New Peroxisome Proliferator-Activated Receptor Agonist (GQ-11) Improves Wound Healing in Diabetic Mice

Abstract

Objective: Chronic wounds associated with diabetes are an important public health problem demanding new treatments to improve wound healing and decrease amputations. Monocytes/macrophages play a key role in sustained inflammation associated with impaired healing and local administration of peroxisome proliferator-activated receptor (PPAR)γ agonists may modulate macrophage, improving healing. In this study, we investigated the effects of GQ-11, a partial/dual PPARα/γ agonist, on macrophage function and wound healing in diabetes. Approach: Wounds were surgically induced at the dorsum of C57BL/6J and BKS.Cg-Dock7^m +/+ Lepr^db/J (db/db) mice and treated with hydrogel (vehicle), pioglitazone or GQ-11, for 7 or 10 days, respectively. After treatment, wounds were analyzed histologically and by quantitative PCR (qPCR). In addition, bone marrow-derived macrophages (BMDM) were cultured from C57BL/6J mice and treated with vehicle, pioglitazone, or GQ-11, after challenge with lipopolysaccharide or interleukin-4 to be analyzed by qPCR and flow cytometry. Results: GQ-11 treatment upregulated anti-inflammatory/pro-healing factors and downregulated pro-inflammatory factors both in wounds of db/db mice and in BMDM. Innovation: Wounds of db/db mice treated with GQ-11 exhibited faster wound closure and re-epithelization, increased collagen deposition, and less Mac-3 staining compared with vehicle, providing a new approach to treatment of diabetic wound healing to prevent complications. Conclusion: GQ-11 improves wound healing in db/db mice, regulating the expression of pro- and anti-inflammatory cytokines and wound growth factors, leading to increased re-epithelization and collagen deposition.

Keywords: biomarkers; cell biology; diabetes; wound closure.

Dulcineia S.P. Abdalla, PhD

Timothy J. Koh, PhD

Figure 1.

GQ-11 increases anti-inflammatory cytokine expression and decreases pro-inflammatory cytokine expression in BMDM. (A) Fold change of Il-1β and Tnf-α after challenge with LPS. (B) Fold change of Il-10, Arg-1, and Tgf-b after LPS challenge. (C) Fold change of Il-1β and Tnf-α after IL-4 challenge. (D) Fold change of Il-10, Arg-1 and Tgf-b after IL-4 challenge. BMDM were pretreated for 24 h with vehicle (DMSO 0.01%), pioglitazone (PIO 10 μM), or GQ-11 (10 μM) and challenged with LPS (100 ng/mL) or IL-4 (20 ng/mL). mRNA expression of BMDM was quantified by qPCR. Negative controls [CTRL (−)] are represented by nontreated and nonchallenged cells. Positive controls [CTRL (+)] are represented by nontreated but challenged cells. Data are expressed as the mean ± SD of biological triplicates. Statistical analyses were performed using ANOVA/Tukey's multiple comparison tests. *p < 0.05. ANOVA, analysis of variance; BMDM, bone marrow-derived macrophages; DMSO, dimethyl sulfoxide; IL, interleukin; LPS, lipopolysaccharide; mRNA, messenger RNA; qPCR, quantitative PCR; SD, standard deviation; TGF, transforming growth factor; TNF, tumor necrosis factor.

Figure 2.

GQ-11 decreases pro-inflammatory cytokine release and increases anti-inflammatory cytokine release by BMDM. (A) Concentrations of IL-6, MCP-1, and TNF-α protein of BMDM supernatant. (B) IL-10 concentration from supernatant of BMDM pretreated for 24 h with vehicle (DMSO 0.01%), pioglitazone (PIO 10 μM), or GQ-11 (10 μM) and challenged with LPS (100 ng/mL). (C) IL-10 concentration in supernatant of BMDM pretreated for 24 h with vehicle (DMSO 0.01%), pioglitazone (PIO 10 μM), or GQ-11 (10 μM) and challenged with IL-4 (20 ng/mL). Total protein of BMDM supernatant was accessed by flow cytometry, with a CBA. Negative controls [CTRL (−)] are represented by nontreated and nonchallenged cells. Positive controls [CTRL (+)] are represented by nontreated but challenged cells. Data are expressed as the mean ± SD of biological triplicates. Statistical analyses were performed using ANOVA/Tukey's multiple comparison tests. *p < 0.05. CBA, cytometric bead array; MCP-1, monocyte chemoattractant protein-1.

Figure 3.

GQ-11 increases the expression of anti-inflammatory cytokines and decreases the expression of pro-inflammatory cytokines in wounds of db/db mice. (A) Fold change of Ppar-α and Ppar-γ in wounds of nondiabetic and diabetic db/db mice. (B) Fold change of Il-10, Tgf-β, and Vegf in nondiabetic and db/db mice. (C) Fold change of Il-6, Tnf-α, and Il-1β in nondiabetic and db/db mice. mRNA expression was quantified by qPCR. Data are expressed as the mean ± SD of six mice per group. Statistical analyses were performed using ANOVA/Tukey's multiple comparison tests. *p < 0.05.

Figure 4.

GQ-11 improves wound closure in db/db mice. (A) Representative lesions at scapula of nondiabetic mice (postsurgery and after 7 days), decrease of wound area along 7 days postsurgery and total wound closure. (B) Representative lesions at scapula of diabetic db/db mice (postsurgery and after 10 days), decrease of wound area along 10 days postsurgery and total wound closure. Mice were submitted to surgical procedure to induce scapula lesions and were treated at the 3rd day postsurgery with vehicle (CTRL—Pluronic Gel^®), pioglitazone (PIO—2 mM), or GQ-11 (2 mM) for 4 days (nondiabetic mice) and 7 days (db/db mice). Positive controls [CTRL (+)] are represented by wounds of nondiabetic mice at the 3rd day postsurgery without treatment. Data are expressed as the mean ± SD of six mice per group. Statistical analyses were performed using ANOVA/Tukey's multiple comparison tests. *p < 0.05.

Figure 5.

GQ-11 increases re-epithelization and collagen deposition in wounds of db/db mice. (A) Representative sections of wounds edge of nondiabetic mice, HE stained, and the respective thickness measurement and re-epithelization calculation. (B) Representative sections of wounds edge of db/db mice, HE stained and the respective thickness measurement and re-epithelization calculation. (C) Representative sections of wounds edge of nondiabetic mice, Trichrome stained, and respective collagen deposition. (D) Representative sections of wounds edge of db/db mice, Trichrome stained, and respective collagen deposition. Mice were submitted to surgical procedure to induce scapula lesion and were treated at the 3rd day postsurgery with vehicle (CTRL—Pluronic Gel), pioglitazone (PIO—2 mM), or GQ-11 (2 mM) for 4 days (nondiabetic mice) and 7 days (db/db mice). Positive controls [CTRL (+)] are represented by wounds of db/+ mice at the 3rd day postsurgery without treatment. Histological sections were collected with frozen samples with cryostat. Magnification 2 × . Data are expressed as the mean ± SD of six mice per group. Statistical analyses were performed using ANOVA/Tukey's multiple comparison tests. *p < 0.05. HE, hematoxylin-eosin.

Figure 6.

GQ-11 decreases macrophage infiltration in wounds of db/db mice. (A) Representative sections of Mac-3 staining in wounds of nondiabetic mice. (B) Representative sections of Mac-3 staining in wounds of db/db mice. Mice were submitted to surgical procedure to induce scapula lesion and were treated at the 3rd day postsurgery with vehicle (CTRL—Pluronic Gel), pioglitazone (PIO—2 mM), or GQ-11 (2 mM) for 4 days (nondiabetic mice) or 7 days (db/db mice). Positive controls [CTRL (+)] are represented by wounds of db/+ mice at the 3rd day postsurgery without treatment. Histological sections were collected with frozen samples with cryostat and staining was performed by immunohistochemistry with specific antibodies against Mac-3 (1:50). Magnification 10 ×.