroid 11β-hydroxylase (CYP11B1) are key mediators of local GC reactivation and production, respectively, and are upregulated in diabetic wounds. Experimental Approach: To investigate the therapeutic potential of metyrapone (MET), a dual 11β-HSD1/CYP11B1 inhibitor, as a topical pro-healing agent we used a full-thickness excisional wound model in alloxan-induced diabetic mice. Key Results: Daily topical application of MET (1 mg/wound) for 14 days accelerated wound closure, reduced neutrophilic infiltration, restored myofibroblast differentiation, and enhanced collagen deposition. MET suppressed the expression of several pro-inflammatory genes and production of cytokines, such as IL-1β and IL-6, while reducing local GC production in the skin, without affecting its systemic levels. In vitro , MET enhanced fibroblast migration without altering proliferation. Gene silencing via siRNA revealed that both 11β-HSD1 and CYP11B1 contributed to local GC production. Conclusions and Implications: Collectively, these findings support MET as a promising topical therapeutic strategy to improve wound healing in diabetic patients. INTRODUCTION Impaired wound healing is a common complication in diabetic patients, frequently leading to lower limb amputations (Hoffstad et al., 2015). Despite significant advancements, understanding the molecular mechanisms of chronic wounds remains challenging due to their complex pathophysiology. Many available treatments are costly or ineffective, underscoring the need for novel therapeutic approaches (Vas et al ., 2020). Chronically, elevated glucose levels induce inflammation through the deposition of advanced glycation end-products (AGEs), which activate immune cells and lead to oxidative stress and pro-inflammatory cytokines release, resulting in persistent low-grade inflammation (Goova et al., 2001; Peppa et al., 2003; Cai et al., 2007). In this context, sustained metabolic stress disrupts the hypothalamic-pituitary-adrenal (HPA) axis, increasing glucocorticoid (GC) secretion systemically. However, excessive GC levels hinder wound healing by altering cutaneous homeostasis, inducing apoptosis in fibroblasts and keratinocytes, reducing collagen deposition, and impairing reepithelialization and angiogenesis mechanisms (Choe et al., 2018; Carolina et al., 2018; Shin et al., 2019). Accordingly, systemic inhibition of GC production or glucocorticoid receptor (GR) activation has been shown to accelerate wound healing in murine models (Vukelic et al., 2011; Almeida et al., 2016; Bitar et al., 1999). Beyond their systemic effects, glucocorticoids are also locally produced in the skin. Fibroblasts, keratinocytes, and melanocytes produce GC - cortisol (in humans) and corticosterone (in rodents) - due to the expression of the proteins directly involved in its synthesis. Active GC can be either synthetized de novo via an-11β-hydroxylase-1 (CYP11B1)-dependent process or via reactivation from inactive forms - cortisone or 11-dehydrocorticosterone, respectively - by the enzyme 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1). Other factors including steroidogenic factor 1 (SF1), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme (CYP11A1) also regulate tissue-specific GC effects (Slominski et al., 2000; Ito et al., 2005; Patel et al., 2011; Slominski et al., 2006). Steady-state local GC production in the skin is essential for maintaining the epidermal barrier, and its deficiency is linked to inflammatory disorders (Lee et al., 2020; Slominski et al., 2017; Phan et al., 2021). However, pharmacological inhibition and genetic ablation of 11β-HSD1 have been shown to be beneficial in certain contexts, such as wound healing (Tiganescu et al., 2013; Small et al., 2005; Brazel et al., 2020; Terao et al., 2011). Thus, despite substantial evidence demonstrating dysregulation of local GC production in several pathological skin conditions, including wounds from patients and murine models of type 2 diabetes, and the well-established role of cortisol/corticosterone downregulation in improving wound healing (Vileikyte et al., 2018; Morgan et al., 2014; Tsai et al., 2023; Ajjan et al., 2022), the topical effects of local GC synthesis inhibition by metyrapone (MET) on diabetic wounds are unknown. Therefore, this study aimed to evaluate the therapeutic potential of MET, a dual 11β-HSD1/CYP11B1 inhibitor (Sampath-Kumar et al., 1997; Tobes et al., 1985), in cutaneous wound healing using a type 1 diabetic mouse model. METHODS Animals Male C57BL/6 mice, aged 8-12 weeks and weighing 20-25 g, were obtained from the Laboratory Animal Breeding Center of UFRJ (NCAL, RJ, Brazil). The animals were housed at a constant temperature of 25°C under a 12 h light/dark cycle with free access to food and water. All experiments were conducted according to the ethical guidelines of the Institutional Animal Care Committee of the Health Science Center of the Federal University of Rio de Janeiro (CONCEA: 01200.001568/2013-87, protocol code 012/20). Experimental model of type 1 diabetes mellitus Type 1 diabetes mellitus (T1DM) induction was performed by a single dose of alloxan (65 mg/kg, intravenously). Animals were fasted for 12 h before the injection. After 7 days, blood glucose was measured by an AccuCheck Active glucometer (Roche Diagnostics, São Paulo, Brazil) to confirm hyperglycemia. Animals presenting blood glucose > 350 mg/dl were considered diabetic. In another set of experiments, the blood glucose was also measured on day 7 after wounding (Fig. 1A-B). Full-thickness excisional wound model Full-thickness excisional wounds were created on the shaved back skin using a 10 mm biopsy punch under anesthesia (112.5 mg/kg ketamine and 7.5 mg/kg xylazine intraperitoneally - Cristália, Brazil) (Wong et al., 2011). Post-surgery, the animals were housed individually and monitored for 14 days. Skin samples from the wound sites were harvested on days 3, 7, and 14 for further analysis. Treatments Metyrapone (MET; 0.25, 0.5, or 1 mg/wound; Sigma-Aldrich, St. Louis, MO) was diluted in 30 µL of 1× phosphate-buffered saline (PBS) by vigorous agitation at room temperature immediately prior to treatment. To avoid precipitation, the solution was rapidly applied topically once daily for 14 consecutive days in the morning (8:00–10:00 a.m.). Control groups received the vehicle alone. Morphometric analysis Wound areas were measured on days 0, 3, 7, 10, and 14. Digital photographs were taken from 10 cm of distance fixed by tripod for the camera, and wound areas were measured using ImageJ software (National Institutes of Health - NIH, Bethesda, MD). The initial wound area on day 0 was considered 100%, and subsequent measurements were calculated as a percentage of the initial wound area. Data are presented as the mean ± standard error of the mean for each group. Histological procedures Skin tissue samples were fixed in 5% buffered formalin (Sigma-Aldrich, St. Louis, MO) for 24 h, dehydrated in ethanol (92.8%), cleared in xylene, and embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin (HE) or Picrosirius Red for histological examination. The bright field analysis was performed using a digital camera connected to a microscope (Olympus DP72 - Olympus, Tokyo, Japan) and the Cell Sens Standard software (Olympus). Immunofluorescence Wound immunofluorescence assays were performed in paraffin sections prepared as previously described. After deparaffinization, the slides were washed in citrate buffer (pH 6.0) for antigen retrieval. The samples were then blocked with 3% bovine serum albumin (BSA) in tris-buffered saline com tween 20 (TBS-T) wash buffer and incubated with an antibody against α-SMA (1:500 – Abcam, Cambridge, UK). After incubation, the slides were washed and stained with an anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1-1000 - Cell Signaling, Danvers, MA), and nuclei were stained with ProLong™ Gold Antifade Mounting (Invitrogen, Boston, MA), according to the manufacturer’s instructions. Negative controls were incubated with the secondary antibody only. Specimens were examined with an Axiovert 100 microscope (Zeiss, Germany), and images were captured with an Olympus DP72 digital camera (Olympus). Corticosterone quantification Whole blood was collected from each mouse on day 14 in the morning and centrifuged for 10 min at 3000 rpm. Wound samples were also collected, homogenized in 1% Triton diluted in PBS, sonicated for 5 min, and centrifuged for 15 min at 10000 g. Plasma and wound corticosterone levels were measured using a Corticosterone ELISA kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s instructions. Scratch assay Immortalized murine fibroblasts (L929) were cultured in a 24-well plate (2.5×10 5 cells/well) with RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS - Gibco, New York, NY), Hepes (20 mM – Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin. The plates were incubated for 24 h at 37º C with 5% CO 2 to allow the cells reach confluence. Subsequently, the cells were then treated with mitomycin C (20 µM/mL – Sigma-Aldrich) for 2 h to inhibit proliferation. The monolayer was then wounded using a 200 µL tip, and the wells were gently washed to remove cell debris. The medium was replaced with DMEM (Gibco) - high glucose (4.5 g/L) or low glucose (1 g/L) – supplemented with 1% SFB (Gibco). The cells were incubated with MET (20-60 µM) diluted in 0.3% DMSO for 24 h in the BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA). Pictures of the scratched areas were taken at 0, 6, 12, 18, and 24 h post wounding. The areas were measured using the ImageJ software and the fibroblast migration was expressed as the number of cells migrated to the open area. Proliferation assay (Ki-67) Immortalized murine fibroblasts (10 5 cells - L929) were cultured as previously described for 24 h with MET (20-60 µM). Then, the cells were fixed with 4% paraformaldehyde, permeabilized with TBS-T, blocked with 3% BSA and incubated overnight with the following antibodies: Ki-67 (1:1000 - Abcam) and β-actin (1:500 - Sigma). After that, the cells were incubated with anti-Rabbit Alexa Fluor 488-conjugated (1:1000 - Cell Signaling) and anti-Mouse PE-conjugated (1:1000 – Invitrogen) secondary antibodies for 2 h. The slides and the fluorescence images were performed as previously described. Western blotting Cell lysates and wound homogenates (30 mg of skin tissue; 10–30 µg of protein loaded per gel lane) were prepared using NuPAGE™ reagents (Invitrogen) and denatured at 95 °C for 5 min prior to SDS–PAGE. After electrophoresis, proteins were transferred onto PVDF membranes. Membranes were blocked with TBS-T containing 5% BSA for 1 h and then incubated with primary antibodies overnight at 4 °C. Then, the membranes were washed and incubated for 2 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology). Immunoreactive bands for GRα (4µg/mL – Invitrogen), CYP11B1/B2 (1:1000 - Invitrogen), 11β-HSD1 (1:1000 - Cell Signaling), α-SMA (1:10000 – Abcam, Cambridge, UK), Collagen I (1:1000 - Abcam) and β-actin (1:1000 - Cell Signaling) were visualized using an enhanced chemiluminescence reagent (Amersham ECL, Biosciences), and images were captured using a Healthcare ImageQuant LAS 5000 (GE Healthcare Life Sciences, France). Densitometric analysis was conducted with ImageJ software (NIH), and results were expressed as the ratio of target protein to β-actin (housekeeping protein). Total RNA extraction and quantitative real-time RT-PCR Total RNA was extracted from wound tissues collected on days 3 and 7 using TRIzol (Invitrogen) reagent according to the manufacturer’s instructions. Two micrograms of RNA were used for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). Quantitative real-time RT-PCR was performed using the SYBR-green (Applied Biosystems, Waltham, MA) fluorescence quantification system. PCR cycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min, with a standard denaturation curve. Primer sequences are provided in the supplementary materials (Supp. Table 1). Relative expression levels of target genes were normalized to Gapdh and calculated using the comparative Ct method. Cytokines and protein quantification The cytokines IL-1β, IL-4, IL-6, IFNy, and TGF-β1, the enzymes MMP-3 and MMP-9, and TIMP-1 were quantified in wound homogenates using ELISA kits (R&D Systems, Minneapolis, MI) following the manufacturer’s instructions. Total protein content was measured using a BCA kit (Thermo Fisher), and results were expressed as ng of cytokine/mg of protein or pg of enzyme/mg of tissue. References for all kits are provided in the supplementary materials (Supp. Table 2). Flow cytometry Flow cytometry of wound tissues was performed as previously described (Brubaker et al., 2011). Briefly, wound samples were incubated for 1:30 h with a dispase I enzyme solution (1 mg/mL; Roche Diagnostics) and then with an enzyme cocktail (1 mg/ml - Sigma-Aldrich) for 1 h, both diluted in RPMI medium without serum. Then, the suspensions were flushed through a 40 µm cell-strainer (Guangzhou Jet Bio-Filtration Co., China). Cell suspension (10 6 cells/mL) from each wound was resuspended in PBS, blocked with 10% mouse serum, and then stained for different populations using specific antibodies described in supplemental materials (Supp. Table 3). Samples were acquired with BD LSRFortessa TM (BD Biosciences, San Jose, CA) and then analyzed with FlowJo software (BD Biosciences). siRNA silencing Murine fibroblasts (1.5×10 6 - L929) were transfected using the Amaxa TM Cell Line Nucleofector TM Kit V (Lonza, Canada). Electroporation was conducted using the Program T-030 and 2 µg of pmaxGFP vector was used to evaluate transfection efficiency. Hsd11b1 and Cyp11b1/b2 siRNAs (100 pmol - Invitrogen) were employed to inhibit fibroblast corticosterone production, while scramble siRNA served as a transfection control. Once the cells reached confluence, a scratch assay was performed as previously described, with the exception that the FBS concentration was 10% in this experiment. Statistical analysis Statistical analysis was performed using GraphPad Software (La Jolla, CA). Data were analyzed using the nonparametric Student’s t-test, two-way ANOVA with Bonferroni post hoc test, or one-way ANOVA with Tukey post hoc test, as appropriate. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001 compared to controls or # P < 0.05, ## P < 0.01, and ### P < 0.001 compared to other conditions. Results are expressed as mean ± standard error of the mean (SEM). RESULTS Corticosteroidogenic key proteins are upregulated in skin wounds of pharmacologically induced type 1 diabetic mouse To begin with, we aimed to investigate whether the acute model of alloxan-induced type 1 diabetes, in C57BL/6J mice, promotes an increase in steroidogenic activity in skin wounds. Initially, we validate the model, and as expected, seven days after alloxan injection, the diabetic group exhibited significantly elevated blood glucose levels, leading to impaired wound healing (Fig. 1A–E). To evaluate the expression of proteins involved in both the reactivation and de novo synthesis of GC locally, we analyzed skin samples on day 3 post-wounding, corresponding to the peak of inflammation in our model (Wong et al., 2011; Zomer and Trentin, 2018). The Sf1 (Nr5a1) , Star , and Hsd11b1 genes were significantly upregulated in diabetic wounds. Interestingly, Cyp11a1 expression remained unaltered on day 3 of healing, similar to Cyp11b1 (Fig. 1F-J). To confirm whether these changes were maintained at the protein level, we quantified key enzymes of both pathways by Western blotting at the same timepoint. Consistently, the enzyme 11β-HSD1 was markedly increased in diabetic skin lesions, whereas CYP11B1 levels were unchanged, indicating that the reactivation pathway may be more active in the early inflammatory phase. Furthermore, elevated GRα levels in the skin of the diabetic group reinforced enhanced glucocorticoid signaling. (Fig. 1K-N). MET topical treatment ameliorates wound healing in diabetic mice without altering systemic corticosterone levels To inhibit local GC production, we topically administered MET at different concentrations immediately after wounding in diabetic mice, once daily for 14 days. Lower concentrations of MET (0.25 and 0.50 mg/30 µL/wound) did not improve wound healing (Supp. Fig. 1). However, at the concentration of 1 mg/30 µL/wound, MET significantly enhanced wound healing (Fig. 2D-F). Conversely, MET impaired wound closure in euglycemic animals (Fig. 2A-C), as expected, given the critical role of local GC production in maintaining skin homeostasis (Phan et al., 2021). Interestingly, we can observe an inversely proportional area under the curve (AUC) between diabetic and euglycemic MET-treated groups (Fig. 2C and F). To validate our macroscopic findings, we measured the distance between the epithelial lips at the wound edges by histomorphometry on day 7 post wounding. The MET-treated wounds in diabetic group exhibited smaller non-re-epithelialized areas compared to PBS-treated wounds in diabetic animals (Fig. 2G-H). These findings suggest that the pro-healing effects of MET are closely dependent on the tissue’s metabolic status. Given the high concentration of MET used in our experiments, one of our primary concerns was the potential for systemic absorption, which could bypass its intended local effects. To evaluate this, we collected wound and blood samples at the end of the experiments to quantify corticosterone levels by ELISA. In euglycemic wound samples, no significant changes in corticosterone levels were observed, regardless of MET treatment. However, in the diabetic group, MET-treated lesions exhibited a significant reduction in corticosterone levels compared to PBS-treated samples (Fig. 2I). In contrast, the blood corticosterone levels were significantly higher in MET- and PBS- topically treated diabetic mice, with no differences between them, compared to euglycemic groups (Fig. 2J). Glucocorticoids are generally upregulated in diabetic conditions as a consequence of impaired negative feedback, leading to sustained activation of the hypothalamic–pituitary–adrenal (HPA) axis (Bruehl et al., 2007). Likewise, corticosterone exerts direct diabetogenic effects, which increases blood glucose levels establishing a vicious circle. To warrant that MET treatment impacts were only local, we also analyzed blood samples for glucose levels on day 7 after wounding. As expected, the diabetic animals displayed elevated blood glucose levels compared to the euglycemic groups, with no significant changes in the MET-treated mice (Fig. 2K). MET improves tissue formation in the wounds of diabetic mice For the histopathological evaluation of the lesions, we chose to conduct the analyses on day 7, as it represents the proliferative phase during the healing process, and while the inflammatory phase subsides (Cialdai et al., 2022). At this point, we can clearly observe the deleterious effects of T1DM on the tissue repair process in the skin. Firstly, we stained the samples with HE in order to obtain an overview of wound morphology. Regarding the epidermis, the number of keratinocytes in the proliferative layer in both groups treated with MET were robustly increased, as well as in the euglycemic control treated with PBS. Additionally, MET-treated diabetic lesions showed a pronounced enlargement in epidermal thickness compared to the diabetic group treated only with the vehicle (Fig. 3A). In the dermis, diabetic animals exhibited disorganized extracellular matrix (ECM) fibers, with little to no differentiation between the papillary and reticular layers. Treatment with MET completely reversed this phenotype. Interestingly, MET did not affect the dermal architecture in euglycemic mice. Furthermore, in the wounds of diabetic animals treated with MET, there was an apparent decrease in leukocyte infiltrate, particularly in the papillary layer, along with pronounced regeneration of perilesional skin appendages (Fig. 3B). Regarding the hypodermis, a substantial loss of subcutaneous fat deposits was expected, given the cachectic process triggered by the T1DM model. However, lesions in diabetic mice treated with MET appeared to show partial preservation of these deposits within the wound bed (Fig. 3C). Diabetes mellitus impairs the differentiation of fibroblasts into myofibroblasts, thereby compromising collagen deposition and wound contraction (Gurtner et al., 2008). To address this, we assessed total collagen deposition using Picrosirius Red staining. Diabetic wounds treated with MET exhibited increased collagen deposition compared to PBS-treated diabetic animals. Conversely, MET-treated euglycemic mice showed a reduction in collagen fiber deposits (Fig. 3D). Topical MET treatment also restored α-SMA expression in diabetic lesions, indicating the presence of myofibroblasts comparable to those in the euglycemic dermis. However, MET-treated euglycemic wounds displayed reduced α-SMA levels (Fig. 3E). To corroborate these histopathological findings, western blot analysis was performed to quantify type I collagen fibers and α-SMA in wound homogenates. PBS-treated diabetic wounds showed significant reductions in both markers, whereas MET-treated diabetic lesions demonstrated similar levels to those observed in PBS-treated euglycemic controls (Fig. 3F). MET induces fibroblast migration with no impact on cell proliferation in vitro To investigate the mechanisms underlying the improved collagen deposition observed in diabetic wounds treated topically with MET, we conducted in vitro experiments to assess the effects of MET on murine fibroblasts (L929) proliferation and migration. Initially, fibroblasts were incubated with either 20 µM or 60 µM of MET for 24 hours under low- or high-glucose conditions. Following incubation, cells were stained with Ki-67 to evaluate proliferative activity. Surprisingly, MET did not significantly alter the proliferative capacity across the tested conditions (Fig. 4A, C). To examine the impact of MET on migration dynamics, fibroblasts were cultured under high-glucose conditions with 1% FBS supplementation to minimize interference from exogenous cortisol or cortisone. A scratch assay was then performed over 24 hours in the presence of 60 µM MET. Fibroblasts treated with MET exhibited enhanced migration capacity at 12 hours post-scratch (Fig. 4B, D), indicating that MET may promote fibroblast migration activity without affecting cell proliferation. MET restores the inflammatory milieu in diabetic wounds One of the primary characteristics of impaired wound healing is sustained inflammation (Falanga et al., 2022). To evaluate the effects of MET topical treatment on the inflammatory response, we first assessed gene expression using qPCR. As expected, T1DM significantly upregulated several pro-inflammatory genes compared to euglycemic groups, while MET treatment downregulated most of these genes in the wounds of diabetic mice (Fig. 5A). MET-treated diabetic lesions exhibited reduced levels of Cxcl1 and Cxcl2 transcripts; however, Ccl2 expression remained elevated in both diabetic groups (Fig. 5B–D). Additionally, MET downregulated key pro-inflammatory genes, including Il1a , Il1b , Il6 , and Tnfa , while Ifny expression was not significantly affected among the experimental groups (Fig. 5E–I). Interestingly, the anti-inflammatory gene Il10 was also reduced in MET-treated diabetic wounds compared to PBS-treated diabetic group (Fig. 5J). Surprisingly, despite its deleterious effects in euglycemic mice, MET did not alter the expression of these genes in euglycemic lesions. To corroborate the effects of MET on the inflammatory milieu , we also quantified cytokine levels by ELISA. The results mirrored the gene expression patterns, with IL-1β, IL-6, and IFNγ levels following similar trends, with no differences observed among groups for IL-4 amounts. Furthermore, diabetic lesions treated with PBS showed elevated levels of TGF-β1, which were unaffected by MET treatment (Fig. 5K–O). Persistent inflammation during the proliferative phase disrupts the balance between matrix metalloproteinases (MMPs) and their inhibitors, leading to ECM degradation [35]. MET-treated diabetic animals exhibited a significant reduction in MMP-3 and TIMP-1 levels compared to PBS-treated diabetic mice, whereas MMP-9 levels did not differ among the groups (Fig. 5P–R). MET reshapes myeloid leukocytes frequencies in diabetic wounds Diabetes mellitus commonly disrupts the infiltration and differentiation of myeloid cells during wound healing, promoting neutrophil infiltration while impairing macrophage differentiation. This disruption maintains macrophages in a classically activated, pro-inflammatory phenotype (M1) and inhibits the transition to the alternatively activated subset (M2), a critical step for initiating the proliferative phase of healing (Falanga et al., 2022; Krishnaswamy et al., 2017). In diabetic wounds treated with MET, the frequency of neutrophils (Ly6G⁺Ly6C int cells) was significantly reduced compared to PBS-treated diabetic mice. Conversely, topical MET treatment restored the proportional abundance of monocytes (Ly6G⁻Ly6C high cells) in diabetic lesions. Additionally, MET-treated diabetic mice exhibited an increased presence of Langerhans cells (F4/80⁺CD207⁺MHC-II⁺ cells) in their wound samples (Fig. 6B-D). Topical MET treatment also restored macrophage (Ly6G⁻F4/80⁺MHC-II⁺ cells) proportions in diabetic wounds, decreasing the frequency of M1 macrophages (F4/80⁺MHC-II⁺CD206⁻CD86⁺ cells), and increasing M2 macrophages (F4/80⁺MHC-II⁺CD206⁺ cells) (Fig. 6E-G). Further analysis of M2 macrophage subsets revealed that T1DM significantly reduced the populations of M2a (F4/80⁺MHC-II⁺CD86⁻CD206⁺ cells) and M2b (F4/80⁺MHC-II⁺CD86⁺CD206⁺ cells). However, MET treatment did not restore these M2-associated phenotypes. In the draining lymph nodes (dLNs), similar patterns were observed. Within the myeloid compartment, MET-treated diabetic mice exhibited reduced neutrophil percentages and increased monocyte frequencies compared with PBS-treated diabetic mice. However, no significant changes in Langerhans cell or macrophage proportions were detected among the groups (Fig. 6J-M). In absolute numbers, no significant differences were observed on MET-treated euglycemic or diabetic animals (Supp. Fig. 2A-D). MET reduces CD4 + T lymphocytes in diabetic wounds while it rescues Foxp3 gene expression During wound healing, lymphoid cells are recruited to coordinate the proliferative phase by modulating inflammation through regulatory T cell (Treg) differentiation or inducing keratinocyte activation via gamma delta T cells (Tγδ). Diabetes mellitus disrupts lymphoid cell dynamics, reducing Forkhead Box Protein 3 (FOXP3) and Vγ5 cell populations (Rhoiney et al., 2023; Cheng et al., 2024). It is important to note that the literature employs two nomenclature systems to classify Vγ chains: the Garman system and the Heilig and Tonegawa system (Garman et al., 1986; Heilig and Tonegawa, 1986). In this article, we adhere to the Heilig and Tonegawa system. In diabetic mice treated with MET, the frequency of CD4 + cells in wound tissues decreased compared to PBS-treated diabetic mice, while CD8 + proportions remained unchanged across experimental groups (Fig. 7B-C). As expected, T1DM increased the Vγ4 + population but reduced Vγ5 + dendritic epidermal T cells (DETCs) in the lesions. MET treatment did not restore T γδ cell percentages (Fig. 7D-E). Notably, qPCR analysis revealed that wounds from MET-treated diabetic mice exhibited increased transcription of the Tbx21 gene, which encodes T-bet. However, the Rorc and Gata3 genes showed no significant changes among the groups. Interestingly, MET-treated diabetic animals also demonstrated upregulation of the Foxp3 gene compared to PBS-treated diabetic mice, suggesting that MET may promote Treg differentiation in diabetic lesions (Fig. 7F-I). In the dLNs, no significant differences were observed among the groups regarding CD4 + or CD8 + frequencies or absolute cell numbers, with one exception: an increased number of CD4 + T lymphocytes in the MET-treated euglycemic group (Fig. 7J-M). Inhibition of 11β-HSD1 more effectively enhances fibroblast migration in vitro To determine which pathway plays a dominant role in local GC production, reactivation or de novo synthesis, we performed a scratch assay, using a fibroblast monolayer, previously silenced using siRNA targeting the Cyp11b1 and Hsd11b1 genes. Transfection efficiency was confirmed via GFP + reporter expression, and protein levels of the targeted enzymes were assessed by Western blot. Transfection rates were comparable across all conditions. Notably, Cyp11b1 silencing was more effective, reducing protein expression by approximately 50%, whereas Hsd11b1 silencing led to a 25% reduction in protein levels (Fig. 8A–D). Despite the smaller reduction in 11β-HSD1 expression, its gene silencing resulted in a significant enhancement of fibroblast migration compared to CYP11B1 knockdown. Although silencing of both enzymes enhanced fibroblast migration, knockdown of 11β-HSD1 produced a significantly stronger effect than CYP11B1 silencing. (Fig. 8E–G). These findings suggest that 11β-HSD1 plays a major role in reducing fibroblast migratory capacity. Nevertheless, both enzymes demonstrate potential as therapeutic targets for wound management, and their synergistic effects highlight MET as a promising candidate for clinical trials. DISCUSSION Since the first evidence of GC production in the skin was described by Ralf Paus’s group in 2005 (Ito et al., 2005), numerous mechanisms involving local steroidogenesis have been proposed, ranging from its role in maintaining cutaneous homeostasis to its involvement in aging (Tiganescu et al., 2011; Tiganescu et al., 2013). In the context of wound healing, several studies have highlighted the beneficial effects of locally inhibiting GC signaling, employing various strategies to impair local GC production or blocking GRα activity (Tiganescu et al., 2013; Morgan et al., 2014). The main strategy used to reduce local GC availability in the skin and enhance wound healing has been the use of genetically modified mice, particularly 11β-HSD1 knockout mice (Small et al., 2005; Terao et al., 2011; Tiganescu et al., 2013). As a pharmacological alternative to these genetic approaches, in this study we propose the topical application of MET to suppress local GC production and promote wound healing. By simultaneously targeting both steps in the steroidogenic pathway, MET can more effectively suppress GC production within the skin. Moreover, the topical application of MET limits systemic exposure, thereby reducing the risk of undesirable side effects. Furthermore, MET is already used clinically to treat various conditions, such as Cushing’s syndrome, treatment-resistant depression, and post-traumatic stress disorder (Daniel et al., 2015; Sanches et al., 2021). Additionally, its pharmacokinetics and pharmacodynamics are well characterized, supporting its potential for repurposing in dermatological applications. In this study, we demonstrated for the first time the topical effects of MET in modulating inflammation and reshaping the immune response in a murine diabetic wound model. As a pro-healing agent, MET significantly reduced corticosterone levels in wounded skin without affecting systemic concentrations, thereby promoting wound healing in diabetic mice. Notably, MET-treated diabetic lesions exhibited healing rates comparable to those of PBS-treated euglycemic controls. It is important to mention that Vukelic and colleagues (2011) previously demonstrated that MET possesses pro-regenerative properties, diminishing cortisol levels and improving wound closure in human skin explants derived from reduction mammoplasty and abdominoplasty specimens of healthy women. In the same study, a partial-thickness wound model in euglycemic female pigs treated with MET further corroborated the ex vivo findings (Vukelic et al., 2011). Interestingly, in our experiments with euglycemic mice, the topical application of MET appeared to impair wound healing. However, it is well established that local GC production at physiological concentrations is essential for epithelial barriers homeostasis. Corroborating, keratinocyte-specific deletion of Cyp11b1 increases susceptibility to inflammatory skin disorders in mice (Phan et al., 2021). Furthermore, at physiological concentrations, cortisol is pivotal in maintaining a healthy fibroblast phenotype (Faust et al., 2024). Similarly, both lesional and non-lesional psoriatic skin exhibit impaired local de novo GC synthesis and reduced GR expression (Hannen et al., 2017). In concern of fibroblast function , MET 60 μM accelerated fibroblast migration in vitro , even in a high glucose medium supplemented with 1% FBS, a condition commonly used to restrict fibroblast migratory capacity. Indeed, human dermal fibroblasts exhibit significantly impaired migration under low-serum or high-glucose conditions, both of which are known to repress key signaling pathways involved in cell motility, such as c-Jun N-terminal kinase (JNK) phosphorylation (Xuan et al., 2014). On the other hand, MET did not alter fibroblast proliferation rates in both concentrations used, 20 μM or 60 μM. These concentrations were selected based on the higher affinity of MET for CYP11B1, whereas inhibition of 11β-HSD1 occurs only at concentrations above 30 μM in a dose-dependent manner (Sampath-Kumar et al., 1997). To investigate which enzyme might be most effective in providing GC locally and consequently impairing wound healing, we used siRNA-mediated gene silencing and demonstrated that knockdown of both Cyp11b1 and Hsd11b1 enhanced fibroblast migratory capacity. However, Hsd11b1 knockdown was more effective in accelerating wound healing in vitro . In agreement with this, 11β-HSD1-deficient mice exhibit accelerated wound healing accompanied by heightened fibroblast’s migration and differentiation (Terao et al., 2011). Interestingly, 11β-HSD1 activity can also be therapeutically increased to retard wound healing under specific conditions, such as in burn injuries. For instance, slow-release prednisone delays wound closure by reducing inflammation, myofibroblast differentiation, and collagen production, thereby mitigating scar stiffness and the formation of adherent scars, major complications in the recovery of extensively burned patients (Tsai et al., 2023). Wound healing is a complex process critically dependent on the initial inflammatory phase, which triggers the subsequent stages characterized by efficiently fibroblasts and keratinocytes activation. Pro-inflammatory cytokines such as IL-1 family members, IL-6, and IL-8 are pivotal for initiating the wound healing dynamics (Macleod et al., 2021; Johnson et al., 2020; Rennekampff et al., 2000). In diabetic skin, however, a pre-existing state of chronic low-grade inflammation, characterized by elevated TNFα levels and accompanied by GC enrichment even before tissue injury, attenuates the early inflammatory response by suppressing the acute cytokine burst post-wounding. Paradoxically, this leads to a prolonged and dysregulated production of inflammatory mediators during the later stages of wound healing, contributing to delayed tissue repair (Peña and Martin, 2024). It is this level of fine-tuning that renders the development of new therapeutic strategies especially challenging. And it is in this context that MET, despite the pro-inflammatory effects [due to the GC synthesis/reactivation inhibition], at a specific concentration, helps to rebalance the inflammatory process after skin wounding. Based on what has been said, at first glance, it may seem counterintuitive that a pro-inflammatory approach could enhance wound healing in inflamed lesions. However, in a previous study by our group, topical supplementation with ADP, a pro-inflammatory purinergic nucleotide, on diabetic wounds using the same murine model, yielded outcomes similar to those observed in MET-treated diabetic mice, including accelerated wound closure, reduced pro-inflammatory cytokine production, and restoration of immune cells to a more pro-regenerative state (Borges et al., 2021). Consistent with these findings, MET not only promoted M2 polarization but also reduced neutrophil infiltration in diabetic wounds. Although neutrophils play a critical role in the early stages of the inflammatory phase, their persistence into the subsequent stages can be detrimental to wound closure, largely due to the formation of neutrophil extracellular traps (NETs) (Wong et al., 2015). Similar to neutrophils, M1 macrophages are essential in the early inflammatory phase, promoting a robust immune response following tissue injury. However, timely polarization toward the M2 phenotype is equally critical to support resolution of inflammation and tissue regeneration (Sharifiaghdam et al., 2022). Beyond their classical role in antigen presentation to T lymphocytes, LCs are also essential for wound healing. During skin repair, LCs are found near endothelial tip cells, and their absence impairs endothelial cell proliferation, resulting in abnormal blood vessel morphology (Peña and Martin, 2024; Vasko et al., 2022). Corroborating that, biopsies from diabetic foot ulcers have shown a marked reduction in LCs in non-healing lesions compared to the healing ones, further supporting the critical role of LCs in the wound healing process (Stojadinovic et al., 2013). In this regard, it is noteworthy that MET treatment also increased the number of LCs in wounds from diabetic mice, suggesting a potential mechanism by which MET may contributes to improved vascularization during tissue repair. γδ T cells are highly enriched within the epidermal layers and contribute to skin homeostasis both at steady state and during wound healing, acting as primary sources of IL-17 and keratinocyte growth factors (Jameson et al., 2002; Konieczny et al., 2022). In our model, T1DM markedly reduced Vγ5 T cell frequencies, however MET treatment failed to restore the Vγ4/Vγ5 ratio in diabetic wounds. Among conventional T cell populations, MET did not alter CD8⁺ T cell frequencies. However, diabetic lesions treated with MET exhibited a marked reduction in CD4⁺ T lymphocytes. In contrast, MET restored Foxp3 gene expression in diabetic wounds, suggesting a potential regulatory T cell-mediated mechanism contributing to its therapeutic effect. Nonetheless, it is important to emphasize that the role of Tregs in wound healing remains controversial in the literature. Barros and colleagues (2019) demonstrated that diabetic CCR4-deficient mice exhibited accelerated wound closure, likely due to impaired Treg migration and homing to the skin (Barros et al., 2019). In contrast, the application of a ROS-scavenging hydrogel conjugated to CCL22—one of the natural ligands of CCR4—was shown to enhance wound healing and hair follicle regeneration in diabetic mice through Treg recruitment (Qu et al., 2024). Similarly, another study employing a comparable strategy using IL-33 to induce Treg differentiation and expansion also reported pro-regenerative effects (Wang et al., 2022). Finally, our findings suggest that MET exerts beneficial effects on the healing of diabetic wounds and emerges as a promising candidate for clinical trials. However, several limitations of our study should be acknowledged. First, all experiments were conducted in mice or mouse-derived lineage fibroblasts, which may not fully recapitulate human wound healing. Second, MET was evaluated exclusively in a T1DM model. In future investigations, we intend to assess MET’s pro-healing potential in type 2 diabetes models, such as the diet-induced obese mouse model. Third, CYP11B1 protein expression was evaluated using a polyclonal antibody, which may exhibit lower specificity and potential cross-reactivity in murine tissues. Although mRNA quantification showed a consistent expression profile, confirming our findings, the use of more specific monoclonal antibodies would further strengthen protein-level validation. 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Journal of Dermatological Science , 90, 3–12. https://doi.org/10.1016/j.jdermsci.2017.12.009 Acknowledgements This work was supported by funds from the Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPq – Process number: 314226/2023-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PROBRAL Call – Process number: 88881.371287/2019-01), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ – Process number: E-26/201.161/2022). We extend our gratitude to Dr. Cynthia Pecli e Silva, Dr. Amanda Revoredo Vicentino and Dr Bruno Lourenço Diaz for their contributions to this study, and especially to the Optical Microscopy Platform (PLAMOL) of the Biophysics Institute Carlos Chagas Filho of UFRJ, where the images here presented were acquired. CONTRIBUTIONS Vanderlei da S. Fraga-Junior contributed to the conceptualization, formal analysis, investigation, methodology, original draft writing, and editing of the manuscript. Willian Rodrigues Ribeiro , Edson José de Oliveira-Junior , Jeferson Kelvin A. de Oliveira Silva , Matheus Palazzo , Evelyn M. do Nascimento , Matheus A. de Pinho , Renato Sampaio Carvalho , Claudia Mermelstein , and Anna Christina Borges conducted the molecular and the histological analyses of the study. Ana Paula Cabral de Araujo Lima conducted the siRNA assay methodology. Ingrid Waclawiak and Fábio Canto performed the flow cytometry acquisition and formal analyses. Thomas Brunner made essential contributions to the conceptualization and development of the research. Claudia Farias Benjamim conceived the project, managed resources, supervised the study, and contributed to original draft writing as well as editing. Claudia Farias Benjamim serves as the guarantor of this work, with full access to all data, and assumes the responsibility for the integrity and accuracy of the data analysis. CONFLICT OF INTEREST STATEMENT The authors declare that they do not have any competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability STATEMENT Raw data will be made available on request. Figure legends Figure 1. Pharmacologically induced T1DM increases local glucocorticoid production pathways in mouse cutaneous lesions. (A) Schematic representation of an excisional full-thickness wound model in diabetic mice induced with alloxan. (B) Blood glucose levels 7 days post-induction of T1DM (n = 11). (C) Representative images and (D) quantification of wound closure in euglycemic and diabetic mice subjected to excisional full-thickness wounding. (E) Area under the curve analysis of open wound area measured on days 0, 3, 7, 10, and 14. The wound area on day 0 was set at 100%, and subsequent measurements at each time point were expressed as a percentage (%) of the initial value (n = 4). (F–J) Wounds collected on day 3 were analyzed by qPCR to quantify transcripts of key glucocorticoid-related genes (n = 5). All qPCR data were normalized to the relative quantification (RQ) of unwounded euglycemic skin controls, indicated in the graphs by a red dotted line. (K–N) Western blot analysis of CYP11B1, 11β-HSD1, and GRα expression in wound tissues (n = 3). Data are presented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001, determined by Student’s t-test or two-way ANOVA with Bonferroni post hoc test, compared to euglycemic mice. Representative results from three independent experiments. Figure 2. MET topical treatment reduces locally corticosterone production and improves wound healing in diabetic mice. (A, B) Representative images and quantification of wound closure in euglycemic mice and (D, E) diabetic mice subjected to excisional full-thickness wounding and treated topically with MET (1 mg/wound) or PBS (control) once daily for 14 days. (C, F) Respectively area under the curve analysis of the open wound area, measured at days 0, 3, 7, 10, and 14. The wound area on day 0 was set at 100%, with subsequent measurements expressed as a percentage (%) of the initial value (n = 4). (G) Representative midline sections of diabetic wounds stained with hematoxylin and eosin (H&E) at day 7 (20 x magnification; scale bars = 1 mm). (H) Quantification of the non-reepithelialized area between the edges of migrating epithelial lips (n = 3). (I) Wounds and (J) serum samples collected at day 14 were analyzed by ELISA for corticosterone levels (n = 6–9). (K) Serum samples collected at day 7 were also analyzed for blood glucose levels (n = 5–6). Data are presented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, determined by one-way ANOVA with Tukey post hoc test or two-way ANOVA with Bonferroni post hoc test. Comparisons are made to PBS-treated euglycemic mice. Representative results from three independent experiments. Figure 3. MET enhances tissue repair and ECM reorganization in diabetic wounds. (A–C) Representative panels of wounds collected at day 7, stained with hematoxylin and eosin (H&E), highlighting the epidermis, dermis, and hypodermis in each group (200 x magnification; scale bar = 100 µm). White arrows indicate the proliferative layer, black arrows indicate infiltrating leukocytes, and asterisks indicate the dermal papillary layer. (D) Representative images of collagen deposition (red staining) in lesions stained with Picrosirius Red at day 7 of healing (200 x magnification; scale bar = 100 µm). (E) Representative immunofluorescent staining for α-SMA (green) and DAPI (blue) in wounds harvested on day 7, the insert represents the negative staining control (200 x magnification; scale bar = 100 µm). (F–H) Western blot analysis of type I collagen fibers and α-SMA expression in wound tissues at day 7 (n = 3–4). Data are presented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, determined by one-way ANOVA with Tukey post hoc test. Comparisons are made to PBS-treated euglycemic mice. Representative results from two independent experiments. Figure 4. MET induces fibroblast migration without affecting proliferation in vitro . Representative images of (A) murine fibroblasts (L929) incubated for 24 hours with MET (60 µM) in either high- or low-glucose medium and immunostained for Ki-67 (green), DAPI (blue), and β-actin (red). The insert box represents the negative staining control. (400 x magnification; scale bar = 50 µm). (B) Representative images of a scratch assay performed with murine fibroblasts (L929) incubated for 24 hours with MET (60 µM) in high-glucose medium, and (C) a graph depicting the number of migrating cells counted at the center of the wound area at 0-, 6-, 12-, and 24-hours post-scratch (40 x magnification; scale bar = 200 µm). (D) Quantification of cell counts of murine fibroblasts (L929) incubated for 24 hours with MET (20 or 60 µM) in high-glucose medium. Data are presented as mean ± standard error of the mean. *P < 0.05, determined by two-way ANOVA with Bonferroni post hoc test, compared to the control group. Representative results from three experiments. Figure 5. MET reshapes the inflammatory milieu in diabetic wounds. (A) Representative heatmap and (B–J) graphs showing the expression of key inflammatory genes in wound tissues collected at day 7 post-wounding (n = 5–6). All qPCR data were normalized to the relative quantification (RQ) of unwounded euglycemic skin controls, indicated in the graphs by a red dotted line. (K–O) Cytokine levels and (P-R) metalloproteinase and TIMP-1 levels in wound tissues collected at day 7 of healing (n = 4–5). Data are presented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, or # P < 0.05, ## P < 0.01, determined by one-way ANOVA with Tukey post hoc test. * Comparisons are made to PBS-treated euglycemic mice, and # comparisons are made to PBS-treated diabetic mice. Representative results from three independent experiments. Figure 6. MET restores Ly6G + LyC6C int and Ly6G - Ly6C + proportions in diabetic wounds. Wounds and skin-draining lymph nodes (dLNs) were harvested at day 7 post-wounding, and cell suspensions were analyzed by flow cytometry. (A) Gating strategy showing representative dot and contour plots. Scatter plots depict the proportions of various innate leukocyte subsets analyzed from (B–I) wound samples and (J–M) dLNs (n = 3–5). Data are presented as mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, or # P < 0.05, ## P < 0.01, determined by one-way ANOVA with Tukey post hoc test. * Comparisons are made to PBS-treated euglycemic mice, and # comparisons are made to PBS-treated diabetic mice. Representative results from three independent experiments. Figure 7. MET decreases CD4 + T lymphocytes in diabetic wounds improving Foxp3 gene expression. Wounds and skin-draining lymph nodes (dLNs) were harvested at day 7 post-wounding, and cell suspensions were analyzed by flow cytometry. (A) Gating strategy showing representative dot and contour plots. Scatter plots depict the proportions of lymphoid subsets analyzed from (B–E) wound samples. (F-I) Graphs showing the expression of transcription factors genes directly involved in T lymphocyte differentiation in wound tissues collected at day 7 post-wounding (n = 5–6). All qPCR data were normalized to the relative quantification (RQ) of unwounded euglycemic skin controls, indicated in the graphs by a red dotted line. Scatter plots depict the proportions of lymphoid subsets in the dLNs (J, K) in percentage and (L, M) in absolute numbers. *P < 0.05, **P < 0.01, ***P < 0.001, or # P < 0.05, ## P < 0.01, ### P < 0.001, determined by one-way ANOVA with Tukey post hoc test. * Comparisons are made to PBS-treated euglycemic mice, and # comparisons are made to PBS-treated diabetic mice. Representative results from three independent experiments. Figure 8. Silencing of Cyp11b1 and Hsd11b1 enhances fibroblast migration . (A) Representative images of murine fibroblasts (L929) transfected with siRNA targeting Cyp11b1 or Hsd11b1 individually; cells transfected with scramble siRNA served as controls (40 x magnification; scale bar = 200 µm). (B–D) Western blot analysis of CYP11B1 and 11β-HSD1 expression following electroporation. (E) Representative images of the scratch assay performed 36 hours post-transfection. (F and G) Quantification of wound closure, expressed as the percentage of closure over time and area under the curve (AUC) analysis (40 x magnification; scale bar = 200 µm). *P < 0.05, **P < 0.01, ***P < 0.001, or # P < 0.05, ## P < 0.01, determined by one-way ANOVA with Tukey post hoc test. * Comparisons are made to scramble control, and # comparisons are made to Cyp11b1 silenced cells. Representative results from three experiments. Figure 9. Graphical summary of MET topical effects on wound healing. Metyrapone topical treatment ameliorates wound healing in type 1 diabetic mice improving their tissue repair due to corticosterone local inhibition. Bullet Point Summary What is already known? • Chronic diabetic wounds have impaired healing partly due to local glucocorticoid excess. • The enzyme 11β-HSD1 increases local GC availability and is upregulated in diabetic skin and during delayed healing. • Genetic ablation of GC production enzymes improves wound healing in preclinical models. What this study adds? Metyrapone (MET) accelerates wound closure in a diabetic mouse model when applied topically. MET reduces local GC production in wounds without affecting systemic GC levels. What is the clinical significance? Topical MET may offer a safer alternative to treat diabetic ulcers. This work supports broader clinical investigation of MET as a topical inhibitor of GC production in diabetic wound care. Information & Authors Information Version history V1 Version 1 10 March 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Vanderlei Fraga-Junior 0000-0003-3155-2388 Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Ingrid Waclawiak Universidade Federal do Rio de Janeiro Instituto de Microbiologia Professor Paulo de Goes View all articles by this author Willian Rodrigues Ribeiro Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Edson José de Oliveira-Junior Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Jeferson Kelvin Alves de Oliveira Silva Universidade Federal do Rio de Janeiro Instituto de Microbiologia Professor Paulo de Goes View all articles by this author Matheus Palazzo Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Evelyn Mendes do Nascimento Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Matheus Azevedo de Pinho Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Ana Paula Lima Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Renato Sampaio Carvalho Universidade Federal do Rio de Janeiro Instituto de Ciencias Biomedicas View all articles by this author Anna Cristina Neves Borges Universidade Federal do Estado do Rio de Janeiro View all articles by this author Claudia Mermelstein Universidade Federal do Rio de Janeiro Instituto de Ciencias Biomedicas View all articles by this author Fábio Barrozo do Canto Universidade Federal Fluminense Instituto de Biologia View all articles by this author Thomas Brunner Universitat Konstanz Fachbereich Biologie View all articles by this author Claudia Farias Benjamim [email protected] Universidade Federal do Rio de Janeiro Instituto de Biofisica Carlos Chagas Filho View all articles by this author Metrics & Citations Metrics Article Usage 254 views 81 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Vanderlei Fraga-Junior, Ingrid Waclawiak, Willian Rodrigues Ribeiro, et al. Inhibition of skin glucocorticoid synthesis by topical metyrapone administration supports wound healing in diabetic mice. Authorea . 10 March 2026. DOI: https://doi.org/10.22541/au.177314987.71327230/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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