Metformin accelerates wound healing in type 2 diabetic db/db mice

Abstract

Wound healing impairment is increasingly recognized to be a consequence of hyperglycemia‑induced dysfunction of endothelial precursor cells (EPCs) in type 2 diabetes mellitus (T2DM). Metformin exhibits potential for the improvement of endothelial function and the wound healing process. However, the underlying mechanisms for the observed beneficial effects of metformin application remain to be completely understood. The present study assessed whether metformin, a widely used therapeutic drug for T2DM, may accelerate wound closure in T2DM db/db mice. Genetically hyperglycemic db/db mice were used as the T2DM model. Metformin (250 mg/kg/day; intragastric) was administered for two weeks prior to EPC collection and wound model creation in db/db mice. Wound healing was evaluated by alterations in the wound area and the number of platelet endothelial cell adhesion molecule‑positive cells. The function of the isolated bone marrow‑derived EPCs (BM‑EPCs) was assessed by a tube formation assay. The number of circulating EPCs, and the levels of intracellular nitric oxide (NO) and superoxide (O2‑) were detected by flow cytometry. Thrombospondin‑1 (TSP‑1) expression was determined by western blot analysis. It was observed that treatment with metformin accelerated wound healing, improved angiogenesis and increased the circulating EPC number in db/db mice. In vitro, treatment with metformin reversed the impaired BM‑EPC function reflected by tube formation, and significantly increased NO production while decreasing O2‑ levels in BM‑EPCs from db/db mice. In addition, TSP‑1 expression was markedly attenuated by treatment with metformin in cultured BM‑EPCs. Metformin contributed to wound healing and improved angiogenesis in T2DM mice, which was, in part, associated with stimulation of NO, and inhibition of O2‑ and TSP‑1 in EPCs from db/db mice.

Figure 1.

Illustration of experimental protocols. The blood glucose of db/db diabetic mice was monitored. Treatment with metformin (250 mg/kg/day; intragastric) was administered for 14 consecutive days. Wound healing and BM-EPC function were measured. BM-EPC, bone marrow-endothelial precursor cell.

Figure 2.

Alterations in blood glucose concentration and body weight of db/db mice. (A) In db/db mice, blood glucose was significantly elevated compared with the control. ***P<0.001 vs. Con. Metformin (250 mg/kg/day for 14 days; intragastric) significantly decreased (B) blood glucose, although it did not alter (C) body weight in db/db mice. ***P<0.001 vs. Con; ^###P<0.001 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=7 mice/group). Con, control; Met, metformin.

Figure 3.

Metformin therapy accelerates wound closure in db/db mice. A dorsal skin wound was created via a 6-mm circular punch biopsy and digital images of the wound were captured every 2 days until day 14. (A) Representative images of wound healing. (B) Metformin therapy accelerated wound closure compared with control in db/db mice. *P<0.05 vs. Con; ^#P<0.05 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=5 mice/group). Con, control; Met, metformin.

Figure 4.

Metformin therapy enhances wound angiogenesis in db/db mice. A dorsal skin wound was created via a 6-mm circular punch biopsy and wound angiogenesis was measured on days 7 and 14. (A) Representative images of CD31 staining on day 7 and (B) quantitative analysis. (C) Representative images of CD31 staining on day 14 and (D) quantitative analysis. Red arrows indicate CD31-positive capillaries (magnification, ×200; scale bar, 50 µm). Quantitative analysis of capillaries in each field demonstrated that wound capillaries in metformin-treated db/db mice were increased on days 7 and 14 when compared with the untreated db/db mice. ***P<0.001, **P<0.01 vs. Con; ^##P<0.01, ^#P<0.05 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=5 mice/group). Con, control; Met, metformin; hpf, high-power field.

Figure 5.

Metformin therapy ameliorates BM-EPC function in db/db mice. (A) Representative images of the tube formation assay of BM-EPCs. The number of tubes in each sample was calculated from 5 fields (magnification, ×50; scale bar, 100 µm) at random. Treatment with metformin ameliorated the tube formation of BM-EPCs. (B) EPC numbers were detected by flow cytometry and the percentage of Sca-1⁺/Flk-1⁺ cells was calculated. Metformin elevated the circulating EPC number in db/db mice. *P<0.05 vs. Con; ^#P<0.05 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=7 mice/group). Con, control; Met, metformin; BM-EPC, bone marrow-endothelial precursor cell; PE, phycoerythrin; FITC, fluorescein isothiocyanate; Sca-1, ataxin-1; Flk-1, vascular endothelial growth factor receptor 2; lpf, low-power field.

Figure 6.

Metformin therapy enhances intracellular NO and suppresses intracellular O₂⁻ levels in BM-EPCs from db/db mice. (A) The intracellular NO level was determined by DAF-FM-staining and flow cytometry. Metformin increased the NO level in BM-EPCs from db/db mice. (B) DHE fluorescence intensity was determined by flow cytometry. Metformin decreased the intracellular O₂⁻ level in BM-EPCs from db/db mice. **P<0.01 vs. Con; ^##P<0.01, ^#P<0.05 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=6–7 mice/group). Con, control; Met, metformin; BM-EPC, bone marrow-endothelial precursor cell; DAF-FM, 4-amino-5-methylamino-2′, 7′-difluorofluorescein; DHE, dihydroethidium; O₂⁻, superoxide; NO, nitric oxide.

Figure 7.

Metformin inhibits the secretion of TSP-1 in BM-EPC culture media from db/db mice. BM-EPCs were isolated and cultured from anesthetized mice. TSP-1 in BM-EPC culture media was subjected to western blotting, and metformin significantly decreased the expression of TSP-1 in BM-EPCs from db/db mice. *P<0.05 vs. Con; ^#P<0.05 vs. db/db. Values are expressed as the mean ± standard error of the mean (n=3 mice/group). Con, control; Met, metformin; TSP-1, thrombospondin-1.