[Effects and mechanisms of broccoli-derived extracellular vesicles on wound healing of full-thickness skin defects in diabetic mice].

Objective: To explore the effects and mechanisms of broccoli-derived extracellular vesicles (BEVs) on wound healing of full-thickness skin defects in diabetic mice. Methods: This study was an experimental study using a group design and a repeated-measures design. BEVs were isolated and purified using ultrafiltration concentration combined with size-exclusion chromatography, and were successfully identified. According to the random number table method, mouse RAW264.7 cells were divided into a control group cultured under routine condition, as well as a lipopolysaccharide (LPS) group and a BEV group, in which cells were first stimulated with LPS for 12 hours and then respectively cultured under routine condition and with BEV. After 24 hours of culture, Western blotting was used to detect the protein expression levels of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) in cells. Immunofluorescence method was used to detect the protein expression levels of CD86 and CD206 in cells. The level of reactive oxygen species in cells was measured using the 2',7'-dichlorodihydrofluorescein diacetate fluorescence probe assay. The mRNA expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in cells were detected using the real-time fluorescence quantitative reverse transcription polymerase chain reaction method. The sample sizes for all of the above experiments were 3. Twenty-four 7-week-old male db/db mice were used, and a full-thickness skin defect wound was created in the dorsal region. The mice were then assigned to control group and BEV group according to the random number table method, with 12 mice in each group. At post injury day (PID) 0 (immediately), 3, 6, and 9, normal saline and 1×1010 particles/mL BEVs solution were injected into the wound sites of mice in control group and BEV group, respectively. Wound healing was assessed at PID 0, 3, 6, 9, and 12, and wound healing rates were calculated at PID 3, 6, 9, and 12. At PID 6, the proportion of CD86- and CD206-positive areas in the wound tissue was assessed using the immunofluorescence method, and the level of reactive oxygen species in the wound tissue was measured using the dihydroethidium fluorescence probe assay. Results: After 24 hours of culture, compared with those in control group, the protein expression levels of iNOS and CD86 of cells in LPS group were significantly elevated (P<0.05), and the level of reactive oxygen species was significantly increased (P<0.05). Compared with those in LPS group, the cells in BEV group showed significantly reduced protein expression levels of iNOS and CD86 (P<0.05), significantly increased protein expression levels of Arg-1 and CD206 (P<0.05), significantly decreased level of reactive oxygen species (P<0.05), and significantly increased mRNA expression levels of Nrf2 and HO-1 (P<0.05). From PID 0 to 12, wounds of mice in both control group and BEV group gradually healed. Specifically, at PID 3, 6, 9, and 12, the wound healing rates of mice in BEV group were significantly higher than those in control group (with t values of 5.98, 5.79, 7.40, and 8.67, respectively, P<0.05). At PID 6, the proportion of CD86-positive area in the wound tissue of mice in BEV group was (0.60±0.29)%, which was significantly lower than (1.61±0.19)% in control group (t=7.20, P<0.05); the proportion of CD206-positive area in the wound tissue of mice in BEV group was (3.42±0.77)%, which was significantly higher than (0.66±0.20)% in control group (t=8.48, P<0.05); the level of reactive oxygen species in the wound tissue of mice in BEV group was significantly lower than that in control group (t=8.38, P<0.05). Conclusions: BEVs can restore the "immuno-oxidative" homeostasis of full-thickness skin defect wounds in diabetic mice by activating the Nrf2/HO-1 axis, inducing the polarization of macrophages from the M1 phenotype to the M2 phenotype, and decreasing the level of reactive oxygen species, thereby significantly accelerating the wound healing process.