[Influence and mechanism of PBNP combined with mouse ADSCs on full-thickness skin defect wounds in diabetic mice].

Objective

To investigate the influence and mechanism of Prussian blue nanoparticles (PBNPs) combined with mouse adipose-derived mesenchymal stem cells (ADSCs) on full-thickness skin defect wounds in diabetic mice.

Methods

This study was an experimental study using group design and repeated-measures design. PBNPs were prepared by hydrothermal synthesis, and their morphology was characterized by transmission electron microscopy. ADSCs were isolated from five male 6-8-week-old Institute of Cancer Research (ICR) mice, USA via collagenase digestion. The cells were divided into control group cultured under routine conditions, high-glucose group cultured with glucose in a final molarity of 30.0 mmol/L, and low-PBNP group and high-PBNP group pretreated with 10 or 20 μg/mL PBNP for 12 h, respectively, followed by the same treatment as in high-glucose group. After 24 h of culture, cell viability was assessed using the cell counting kit-8, the proportion of senescent cells in the cells was detected by β-galactosidase staining, and the protein expression levels of senescence-associated proteins p16 and p21 were determined by Western blotting. Twenty-four male 6-8-week-old ICR mice were used to establish the diabetic model. A full-thickness skin defect wound was then created on the back of each mouse. The injured mice were divided into four groups (with 6 mice in each group) according to the random number table method, including control group with wounds treated with normal saline, ADSC group with wounds treated with normal saline containing 5×106 ADSC (the same number of cells used below), low-PBNP group and high-PBNP group with wounds treated with normal saline containing ADSC pretreated with 10 and 20 μg/mL PBNP for 12 h, respectively. Wound healing was observed at post-injury day (PID) 0 (immediately), 3, 7, 10, and 14. The percentage of remaining wound area was calculated at PID 3, 7, 10, and 14. At PID 7, the protein expression levels of the cell proliferation markers Ki67 and vascular endothelial growth factor (VEGF) in wound tissue were detected by immunofluorescence method. At PID 14, the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10 in wound tissue were determined by enzyme-linked immunosorbent assay.

Results

PBNPs exhibited a dispersed regular cubic structure. After 24 h of culture, compared with those in control group, cells in high-glucose group showed a significantly lower cell viability (P<0.05), a significantly higher proportion of senescent cells (P<0.05), and significantly higher protein expression levels of p16 and p21 (P<0.05). Compared with those in high-glucose group, cells in low-PBNP and high-PBNP groups showed significantly higher cell viability (P<0.05), significantly lower proportions of senescent cells (P<0.05), and significantly lower protein expression levels of p16 and p21 (P<0.05). From PID 0 to 14, the wounds of mice in all four groups healed gradually. At PID 3, 7, 10, and 14, the percentages of remaining wound area of mice in high-PBNP group were (75.3±3.1)%, (46.7±2.5)%, (24.0±5.2)%, and (8.0±1.0)%, respectively, all significantly lower than (85.0±2.0)%, (62.7±3.1)%, (46.7±3.8)%, and (19.3±2.1)% in ADSC group (P<0.05). At PID 7, the percentage of remaining wound area of mice in ADSC group was significantly lower than that in control group ((77.3±3.2)%, P<0.05). At PID 7, the protein expression levels of Ki67 and VEGF of cells in ADSC group were significantly higher than those in control group (P<0.05), the protein expression levels of Ki67 of cells in low-PBNP and high-PBNP groups and the protein expression level of VEGF of cells in high-PBNP group were significantly higher than those in ADSC group (P<0.05). At PID 14, compared with those in control group, wound tissue in ADSC group showed a significantly higher expression level of IL-10 (P<0.05) and significantly lower expression levels of TNF-α, IL-1β, and IL-6 (P<0.05); compared with those in ADSC group, wound tissue in low-PBNP and high-PBNP groups showed significantly higher expression levels of IL-10 (P<0.05) and significantly lower expression levels of TNF-α, IL-1β, and IL-6 (P<0.05).

Conclusions

PBNP can inhibit high glucose-induced senescence of mouse ADSCs. PBNP combined with mouse ADSCs is more effective than mouse ADSCs alone in promoting cell proliferation and angiogenesis in full-thickness skin defect wounds of diabetic mice, inhibiting the release of inflammatory cytokines, and accelerating wound healing.