Overexpression of IL-10 in Adipose Mesenchymal Stem Cells Promotes Wound Healing in Diabetic Mice

Background: Diabetic ulcer is a serious chronic non-healing wound, which often leads to amputation or even death, causing great damage to the patients and their families. In recent years, stem cells are increasingly studied for tissue repair. Adipose tissue-derived mesenchymal stem cells (Adipose-derived mesenchymal stem cells, ADSCs) have the advantages of wide source and easy access. ADSCs can accelerate wound healing and reduce scar hyperplasia, especially for chronic wounds, patients cannot heal with traditional treatments, and ADSCs bring hope to these patients. Our study intends to overexpress IL-L 10 in ADSCs by genetic engineering technology, transplant ADSC-IL 10 to diabetic mouse wounds, and use the role of IL-10 in promoting M2 macrophages transformation, combined with ADSCs to achieve rapid healing of diabetic ulcer wounds. Methods : The expression of IL-10 protein in the supernatant of ADSC-IL10 was assessed using an ELISA kit. Flow cytometry was employed to analyze the surface stem cell markers (CD44, CD73, CD90, CD105) of ADSC-IL10. Additionally, the migration and proliferation of ADSC-IL10 were evaluated through cell scratch assays and MTT assays. The lipogenic marker PPARγ and the osteogenic marker RUNX2 were detected using fluorescent real-time quantitative PCR. Conditioned media from ADSC-IL10 (ADSC-IL10-CM) and ADSC-PCDH (ADSC-CM) were employed to culture mouse peritoneal macrophages (Raw 264.7). Following a 12-hour incubation period, the Raw 264.7 cells were harvested for mRNA extraction. The cell types of Raw cells were identified through qPCR, with the M1 phenotype determined by CD86 expression and the M2 phenotype by Arg-1 and CD206. The expression levels of inflammatory factors, including IL-1β, IL-6, IL-10, MCP-1, and growth factors such as EGF, VEGF, and TGFβ-1, were quantified using qPCR. The transwell method was utilized to assess the impact of ADSC-IL10-CM and ADSC-CM on the migration of normal skin fibroblasts. The scribing method was applied to examine the effect of ADSC-CM on the migration of human immortalized epidermal cells. A diabetic mouse model was induced by a high-fat, high-sugar diet combined with streptozocin. In the ADSC-IL10 group, 1×10^6 ADSC-IL10 cells were transplanted onto a 1.5cm×1.5cm wound surface, and similarly, 1×10^6 ADSC-PCDH cells were transplanted in the ADSC-PCDH group. The control group received an equal volume of 0.9% normal saline. The wound healing process in mice was observed and documented at various time points. Tissue samples from the wounds on days 3 and 7 were subjected to histological staining and qPCR analysis. HE staining was used to monitor wound healing progress, while immunofluorescence (CD206, Arg-1) was utilized to quantify the presence of M2-type macrophages in mouse skin. The expression levels of CD206 and Arg-1 genes were measured by qPCR to ascertain the macrophage phenotypes within the wound tissues. Levels of inflammatory factors such as IL-1β, IL-6, IL-10, MCP-1 were determined to evaluate the inflammatory status of the wound tissues. Additionally, the expression of EGF, VEGF, and TGFβ-1 growth factors within the wound tissues was assessed. Conclusion : The overexpression of IL-10 does not alter the biological characteristics of ADSCs. When transplanted into diabetic mice, ADSC-IL10 can accelerate wound healing more effectively than ADSCs alone. The mechanism through which ADSC-IL10 enhances wound healing may encompass several aspects: it stimulates the expression of M2-type macrophages, suppresses the secretion of pro-inflammatory factors such as IL-1β, IL-6, and MCP-1, and promotes the production of growth factors like EGF, TGFβ-1, and VEGF. Additionally, it encourages the migration of skin fibroblasts and epidermal cells to the wound sites in diabetic mice.