Objective: To investigate the effects and mechanism of copper oxide nanozymes on wound healing of full-thickness skin defects in diabetic mice. Methods: (1) Copper oxide nanozymes were synthesized through the reaction of copper chloride and L-ascorbic acid. Transmission electron microscope was used for observing the particle size and morphology of copper oxide nanozymes, and dynamic light scattering particle size analyzers and Zeta potentiometer were used to analyze the hydrated particle size and surface potential of copper oxide nanozymes, respectively. (2) The hydrogen peroxide detection kit, superoxide anion determination kit, and 3, 3', 5, 5'-tetramethylbenzidine were used to test the hydrogen peroxide, superoxide anion, and hydroxyl radicals scavenging ability of 150 ng/mL copper oxide nanozymes, respectively, and the scavenging proportions of hydrogen peroxide, superoxide anion, and hydroxyl radicals were calculated. The sample numbers were all 3. (3) Mouse fibroblast cell line 3T3 cells were divided into blank control group, simple hydrogen peroxide group, and hydrogen peroxide+ copper oxide group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in hydrogen peroxide+ copper oxide group were pre-treated with copper oxide nanozymes in final mass concentration of 25 ng/mL for 30 minutes, and then hydrogen peroxide in final molarity of 250 μmol/L was added into simple hydrogen peroxide group and hydrogen peroxide+ copper oxide group. Cells in blank control group were routinely cultured. After 24 hours of culture, 2', 7'-dichlorodihydrofluorescein diacetate fluorescence probe was used to detect the level of reactive oxygen species (indicated by green fluorescence intensity) in cells and cell counting kit-8 assay was performed to detect and calculate the cell survival rate. (4) Ten male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into phosphate buffer saline (PBS) group and copper oxide group, with 5 mice in each group. The mice in the copper oxide group were injected with 800 ng/kg copper oxide nanozyme at a concentration of 200 ng/mL via the caudal vein, and the mice in PBS group were treated with the same volume of PBS. The mice in the two groups were treated once a day for seven consecutive days. On the eighth day, 5 mice from each group were conducted and blood samples were taken for analysis of blood panel and serum biochemistry, and then the heart, liver, spleen, lung, and kidney were harvested for histopathological observation by hematoxylin-eosin (HE) staining after the mice were sacrificed. (5) Twenty mice were divided into PBS group and copper oxide group, with 10 mice in each group. Diabetes was induced by streptozotocin and high-sugar and high-fat diet and a full-thickness skin defect wound with diameter of 6 mm was reproduced on the back of each diabetic mouse. Immediately after injury, 20 μL PBS and 20 μL copper oxide nanozymes at the concentration of 200 ng/mL were added respectively to the wounds of mice in PBS group and copper oxide group, with the treatment being continued for twelve consecutive days. Three mice were selected from each group, and the wound healing was observed on post injury day (PID) 0 (immediately), 3, 6, 9, and 12 and the un-healed area was calculated. On PID 6, three mice from each group that were not for wound observation were sacrificed, and the content of interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 in the wound tissue were determined by enzyme-linked immunosorbent assay. On PID 12, the rest 7 mice in each group were sacrificed for observation of the length of regenerated epidermis in wound tissue by HE staining, and the level of reactive oxygen species (indicated as red fluorescence intensity) in wound tissue by dihydroethidium staining. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, independent sample t test, and Bonferroni test. Results: (1) The prepared copper oxide nanozymes were uniform in size with an average diameter of 3.5-4.0 nm in dry state, the hydrated particle size of 4.5 nm, and the surface potential of (-9.8±0.3) mV. By comprehensive judgment, copper oxide nanozymes had been successfully prepared. (2) After being treated with copper oxide nanozyme for 2 hours, 10 minutes, and 5 minutes, respectively, the scavenging proportions of hydrogen peroxide, superoxide anion, and hydroxyl radicals were (77±5)%, (45±5)%, and (84±4)%, respectively. (3) After 24 hours of culture, the cells in simple hydrogen peroxide group showed a significantly increased level of reactive oxygen species with abnormal morphology and decrease in cell number, while the cells in hydrogen peroxide+ copper oxide group showed a remarkably decreased level of reactive oxygen species with normal morphology similar to that of blank control group. The cell survival rate in simple hydrogen peroxide group was obviously reduced compared with the rates in blank control group and hydrogen peroxide+ copper oxide group (P<0.01), while there was no significant difference in cell survival rate between hydrogen peroxide+ copper oxide group and blank control group. (4) After 7 days of injection, there were no obvious differences in liver and kidney function indexes and blood panel indexes between mice in PBS group and copper oxide group. No necrosis, hyperaemia or hemorrhage in heart, liver, spleen, lung, or kidney was observed in mice in copper oxide group, which was similar to that in PBS group. (5) Compared with that of PBS group, wounds of mice in copper oxide group showed an accelerated healing trend with less redness. On PID 6, 9, and 12, the areas of un-healed wound of mice in copper oxide group (28.8±1.9), (17.6±3.8), and (10.4±1.8) mm(2), respectively, significantly lower than (38.0±4.3), (30.2±3.0), and (24.2±3.0) mm(2) in PBS group (t=3.706, 5.075, 5.558, P<0.01). On PID 6, the content of IL-1β, TNF-α, and IL-6 in wounds of mice in copper oxide group were significantly lower than that in PBS group (t=6.115, 11.762, 11.725, P<0.01). On PID 12, the length of regenerated epidermis in wounds of mice in copper oxide group was obviously longer than that in PBS group, the level of reactive oxygen species in wounds of mice in copper oxide group was obviously lower than that in PBS group. Conclusions: Copper oxide nanozyme not only has good biocompatibility, but also has efficient reactive oxygen species scavenging activity. It can eliminate the over-expressed reactive oxygen species in the full-thickness defect wounds of diabetic mice, reduce oxidative stress and inflammation, thus promoting wound repair.
目的： 探讨氧化铜纳米酶对糖尿病小鼠全层皮肤缺损创面修复的作用及其机制。 方法： (1)采用氯化铜与L-抗坏血酸反应的方法合成氧化铜纳米酶。采用透射电子显微镜观察氧化铜纳米酶大小和形貌，动态光散射粒度分析仪和纳米粒度电位仪分别分析其水合粒径和表面电位。(2)采用过氧化氢检测试剂盒、超氧阴离子检测试剂盒、3，3′，5，5′-四甲基联苯胺显色剂分别测定150 ng/mL氧化铜纳米酶的过氧化氢、超氧阴离子、羟自由基清除能力，计算过氧化氢、超氧阴离子、羟自由基清除比例(样本数均为3)。(3)将小鼠成纤维细胞系3T3细胞采用随机数字表法(分组方法下同)分为空白对照组、单纯过氧化氢组和过氧化氢＋氧化铜组，每组3孔。将终质量浓度25 ng/mL的氧化铜纳米酶加入过氧化氢＋氧化铜组细胞中预处理30 min。然后，在单纯过氧化氢组和过氧化氢＋氧化铜组细胞中各加入终物质的量浓度250 μmol/L过氧化氢，空白对照组常规培养。培养24 h后，采用2′，7′-二氯二氢荧光素二乙酸酯荧光探针检测细胞内活性氧水平(以绿色荧光强度表示)，细胞计数试剂盒8法检测并计算细胞存活率。(4)取10只6～8周龄雄性BALB/c小鼠(性别、鼠龄下同)，分为磷酸盐缓冲液(PBS)组与氧化铜组，每组5只。氧化铜组小鼠经尾静脉注射200 ng/mL的氧化铜纳米酶800 ng/kg，PBS组小鼠注射等体积的PBS。2组小鼠均每天注射1次，连续注射7 d。于第8天，取每组5只小鼠，采血行血细胞与血清生化分析，处死后收集心、肝、脾、肺和肾行苏木精-伊红(HE)染色后组织病理学观察。(5)取20只小鼠分为PBS组与氧化铜组，每组10只。采用链脲佐菌素及高糖高脂饮食法诱导糖尿病，并在其背部制作直径6 mm的全层皮肤缺损创面。伤后即刻，PBS组小鼠创面滴加20 μL PBS，氧化铜组小鼠创面滴加20 μL 200 ng/mL的氧化铜纳米酶，连续处理12 d。每组选定3只小鼠分别于伤后0(即刻)、3、6、9、12 d观察创面愈合情况，并计算创面未愈合面积。伤后6 d，每组取未行创面观察的3只小鼠，处死后酶联免疫吸附测定法测定创面组织中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、IL-6含量。伤后12 d，处死2组各剩余7只小鼠，行HE染色并观察创面新生上皮长度，行二氢乙锭荧光探针染色观察活性氧水平(以红色荧光强度表示)。对数据行单因素方差分析、重复测量方差分析、独立样本t检验、Bonferroni检验。 结果： (1)制备的氧化铜纳米酶大小均匀，在干燥状态下平均直径为3.5～4.0 nm，水合粒径约为4.5 nm，表面电位为(-9.8±0.3)mV。综合判定，成功制备了氧化铜纳米酶。(2)氧化铜纳米酶分别处理2 h、10 min、5 min后，过氧化氢、超氧阴离子、羟自由基清除比例分别为(77±5)%、(45±5)%、(84±4)%。(3)培养24 h后，单纯过氧化氢组细胞内活性氧水平急剧增高，细胞形态异常且数量减少；过氧化氢＋氧化铜组细胞内活性氧水平明显降低，细胞形态与空白对照组相比无明显变化。单纯过氧化氢组细胞存活率明显低于空白对照组和过氧化氢＋氧化铜组(P<0.01)，而过氧化氢＋氧化铜组细胞存活率与空白对照组相近。(4)注射7 d后，PBS组和氧化铜组小鼠肝功能指标、肾功能指标、血细胞相关指标均相近；氧化铜组小鼠的心、肝、脾、肺和肾中，均未观察到组织坏死、充血或出血，与PBS组无明显差别。(5)氧化铜组小鼠创面相比PBS组表现出更快的愈合趋势，创面红肿情况较轻。氧化铜组小鼠伤后6、9、12 d创面未愈合面积分别为(28.8±1.9)、(17.6±3.8)、(10.4±1.8)mm(2)，明显小于PBS组的(38.0±4.3)、(30.2±3.0)、(24.2±3.0)mm(2)(t＝3.706、5.075、5.558，P<0.01)。伤后6 d，氧化铜组小鼠创面IL-1β、TNF-α、IL-6含量均明显低于PBS组(t＝6.115、11.762、11.725，P<0.01)。伤后12 d，氧化铜组小鼠创面新生上皮长度长于PBS组，创面组织活性氧水平明显低于PBS组。 结论： 氧化铜纳米酶不仅具有良好的生物相容性，同时具有高效的活性氧清除活性，能够消除糖尿病小鼠全层皮肤缺损创面过量表达的活性氧，降低氧化应激、减轻炎症，促进创面修复。.
Keywords: Copper oxide nanozyme; Diabetes mellitus; Oxidative stress; Wound healing.