Pharmaceutical Activation of Nrf2 Accelerates Diabetic Wound Healing by Exosomes from Bone Marrow Mesenchymal Stem Cells

Abstract

Background and objectives: Despite advances in wound treatments, chronic diabetic wounds remain a significant medical challenge. Exosomes from mesenchymal stem cells (MSCs) and small molecule activators of nuclear factor erythroid 2-related factor 2 (Nrf2) have emerged as potential therapies for nonhealing diabetic wounds. This study aimed to evaluate the effects of exosomes from bone marrow-derived MSCs (BMSCs) alone, or in combination with a small molecule activator of Nrf2 on diabetic wound healing.

Methods and results: BMSCs and endothelial progenitor cells (EPCs) were isolated from the femur and tibia bone marrow of Sprague-Dawley (SD) rats and culture-expanded. Exosomes were harvested from the BMSC culture supernatants through ultracentrifugation. The effects of the exosomes and Nrf2 knockdown, alone or in combination, on EPC tube formation were evaluated. Streptozotocin-induced diabetic rats bearing a fresh full-thickness round wound were treated with the exosomes alone, or in combination with a lentiviral shRNA targeting Nrf2 (Lenti-sh-Nrf2) or tert-butylhydroquinone (tBHQ), a small molecule activator of Nrf2. Two weeks later, wound closure, re-epithelization, collagen deposition, neovascularization, and local inflammation were evaluated. BMSC exosomes promoted while Nrf2 knockdown inhibited EPC tube formation. BMSC exosomes accelerated wound closure, re-epithelization, collagen deposition, and neovascularization, and reduced wound inflammation in diabetic rats. These regenerative and anti-inflammatory effects of the exosomes were inhibited by Lenti-sh-Nrf2 but enhanced by tBHQ administration.

Conclusions: BMSC exosomes in combination with a small molecule Nrf2 activator hold promise as a new therapeutic option for chronic diabetic wounds.

Keywords: Bone marrow-derived mesenchymal stem cells; Diabetic wound healing; Exosomes; Nrf2; Tert-Butylhydroquinone.

Fig. 1

Identification of BMSCs and BMSC exosomes. (A) The detection of the surface markers CD105, CD90, and CD45 in isolated BMSCs with flow cytometry. (B) A TEM image of BMSC exosomes. Scale bar=100 nm. (C) The particle size distribution of BMSC exosomes determined with light scattering. (D) The detection of the exosome markers CD9, CD63, and TSG101 in BMSC exosomes with western blot analysis.

Fig. 2

The effects of BMSC exosomes and Nrf2 knockdown, alone or in combination, on EPC tube formation. (A) The detection of the surface markers CD31, CD34, and CD45 in isolated EPCs with flow cytometry. (B) EPCs were transfected with Lenti-sh-Nrf2 or Lenti-sh-NC for 48 hours. The Nrf2 protein levels were determined with western blot analysis. (C, D) EPCs stably expressing sh-Nrf2 or sh-NC were treated with BMSC exosomes (200 μg/ml) for 72 hours. (C) Tube formation was observed under a microscope. Scale bar=100 μm. (D) The relative tube length in each treatment group was determined. N=3, *p<0.05, **p< 0.01.

Fig. 3

The effects of BMSC exosomes alone, or in combination with knockdown or pharmaceutical activation of Nrf2 on wound closure in diabetic rats. STZ-induced diabetic rats received 100 μg/ml subcutaneous BMSC exosomes alone at the wound site, or in combination with 200 μl intravenous Lenti-sh-Nrf2 or Lenti-sh-NC, or 50 mg/kg intraperitoneal tBHQ immediately after the wounding and again a week after. Rats that received PBS were included as control. (A) The photo images of the wounds at day 0, 7, and 14 after the wounding. (B) The wound size at day 0, 7, and 14 after the wounding. N=5; *p<0.05, **p<0.01 vs. PBS control.

Fig. 4

The effects of BMSC exosomes alone, or in combination with knockdown or pharmaceutical activation of Nrf2 on wound tissue regeneration in diabetic rats. (A) The Nrf2 protein levels in the wound tissues at day 14 after the wounding were evaluated with western blot analysis. The STZ-induced diabetic rats were treated after skin wounding as described in Fig. 3. (A, B) Wound re-epithelization and collagen deposition were evaluated with H&E (B) and Masson (C) staining of the wound tissues at day 14 after the wounding. Scale bar=100 μm. N=5; *p<0.05, **p<0.01 vs. PBS control.

Fig. 5

The effects of BMSC exosomes alone, or in combination with knockdown or pharmaceutical activation of Nrf2 on wound neovascularization in diabetic rats. The STZ-induced diabetic rats were treated after skin wounding as described in Fig. 3. The CD31 expression in the wound tissues at day 14 after the wounding was detected with immunohistochemistry. Scale bar=100 μm. N=5; *p<0.05, **p<0.01 vs. PBS control.

Fig. 6

The effects of BMSC exosomes alone, or in combination with knockdown or pharmaceutical activation of Nrf2 on wound inflammation in diabetic rats. The STZ-induced diabetic rats were treated after skin wounding as described in Fig. 3. The levels of TNF-α, IL-1β, IL-4, and IL-10 in the wound tissues at day 14 after the wounding were determined with ELISA. N=5; *p<0.05, **p<0.01 vs. PBS control.