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. 目的： 探讨西兰花来源细胞外囊泡（BEV）对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制。 方法： 该研究为成组设计及重复测量设计实验研究。采用超滤浓缩联合尺寸排阻层析法分离提纯BEV并成功鉴定。采用随机数字表法将小鼠RAW264.7细胞分为常规培养的对照组，以及均经内毒素/脂多糖（LPS）刺激12 h后分别行常规培养、加入BEV培养的LPS组、BEV组。培养24 h后，采用蛋白质印迹法检测细胞中诱导型一氧化氮合酶（iNOS）和精氨酸酶-1（Arg-1）的蛋白表达水平，采用免疫荧光法检测细胞中CD86和CD206的蛋白表达水平，采用2'，7'-二氯二氢荧光素二乙酸酯荧光探针法检测细胞中活性氧水平，采用实时荧光定量反转录PCR法检测细胞中核转录因子红系2相关因子2（Nrf2）和血红素加氧酶-1（HO-1）的mRNA表达水平，上述实验样本数均为3。取24只7周龄雄性db/db小鼠，在其背部制作1个全层皮肤缺损创面后按随机数字表法分为对照组和BEV组，每组12只。伤后0（即刻）、3、6、9 d，分别在对照组、BEV组小鼠创面局部注射生理盐水、1×1010个/mL BEV溶液。伤后0、3、6、9、12 d观察创面愈合情况，计算伤后3、6、9、12 d创面愈合率。伤后6 d，采用免疫荧光法检测创面组织中CD86和CD206阳性面积占比，采用二氢乙锭荧光探针法检测创面组织中活性氧水平。 结果： 培养24 h后，与对照组相比，LPS组细胞中iNOS和CD86的蛋白表达水平均显著升高（P<0.05），活性氧水平显著升高（P<0.05）；与LPS组相比，BEV组细胞中iNOS和CD86的蛋白表达水平均显著降低（P<0.05），Arg-1和CD206的蛋白表达水平均显著升高（P<0.05），活性氧水平显著降低（P<0.05），Nrf2及HO-1的mRNA表达水平均显著升高（P<0.05）。伤后0~12 d，对照组和BEV组小鼠创面均逐渐愈合，其中伤后3、6、9、12 d，BEV组小鼠创面愈合率均显著高于对照组（t值分别为5.98、5.79、7.40、8.67，P<0.05）。伤后6 d，BEV组小鼠创面组织中CD86阳性面积占比为（0.60±0.29）%，较对照组的（1.61±0.19）%显著降低（t=7.20，P<0.05）；BEV组小鼠创面组织中CD206阳性面积占比为（3.42±0.77）%，较对照组的（0.66±0.20）%显著升高（t=8.48，P<0.05）；BEV组小鼠创面组织中活性氧水平较对照组显著降低（t=8.38，P<0.05）。 结论： BEV可通过激活Nrf2/HO-1轴，诱导巨噬细胞由M1型向M2型极化并降低活性氧水平，恢复糖尿病小鼠全层皮肤缺损创面“免疫-氧化”稳态，从而显著加速创面愈合进程。.
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