Effects and mechanisms of broccoli-derived extracellular vesicles on wound healing of full-thickness skin defects in diabetic mice
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摘要:
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,采用免疫荧光法检测细胞中CD86和CD206的蛋白表达,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,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的蛋白表达均显著降低（P<0.05）,Arg-1和CD206的蛋白表达均显著升高（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。 Abstract: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 a combination of ultrafiltration concentration and size-exclusion chromatography, and were successfully identified. According to the random number table method, mouse RAW264.7 cells were divided into control group with routine culture, as well as lipopolysaccharide (LPS) group and BEV group, both of which were stimulated with LPS for 12 hours and then respectively subjected to routine culture and culture with BEV. After 24 hours of culture, Western blotting was used to detect the protein expressions of inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) in cells. Immunofluorescence method was used to detect the protein expressions 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 expressions of nuclear factor E2-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 a 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 percentages of areas positive for CD86 and CD206 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 expressions 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 expressions of iNOS and CD86 (P<0.05), significantly increased protein expressions of Arg-1 and CD206 (P<0.05), significantly reduced level of reactive oxygen species (P<0.05), and significantly increased mRNA expressions 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 percentage of the area positive for CD86 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 percentage of the area positive for CD206 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 those 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 reducing the level of reactive oxygen species, thereby significantly accelerating the wound healing process. -
参考文献
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图 1 西兰花来源细胞外囊泡的表征。1A.西兰花来源细胞外囊泡呈清晰的茶托状结构 透射电子显微镜 ×60 000；1B.西兰花来源细胞外囊泡平均粒径为117.7 nm；1C.蛋白质印迹法检测的西兰花来源细胞外囊泡中CD9、CD63和肿瘤易感基因101（TSG101）的蛋白表达
图 2 3组小鼠RAW264.7细胞培养24 h后iNOS、Arg-1及CD86、CD206的蛋白表达情况。2A、2B.分别为蛋白质印迹法检测的iNOS、Arg-1蛋白表达情况；2C、2D、2E.分别为对照组、LPS组、BEV组CD86蛋白表达情况,图2D的蛋白表达显著高于图2C、2E Alexa Fluor 488-四甲基异硫氰酸罗丹明-4',6-二脒基-2-苯基吲哚 ×200；2F、2G、2H.分别为对照组、LPS组、BEV组CD206蛋白表达情况,图2G的蛋白表达显著低于图2H,与图2F相近 Alexa Fluor 488-四甲基异硫氰酸罗丹明-4',6-二脒基-2-苯基吲哚 ×200
注：iNOS为诱导型一氧化氮合酶,Arg-1为精氨酸酶-1；条带图上方的1、2、3分别表示对细胞行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、加入西兰花来源细胞外囊泡（BEV）培养的LPS组、BEV组；荧光图像中CD86和CD206阳性染色均为绿色,细胞骨架阳性染色为红色,细胞核阳性染色为蓝色
图 3 3组小鼠RAW264.7细胞培养24 h后活性氧水平 2',7'-二氯荧光素-Hoechst 33342 ×100。3A、3B、3C.分别为对照组、LPS组、BEV组,图3B的活性氧水平显著高于图3A、3C
注：对照组细胞常规培养,内毒素/脂多糖（LPS）组、西兰花来源细胞外囊泡（BEV）组细胞均经LPS刺激12 h后分别行常规培养、加入BEV培养；活性氧阳性染色为绿色,细胞核阳性染色为蓝色
图 4 4组小鼠RAW264.7细胞培养24 h后CD86、CD206的蛋白表达及活性氧水平。4A、4B、4C、4D.分别为LPS组、BEV组、BEV+ML385组、ML385组CD86蛋白表达情况,图4B的蛋白表达显著低于图4A、4C、4D Alexa Fluor 488-四甲基异硫氰酸罗丹明-4',6-二脒基-2-苯基吲哚 ×200；4E、4F、4G、4H.分别为LPS组、BEV组、BEV+ML385组、ML385组CD206蛋白表达情况,图4F的蛋白表达显著高于图4E、4G、4H Alexa Fluor 488-四甲基异硫氰酸罗丹明-4',6-二脒基-2-苯基吲哚 ×200；4I、4J、4K、4L.分别为LPS组、BEV组、BEV+ML385组、ML385组活性氧水平,图4J的活性氧水平显著低于图4I、4K、4L 2',7'-二氯荧光素-Hoechst 33342 ×100
注：内毒素/脂多糖（LPS）组、西兰花来源细胞外囊泡（BEV）组细胞均经LPS刺激12 h后分别行常规培养、加入BEV培养；对BEV+ML385组和ML385组细胞先进行ML385预处理24 h,随后处理分别与BEV组和LPS组相同；CD86、CD206和活性氧阳性染色均为绿色,细胞骨架阳性染色为红色,细胞核阳性染色为蓝色
图 5 2组糖尿病小鼠全层皮肤缺损创面伤后不同时间点的愈合情况。5A、5B、5C.分别为对照组伤后0、6、12 d的创面,逐渐缩小；5D、5E、5F.分别为BEV组伤后0、6、12 d的创面,其中图5E、5F创面面积均分别显著小于图5B、5C；5G.创面愈合率比较（样本数为6,
注：伤后0（即刻）、3、6、9 d,分别在对照组、西兰花来源细胞外囊泡（BEV）组小鼠创面局部注射生理盐水、BEV溶液；置于创面外作为比例尺的圆环胶圈内径14 mm、外径18 mm；图5G中处理因素主效应,F=90.70,P<0.001；时间因素主效应,F=653.20,P<0.001；两者交互作用,F=22.32,P<0.001；与对照组相比,aP<0.05
图 6 2组糖尿病小鼠伤后6 d全层皮肤缺损创面组织中CD86、CD206的蛋白表达及活性氧水平。6A、6B.分别为对照组、BEV组创面组织中CD86蛋白表达情况,图6B中CD86阳性面积占比显著低于图6A Alexa Fluor 594-4',6-二脒基-2-苯基吲哚 ×200；6C、6D.分别为对照组、BEV组创面组织中CD206蛋白表达情况,图6D中CD206阳性面积占比显著高于图6C Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×200；6E、6F.分别为对照组、BEV组创面组织中活性氧水平,图6F活性氧水平显著低于图6E 乙锭-4',6-二脒基-2-苯基吲哚 ×200
注：伤后0（即刻）、3、6、9 d,分别在对照组、西兰花来源细胞外囊泡（BEV）组小鼠创面局部均匀注射生理盐水、BEV溶液；CD206阳性染色为绿色,CD86和活性氧阳性染色均为红色,细胞核阳性染色为蓝色
Table 1. 3组小鼠RAW264.7细胞培养24 h后特异性标志物蛋白表达水平比较（
组别 样本数 诱导型一氧化氮合酶 精氨酸酶-1 CD86 CD206 对照组 3 0.095±0.008 1.20±0.08 0.345±0.055 1.08±0.15 LPS组 3 1.000±0.227 1.00±0.18 1.000±0.053 1.00±0.09 BEV组 3 0.617±0.167 1.65±0.21 0.609±0.115 3.69±0.28 F值 23.44 11.90 64.03 286.70 P值 0.002 0.008 0.015 0.002 P1值 0.001 0.307 0.003 0.582 P2值 0.049 0.006 0.028 0.003 注：对照组细胞常规培养,内毒素/脂多糖（LPS）组、西兰花来源细胞外囊泡（BEV）组细胞均经LPS刺激12 h后分别行常规培养、加入BEV培养；F值、P值为3组间各指标总体比较所得；P1值、P2值分别为对照组与LPS组、BEV组与LPS组比较所得 
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Table 2. 4组小鼠RAW264.7细胞培养24 h后Nrf2、HO-1表达及极化和活性氧水平比较（
组别 样本数 Nrf2的mRNA表达 HO-1的mRNA表达 CD86的蛋白表达 CD206的蛋白表达 活性氧水平 LPS组 3 1.000±0.117 1.000±0.224 1.000±0.132 1.00±0.14 1.00±0.07 BEV组 3 2.773±0.308a 3.373±0.284a 0.166±0.022a 4.64±0.17a 0.40±0.11a BEV+ML385组 3 1.123±0.162b 1.259±0.218b 0.851±0.038b 1.12±0.14b 0.93±0.10b ML385组 3 1.058±0.083b 1.212±0.136b 1.023±0.135b 0.93±0.06b 0.95±0.10b F值 62.21 75.18 52.07 542.50 27.57 P值 <0.001 <0.001 <0.001 <0.001 <0.001 注：Nrf2为核转录因子红系2相关因子2,HO-1为血红素加氧酶-1；内毒素/脂多糖（LPS）组、西兰花来源细胞外囊泡（BEV）组细胞均经LPS刺激12 h后分别行常规培养、加入BEV培养；对BEV+ML385组和ML385组细胞先进行ML385预处理24 h,随后处理分别与BEV组和LPS组相同；与LPS组比较,aP<0.05；与BEV组比较,bP<0.05
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