[Effects of mitochondrial transplantation on full-thickness skin defects in diabetic rats]

Objective: To explore the effects of mitochondrial transplantation on full-thickness skin defects in diabetic rats. Methods: This study was an experimental study. Functionally intact mitochondria were extracted from the liver tissue of 6-8-week-old male Sprague Dawley rats (the same age and sex below). Mouse L929 cells and human umbilical vein endothelial cells (HUVECs) were cultured in medium containing high-glucose (50 mmol/L) for 24 hours to induce high-glucose injury. According to the random number table method (the same grouping method below), they were then divided into control group (Ctrl group, treated with conventional medium), growth factor group (GF group, treated with medium containing 20 U/mL recombinant human epidermal growth factor), and mitochondrial group (Mito group, treated with medium containing 12.5 μg/mL exogenous mitochondria). The scratch assay was performed to evaluate the migration rate of mouse L929 cells at 6 hours after scratching (n=3). The length and number of branch nodes of tube formed by HUVECs were measured after 2 and 6 hours of culture (n=3). After 24 hours of culture, the reactive oxygen species (ROS) levels and mitochondrial membrane potential in aforemetioned two types of cells were detected according to the kit instructions (n=6). Eighteen Sprague Dawley rats were selected and a type 1 diabetic rat model was successfully established. Then, the full-thickness skin defects with a diameter of 1 cm were created on their backs. The rats were divided into Ctrl group, GF group, and Mito group (with each group of 6 rats). At post-injury days (PID) 0 (immediately), 3, and 6, the wounds were subcutaneously injected with normal saline (Ctrl group), topically sprayed with an equal amount of rhEGF solution at a dose of 40 U/cm² (GF group), or subcutaneously injected with equal amount of mitochondrial suspension at a dose of 5 μg/g (Mito group), respectively. The percentage of remaining wound area of rats was calculated at PID 3, 6, and 12. At PID 12, the epithelialization and collagen deposition in the wound of rats were detected by hematoxylin and eosin staining and Masson's staining, respectively. Immunofluorescence staining was used to detect the expression of CD31 (a marker for neovascularization) and neurofilament 200 (a marker for nerves) in the wound of rats. The ROS levels, number of apoptotic cells, ATP content in the wound of rats were detected according to the kit instructions. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in the wound of rats were detected by enzyme-linked immunosorbent assay. Results: At 6 hours after scratching, compared with that in Ctrl group, the migration rate of mouse L929 cells in GF group was significantly increased (P<0.05). compared with that in GF group, the migration rate of mouse L929 cells in Mito group was significantly increased (P<0.05); After 2 and 6 hours of culture, compared with those in Ctrl group, the numbers of branch nodes and the lengths of tube formed by HUVECs in both GF group and Mito group were significantly increased (P<0.05). Compared with those in GF group, the numbers of branch nodes and the lengths of tube formation of HUVECs after 2 and 6 hours of culture in Mito group were significantly increased (P<0.05). After 24 hours of culture, compared with those in Ctrl group, the ROS levels in both mouse L929 cells and HUVECs in GF group and Mito group were significantly decreased (P<0.05), while the mitochondrial membrane potentials were significantly increased (P<0.05); compared with those in GF group, the ROS levels in both mouse L929 cells and HUVECs in Mito group were significantly decreased (P<0.05), while the mitochondrial membrane potentials were significantly increased (P<0.05). At PID 3, 6, and 12, the percentages of remaining wound area of rats in Mito group ((46±3)%, (37±3)%, (18±3)%) were significantly lower than those in Ctrl group ((71±4)%, (63±4)%, (47±5)%) and GF group((62±3)%, (54±3)%, (33±4)%), P<0.05. At PID 12, in the wounds of rats in Mito group, the status of epithelialization and collagen deposition, as well as the conditions of angiogenesis and nerve repair, were superior to those in growth factor and control groups. Compared with those in Ctrl group, the ROS levels and number of apoptotic cells in the wounds of rats in GF group and Mito group were significantly decreased (P<0.05), the ATP content significantly increased (P<0.05), and the levels of TNF-α, IL-1β, and IL-6 were all significantly reduced (P<0.05). Compared with those in GF group, the ROS levels and number of apoptotic cells in the wounds of rats in Mito group were significantly decreased (P<0.05), the ATP content significantly increased (P<0.05), and the levels of TNF-α, IL-1β, and IL-6 were all significantly reduced (P<0.05). Conclusions: Mitochondrial transplantation enhances mitochondrial ATP production and reduces the level of oxidative stress in cells under high-glucose injury, which improves the migration capacity of mouse L929 cells and the angiogenesis capacity of HUVECs. Simultaneously, it facilitates epithelialization and collagen deposition in full-thickness skin defect wounds of diabetic rats, reduces the levels of inflammatory cytokines, and inhibits cell apoptosis, thereby accelerating wound healing.