Abstract P2021: Cutaneous Wound Healing in Diabetic Mice is Improved by Topical Mineralocorticoid Receptor Blockade

Cutaneous Wound Healing in Diabetic Mice Is Improved by Topical Mineralocorticoid Receptor Blockade

Chronic refractory wounds are one of the complications of diabetes mellitus that require effective therapy. The dermal-wound-healing property of IL-33 in diabetics is little understood. Therefore, this study aimed to express recombinant humanized mature IL-33 (rhmatIL-33) in Escherichia coli BL21 (DE3) and demonstrate its efficacy on dermal wounds in streptozotocin (STZ)-induced diabetic and nondiabetic mice by the dorsal incisional skin wound model. Results revealed that the rhmatIL-33 accelerated the scratch-healing of keratinocytes and fibroblasts at the cellular level. The wounds of diabetic mice (DM) showed more severe ulceration and inflammation than wild-type mice (WT), and the exogenous administration of rhmatIL-33 increased wound healing in both diabetic and wild-type mice. Compared with the up-regulation of endogenous IL-33 mRNA after injury in WT mice, the IL-33 mRNA decreased after injury in DM mice. Exogenous IL-33 administration increased the endogenous IL-33 mRNA in the DM group but decreased the IL-33 mRNA expression level of the WT group, indicating that IL-33 plays a balancing role in wound healing. IL-33 administration also elevated ILC2 cells in the wounds of diabetic and non-diabetic mice and improve the transcript levels of YM1, a marker of M2 macrophages. In conclusion, Hyperglycemia in diabetic mice inhibited the expression of IL-33 in the dermal wound. Exogenous addition of recombinant IL-33 promoted wound healing in diabetic mice by effectively increasing the level of IL-33 in wound tissue, increasing ILC2 cells, and accelerating the transformation of macrophage M1 to M2 phenotype.

Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

Diabetic wounds are characterized by delayed wound healing due to persistent inflammation and excessive production of reactive oxygen species. Vitamin D, which is well acknowledged to enhance intestinal calcium absorption and increase in plasma calcium level, has recently been shown to display beneficial effects in various vascular diseases by promoting angiogenesis and inhibiting inflammatory responses. However, the role of Vitamin D in diabetic wound healing is still unclear. In the present study, we investigated the role of Vitamin D in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice. Four weeks after injection of STZ, a full thickness excisional wound was created with a 6-mm diameter sterile biopsy punch on the dorsum of the mice. Vitamin D was given consecutively for 14 days by intraperitoneal injection. Vitamin D supplementation significantly accelerated wound healing in diabetic mice and improved the healing quality as assessed by measuring the wound closure rate and histomorphometric analyses. By monitoring the level of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL) 6 (IL-6), IL-1β) in the wounds, reduced inflammatory response was found in VD treatment group. Furthermore, nuclear factor κB (NF-κB) pathway was found to be involved in the process of diabetic wound healing by assessing the relative proteins in diabetic wounds. Vitamin D supplementation obviously suppressed NF-κB pathway activation. These results demonstrated that Vitamin D improves impaired wound healing in STZ-induced diabetic mice through suppressing NF-κB-mediated inflammatory gene expression.

Netrins are secreted chemoattractants with the roles in axon guidance, cell migration and epithelial plasticity. In the present study, we investigated the roles of netrin-1 in the regulation of corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice and cultured corneal epithelial cells. In diabetic mice, the expression of netrin-1 was decreased when compared with that of normal mice. Furthermore, high glucose blocked the wounding-induced up-regulation of netrin-1 expression in corneal epithelial cells. Exogenous netrin-1 promoted the corneal epithelial wound healing in diabetic mice, and facilitated the proliferation and migration by reactivating the phosphorylation of ERK and EGFR in high-glucose treated corneal epithelial cells. Moreover, netrin-1 decreased the neutrophil infiltration and promoted M2 macrophage transition, accompanied with the attenuated expression of pro-inflammatory factors in diabetic mouse corneal epithelium. The promotions of netrin-1 on corneal epithelial wound healing and inflammation resolution were mediated at least through the adenosine 2B receptor. In addition, netrin-1 promoted the regeneration of corneal nerve fibers that was impaired in diabetic mice. Taken together, netrin-1 regulates corneal epithelial wound healing, inflammation response and nerve fiber regeneration in diabetic mice, indicating the potential application for the therapy of diabetic keratopathy.

Impaired skin wound healing is a major medical problem in diabetic subjects. Kinins exert a number of vascular and other actions limiting organ damage in ischaemia or diabetes, but their role in skin injury is unknown. We investigated, through pharmacological manipulation of bradykinin B1 and B2 receptors (B1R and B2R respectively), the role of kinins in wound healing in non-diabetic and diabetic mice. Using two mouse models of diabetes (streptozotocin-induced and db/db mice) and non-diabetic mice, we assessed the effect of kinin receptor activation or inhibition by subtype-selective pharmacological agonists (B1R and B2R) and antagonist (B2R) on healing of experimental skin wounds. We also studied effects of agonists and antagonist on keratinocytes and fibroblasts invitro. Levels of Bdkrb1 (encoding B1R) and Bdkrb2 (encoding B2R) mRNAs increased 1-2-fold in healthy and wounded diabetic skin compared with in non-diabetic skin. Diabetes delayed wound healing. The B1R agonist had no effect on wound healing. In contrast, the B2R agonist impaired wound repair in both non-diabetic and diabetic mice, inducing skin disorganization and epidermis thickening. Invitro, B2R activation unbalanced fibroblast/keratinocyte proliferation and increased keratinocyte migration. These effects were abolished by co-administration of B2R antagonist. Interestingly, in the two mouse models of diabetes, the B2R antagonist administered alone normalized wound healing. This effect was associated with the induction of Ccl2 (encoding monocyte chemoattractant protein 1)/Tnf (encoding tumour necrosis factor α) mRNAs. Thus stimulation of kinin B2 receptor impairs skin wound healing in mice. B2R activation occurs in the diabetic skin and delays wound healing. B2R blockade improves skin wound healing in diabetic mice and is a potential therapeutic approach to diabetic ulcers.

circ-Erbb2ip from adipose-derived mesenchymal stem cell-derived exosomes promotes wound healing in diabetic mice by inducing the miR-670-5p/Nrf1 axis

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice

Chronic non-healing wounds are among the most common complications of diabetes, highlighting the urgent need for effective and accessible therapeutic strategies. Although microwave therapy has shown promise in promoting tissue repair, its underlying mechanisms in diabetic wound healing remain unclear. This study investigated the therapeutic effects of microwave therapy on diabetic wound healing and its potential modulation of the Interleukin-33 (IL-33) /ST2 signaling pathway. Full-thickness excisional wounds were established in C57BL/6 mice, categorized into normal control / wild-type (CON/WT), diabetic (DM), and ST2-deficient (ST2-/-) groups. Mice received optimized microwave treatment (10 watts (W) for 10 min (min) daily), with or without IL-33-enriched macrophage supernatant (IL-33-MS). Wound healing outcomes were evaluated by histology, immunofluorescence, and gene expression analyses. In vitro experiments using RAW264.7 macrophages and HaCaT keratinocytes assessed IL-33 induction, macrophage polarization, and keratinocyte migration. Microwave treatment significantly accelerated wound healing in diabetic mice by enhancing granulation tissue formation, collagen remodeling, neovascularization, and myofibroblast activation. This effect was accompanied by increased IL-33 expression, particularly in macrophages, along with upregulation of M2 markers (CD206, IL-4, YM1) and downregulation of M1 markers (iNOS, Tnf-α). In ST2-/- mice, microwave therapy failed to promote wound repair, indicating that the IL-33/ST2 axis is essential for its pro-healing effect. IL-33-MS promoted wound closure in WT but not in ST2-/- mice. In vitro, microwave exposure upregulated IL-33 in macrophages and enhanced M2 polarization and HaCaT cell migration via ST2-dependent signaling. Microwave therapy facilitates diabetic wound healing by activating the IL-33/ST2 pathway, promoting M2 macrophage polarization, and improving the wound microenvironment. These findings provide mechanistic insight into the immunomodulatory effects of microwave therapy and support its potential as a non-invasive strategy for chronic diabetic wound management.

SIRT1 activation promotes angiogenesis in diabetic wounds by protecting endothelial cells against oxidative stress

Omentin-1 promotes diabetic wound healing by regulating macrophage efferocytosis and M2 polarization.

Diabetic wounds are recalcitrant to healing. One of the important characteristics of diabetic trauma is impaired macrophage polarization with an excessive inflammatory response. Many studies have described the important regulatory roles of microRNAs (miRNAs) in macrophage differentiation and polarization. However, the differentially expressed miRNAs involved in wound healing and their effects on diabetic wounds remain to be further explored. In this study, we first identified differentially expressed miRNAs in the inflammation, tissue formation and reconstruction phases in wound healing using Illumina sequencing and RT-qPCR techniques. Thereafter, the expression of musculus (mmu)-miR-145a-5p (“miR-145a-5p” for short) in excisional wounds of diabetic mice was identified. Finally, expression of miR-145a-5p was measured to determine its effects on macrophage polarization in murine RAW 264.7 macrophage cells and wound healing in diabetic mice. We identified differentially expressed miRNAs at different stages of wound healing, ten of which were further confirmed by RT-qPCR. Expression of miR-145a-5p in diabetic wounds was downregulated during the tissue formation stage. Furthermore, we observed that miR-145a-5p blocked M1 macrophage polarization while promoting M2 phenotype activation in vitro. Administration of miR-145a-5p mimics during initiation of the repair phase significantly accelerated wound healing in db/db diabetic mice. In conclusion, our findings suggest that rectifying macrophage function using miR-145a-5p overexpression accelerates diabetic chronic wound healing.

ObjectiveDiabetic ulcers are serious chronic wounds that are challenging to heal and can lead to amputation or even death. This study aims to utilize interleukin‐10 (IL‐10) overexpressing adipose mesenchymal stem cells to investigate their potential in promoting the healing of diabetic ulcers and to explore their mechanism of action.MethodsThe analysis of stem cell characteristics of ADSC‐IL10 was performed through flow cytometry, cell scratch assay, MTT assay, and adipogenic and osteogenic differentiation assays. The detection of the M1 and M2 phenotypes of mouse peritoneal macrophages (RAW 264.7) under conditioned medium stimulation was carried out using qPCR technology. The assessment of the effects of conditioned media from ADSCs overexpressing IL‐10 (ADSC‐IL10 CM) and conditioned media from adipose‐derived stem cells (ADSC CM) on the migration of normal skin fibroblasts and human immortalized epidermal cells was done using Transwell and cell scratch methods. A diabetic mouse model was induced using a high‐fat/high‐sugar diet plus streptozotocin (STZ) to detect the number of M2 macrophages and the expression levels of inflammatory factors (IL‐1β, IL‐6, IL‐10, and MCP‐1) and growth factors (EGF, VEGF, and TGFβ‐1) in mouse skin tissue.ResultsThe overexpression of IL‐10 did not change the biological properties of ADSCs. In diabetic mice, the transplantation of IL‐10 overexpressing ADSCs for wound healing was more effective than the transplantation of ADSCs alone. ADSCs overexpressing IL‐10 promoted the expression of M2 macrophages marker; inhibited the secretion of proinflammatory factors such as IL‐1β, IL‐6, and MCP‐1; and enhanced the production of growth factors including EGF, TGFβ‐1, and VEGF. Furthermore, it facilitated the migration of skin fibroblasts and epidermal cells from diabetic mice to the wound site.ConclusionADSCs that overexpress IL‐10 promote wound healing in diabetic mice by reducing inflammatory responses, enhancing growth factor secretion, and increasing the migration of fibroblasts and epidermal cells.

BackgroundPatients with diabetic cutaneous ulcers experience financial burden and a lower quality of life and life expectancy. Endothelial progenitor cell (EPC)-derived exosomes facilitate skin wound healing by positively modulating vascular endothelial cell function. Exosomes play their important regulatory role through microRNA (miRNA). We explored the potential role and molecular mechanisms of miRNA in EPC-derived exosome healing of diabetic skin wounds.MethodsExosomes were isolated from the media of EPCs derived from mice bone marrow. High-throughput sequencing was used to detect the expression of exosome miRNA, and miRNA target genes were predicted using online databases. A diabetic mouse skin wound model was established, and wounds were treated with exosomes, miRNA-221-3p, or phosphate-buffered saline.ResultsExosomes from EPCs accelerated skin wound healing in both control and diabetic mice. High-throughput sequencing showed that miRNA-221-3p was highly expressed in EPC-derived exosomes. Skin wound healing in control and diabetic mice was significantly enhanced by EPC-derived exosomes and miRNA-221-3p administration. Immunohistochemical analyses showed that EPC-derived exosomes and miRNA-221-3p increased protein expression levels of the angiogenesis-related factors VEGF, CD31 and cell proliferation marker Ki67. Bioinformatics analyses indicated that miRNA-221-3p may be involved in the AGE-RAGE signaling pathway in diabetic complications, cell cycle, and the p53 signaling pathway.ConclusionWe concluded that miRNA-221-3p is one of the high-expressed miRNAs in EPC-derived exosomes and promoted skin wound healing in diabetic mice. The finding uncovers the molecular mechanism of EPC-derived exosomes and provides a potential novel approach to the clinical treatment of diabetic skin wounds.

Timely repair of damaged skin is very important to maintain the integrity and homeostasis of skin, but the wound healing process is compromised in diabetic patients due to several extrinsic and intrinsic factors thus lead to leg amputation and death eventually. Sirtuins, a family of seven conserved proteins are known to be associated with pathophysiological processes of the skin. The most important among them are sirt1and sirt3 involved in cell regeneration and cell survival. Naphthoquinone derivatives have a wide range of therapeutic properties, but the potential diabetic wound healing activity of lapachol has not been identified yet. The present study thus aimed to investigate the wound healing effects of lapachol in a diabetic mouse model. Diabetic wounded mice were divided into 3 groups; vehicle, lapachol 0.05%, and lapachol 0.1%. Skin samples collected from diabetic wounded mice on different time points after treatment for 10 consecutive days were subjected to downstream analysis by western blot, ELISA and histology. Lapachol treatment was found to enhance the expression of sirt1/sirt3 and other proteins involved in cell migration and blood vessel formation. The tissue development rate was increased by lapachol treatment with better collagen deposition. Interestingly, lapachol treatment also gave rise to a high concentration of growth factors resulting in speedy and timely recovery of injured skin. In summary, our findings suggest that lapachol promotes efficient wound healing in a diabetic mouse model by increasing the expression of sirt1 and sirt3 and other proteins related to wound repair and skin regeneration including α-PAK, RAC1/CDC42, VEGF and growth factors viz PDGF and VEGF. This research work finds a novel potential activator of sirtuins in the form of lapachol and depicts the role of activated sirtuins in diabetic wound healing.