Dimethyl fumarate accelerates wound healing under diabetic conditions

in Journal of Molecular Endocrinology
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Impaired wound healing is a common complication among patients with diabetes mellitus (DM), resulting in high rates of disability and mortality. Recent findings highlighted the critical role of nuclear factor erythroid 2-related factor 2 (NRF2) – a master of cellular antioxidants scavenging excessive DM-induced free radicals – in accelerating diabetic wound healing. Dimethyl fumarate (DMF) is a potent NRF2 activator used for the treatment of multiple sclerosis. However, the effect of DMF on wound healing has not been determined. The present study investigated the effect of DMF on the diabetic and the non-diabetic wound healing in streptozotocin-induced diabetic mice and non-diabetic control mice. DMF activated NRF2 signaling under both conditions. Interestingly, DMF attenuated oxidative damage and inflammation and accelerated wound closure in diabetic mice. However, this effect was not observed in non-diabetic mice. Keratinocytes were treated with normal glucose (NG), high glucose (HG) or hydrogen peroxide (H2O2), in the presence or absence of DMF to assess the role of reactive oxygen species (ROS) – inducible in DM – in mediating DMF-induced protection. Both HG and H2O2 elevated ROS, oxidative damage and inflammation, the effects of which were similarly blunted by DMF. However, in spite of the activation of NRF2, DMF lost this capability under the NG condition. The findings of this study demonstrate that ROS activate the protective effect of DMF on the diabetic wound healing.

 

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    DMF accelerated wound closure under diabetic, but not non-diabetic conditions. (A) Blood glucose levels 1 week after the last injection of STZ. (B) Representative images of wound closure every other day, for a total period of 10 days after the onset of DM. (C) The percentage of wound closure was calculated. (D) Heat map of wound closure. The data are presented as means ± s.d. (n = 6). *P < 0.05 vs Ctrl; P < 0.05 vs DM. Symbols: red solid square, Ctrl; red hollow square, DM; blue solid circle, Ctrl/DMSO; blue hollow circle, DM/DMSO; orange solid up-triangle, Ctrl/DMF-10; orange hollow up-triangle, DM/DMF-10; green solid down-triangle, Ctrl/DMF-20; green hollow down-triangle, DM/DMF-20; magenta solid left-triangle, Ctrl/DMF-40; magenta hollow left-triangle, DM/DMF-40; navy solid right-triangle, Ctrl/DMF-80; navy hollow right-triangle, DM/DMF-80. Ctrl, control; DM, diabetes mellitus; DMF, dimethyl fumarate; DMF-10, DMF at 10 mg/kg; DMF-20, DMF at 20 mg/kg; DMF-40, DMF at 40 mg/kg; DMF-80, DMF at 80 mg/kg; STZ, streptozotocin.

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    DMF attenuated cutaneous oxidative damage and inflammation in diabetic mice, but not in non-diabetic mice. (A) Western blotting was used to determine the levels of NRF2 protein. In addition, the expression of (B) Ho1 mRNA and (C) protein was measured using RT-PCR and Western blotting respectively, as a reflection of the function of NRF2. (D) A commercial kit was used to measure the levels of MDA, and RT-PCR was used to determine the mRNA levels of (E) Vcam-1 and (F) Icam-1. The data were normalized to Ctrl and presented as means ± s.d. (n = 6). *P < 0.05 vs Ctrl; P < 0.05 vs DMF-10; P < 0.05 vs DM; §P < 0.05 vs DMF-10. Bars: red solid, Ctrl; red hollow, DM; blue solid, Ctrl/DMSO; blue hollow, DM/DMSO; orange solid, Ctrl/DMF-10; orange hollow, DM/DMF-10; green solid, Ctrl/DMF-20; green hollow, DM/DMF-20; magenta solid, Ctrl/DMF-40; magenta hollow, DM/DMF-40; navy solid, Ctrl/DMF-80; navy hollow, DM/DMF-80. Ho1, heme oxygenase-1; Icam-1, intercellular adhesion molecule-1; MDA, malondialdehyde; NRF2, nuclear factor erythroid 2-related factor 2; Vcam-1, vascular cell adhesion molecule-1. Other abbreviations are the same as those in Fig. 1. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0102.

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    Oxidative damage, inflammation, and NRF2 signaling was exhibited prominently in keratinocytes. (A) H&E staining was performed in the Ctrl, DM, and DMF-20 or vehicle-treated DM groups to evaluate morphology of the skin. IHC staining was performed using antibodies against (B) 4-HNE, (C) VCAM-1, (D) NRF2, and (E) HO1 to determine the location of oxidative damage, inflammation, and NRF2 signaling. Positively stained areas of the epidermis, excluding the sebaceous gland were quantified. Symbol: black arrow, positively stained keratinocytes. 4-HNE, 4-hydroxynonenal; IHC, immunohistochemical. Other abbreviations are the same as those in Figs 1 and 2.

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    NRF2 signaling was activated by H2O2 or HG to a similar extent. Keratinocytes were subjected to NG, H2O2, or HG in the presence or absence of DMF, to measure the levels of (A) NRF2 protein, and the mRNA and protein expression of (B and C) Ho1, and (D and E) Nqo1. The data were normalized to NG and presented as means ± s.d. (n = 3). *P < 0.05 vs NG; P < 0.05 vs NG/DMF; P < 0.05 vs HG. Bars: red solid, NG; blue solid, NG/DMSO; orange solid, NG/DMF; red hollow, NG/H2O2; blue hollow, NG/H2O2/DMSO; orange hollow, NG/H2O2/DMF; green solid, NG/mannitol; red hollow with diagonal stripes, HG; blue hollow with diagonal stripes, HG/DMSO; orange hollow with diagonal stripes, HG/DMF. H2O2, hydrogen peroxide; HG, high glucose; NG, normal glucose; Nqo1, NAD(P)H dehydrogenase quinone 1; ROS, reactive oxygen species. Other abbreviations are the same as those in Figs 1 and 2. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0102.

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    DMF alleviated oxidative damage and inflammation in keratinocytes following the generation of sufficient levels of ROS by H2O2 or HG. Keratinocytes were treated with DMF or vehicle, in the presence or absence of H2O2 or HG. The levels of (A) ROS and (B) MDA, along with the mRNA and protein expression of (C and D) Vcam-1 and (E and F) Icam-1 were determined. The data were normalized to NG and was presented as means ± s.d. (n = 3). *P < 0.05 vs NG; P < 0.05 vs NG/H2O2; P < 0.05 vs HG. Bars are the same as those in Fig. 4. Abbreviations are the same as those in Figs 1, 2 and 4. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0102.

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    Possible role of ROS in mediating the protection offered by DMF. Under non-diabetic conditions, the wound expresses basal levels of ROS, producing limited levels of free radicals. NRF2 antioxidant signaling is activated in response to basal ROS, scavenging the free radicals and leading to mitigated oxidative damage and inflammation. This mild ROS-induced activation of NRF2 facilitates normal wound healing. On the contrary, under diabetic conditions, ROS are abundantly produced, generating excessive free radicals that exceed the scavenging capacity of NRF2 antioxidant signaling. This effect causes severe oxidative damage and inflammation which impair diabetic wound healing. When treated with DMF, NRF2 signaling is activated under both the diabetic and non-diabetic conditions. Under diabetic conditions, the DMF-elevated antioxidants neutralize ROS-induced excessive free radicals, attenuating oxidative damage and inflammation and accelerating diabetic wound healing. However, under non-diabetic conditions, low levels of free radicals are induced by the lower levels of ROS. This provides insufficient targets for DMF-induced antioxidants. This may explain the non-accelerated wound healing process in the presence of DMF under non-diabetic conditions. Symbols: ↓, activation; , inhibition. Abbreviations are the same as those in Figs 1, 2 and 4. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0102.

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