Acarbose Accelerates Wound Healing via Akt/eNOS Signaling in db/db Mice

Abstract

Refractory wound is a dreaded complication of diabetes and is highly correlated with EPC dysfunction caused by hyperglycemia. Acarbose is a widely used oral glucose-lowering drug exclusively for T2DM. Previous studies have suggested the beneficial effect of acarbose on improving endothelial dysfunction in patients with T2DM. However, no data have been reported on the beneficial efficacy of acarbose in wound healing impairment caused by diabetes. We herein investigated whether acarbose could improve wound healing in T2DM db/db mice and the possible mechanisms involved. Acarbose hastened wound healing and enhanced angiogenesis, accompanied by increased circulating EPC number in db/db mice. In vitro, a reversed BM-EPC dysfunction was observed after the administration of acarbose in db/db mice, as reflected by tube formation assay. In addition, a significantly increased NO production was also witnessed in BM-EPCs from acarbose treated db/db mice, with decreased O₂ levels. Akt inhibitor could abolish the beneficial effect of acarbose on high glucose induced EPC dysfunction in vitro, accompanied by reduced eNOS activation. Acarbose displayed potential effect in promoting wound healing and improving angiogenesis in T2DM mice, which was possibly related to the Akt/eNOS signaling pathway.

Figure 1

Experimental schedule. The blood glucose of db/db diabetic mice was monitored every 7 day until day 21; then acarbose (50 mg/kg/d, i.g.) treatment was conducted for consecutive 14 days. At last, wound healing models were created and BM-EPCs were collected.

Figure 2

Blood glucose and body weight change in db/db mice. (a) Blood glucose was significantly increased in db/db mice compared to control. ^∗P < 0.05 versus control. In db/db mice, acarbose treatment (50 mg/kg/d × 14 d, i.g.) significantly decreased blood glucose (b) but did not modify body weight (c). ^∗∗∗P < 0.001 versus Con; ^#P < 0.05 versus db/db. Values are expressed as the mean ± standard deviation (n = 7 per group). Con, control; Aca, acarbose.

Figure 3

Acarbose therapy accelerated wound closure and enhanced angiogenesis in db/db mice. An approximate 6 mm diameter circle wound was made by punch biopsy on dorsal and wound healing was assessed every 2 days until day 14. (a) Acarbose treatment obviously accelerated wound closure in db/db mice compared to untreated diabetic ones. (b) Acarbose treatment significantly increased wound capillaries compared with the untreated db/db mice on days 7 and 14. (c) Typical photographs of CD31 staining on days 7 and 14; red arrows point to CD31-positive capillaries; boxed regions (100x; scale bar = 100 μm) are shown at higher magnification (200x; scale bar = 50 μm) to the right. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05 versus Con; ^##P < 0.01, ^#P < 0.05 versus db/db. (d) SDF-1α expression in wound site was present predominantly in acarbose-treated group on day 7; red arrows show positive brown staining for SDF-1α; scale bar = 50 μm. Values are expressed as the mean ± standard deviation (n = 5 per group). Con, control; Aca, acarbose.

Figure 4

Acarbose therapy improved BM-EPC function and decreased intracellular reactive oxygen species (ROS) levels in db/db mice. (a) Circulating EPC numbers were detected by flow cytometry and the percentage of Sca-1⁺/Flk-1⁺ cells was calculated. Acarbose significantly increased circulating EPC number in db/db mice. (b) Typical images of tube formation assay of BM-EPCs. The number of tubes in each sample was calculated from 5 low-power fields (50x; scale bar = 100 μm) at random. Acarbose enhanced the capacity of tube formation of BM-EPCs. (c) Intracellular NO level was determined by flow cytometry and the percentage of DAF fluorescence intensity was calculated. Acarbose obviously enhanced NO level in BM-EPCs. (d) DHE fluorescence intensity was determined by flow cytometry. Acarbose suppressed intracellular O₂⁻ level in BM-EPCs. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05 versus Con; ^###P < 0.001, ^##P < 0.01, and ^#P < 0.05 versus db/db. Values are expressed as the mean ± standard deviation (n = 7–9 per group). Con, control; Aca, acarbose.

Figure 5

Acarbose stimulated the expression levels of activated Akt/eNOS in BM-EPCs from db/db mice. BM-EPCs were isolated and cultured from anesthetized mice. Akt and eNOS in BM-EPCs were conducted by western blotting, and acarbose greatly enhanced activated Akt and eNOS expression in BM-EPCs from db/db mice. ^∗∗P < 0.01, ^∗P < 0.05 versus Con; ^#P < 0.05 versus db/db. Values are expressed as the mean ± standard deviation (n = 4 per group). Con, control; Aca, acarbose.

Figure 6

Acarbose alleviated EPC dysfunction, decreased ROS expression, and increased Akt/eNOS phosphorylated-to-total ratio in BM-EPCs induced by high glucose. BM-EPCs were isolated from normal mice and cultured under high glucose (33 mM) together with acarbose (1 μM) for 24 h. (a) Assessment of tube formation ability. (b) Intracellular NO level was determined by flow cytometry and the percentage of DAF fluorescence intensity was calculated. (c) Intracellular DHE fluorescence intensity of EPCs was measured by flow cytometry. Western blot analysis was subjected to detected expression levels of activation Akt (d) and eNOS (e) in EPCs induced by high glucose. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05 versus Con; ^##P < 0.01, ^#P < 0.05 versus HG. Values are expressed as the mean ± standard deviation ((a), (b), (c): n = 7 per group; (d), (e): n = 3-4 per group). Scale bar = 100 μm. Con, control; HG, high glucose; Aca, acarbose.

Figure 7

Acarbose ameliorated EPC function and suppressed intracellular ROS levels via an Akt dependent pathway in vitro. Acarbose (1 μM) and p-Akt inhibitor and MK-2206 (1 μM) were added to the high glucose medium for 24 h. Measurement of tube formation (a), migration (b), and adhesion (c) capacity of BM-EPCs. Determination of intracellular NO level (d) and O₂⁻ level (e). ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05. Values are expressed as the mean ± standard deviation ((a), (b), (c): n = 6 per group; (d), (e): n = 7 per group). Scale bar = 100 μm. Con, control; HG, high glucose; Aca, acarbose; MK, MK-2206.