Effects of interleukin-4-modified gold nanozymes on the full-thickness skin defects in diabetic mice
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摘要:
目的 探讨白细胞介素4修饰的金纳米酶颗粒（IL-4-AuNP）对糖尿病小鼠全层皮肤缺损创面愈合的作用及其机制。 方法 采用实验研究方法。改进文献中的方法合成金纳米酶颗粒（AuNP）及IL-4-AuNP，采用透射电子显微镜拍摄2种颗粒形貌并计算其粒径，采用纳米粒度电位仪和粒度分析仪分别检测2种颗粒的表面电位和水合粒径。采用过氧化氢检测试剂盒和超氧阴离子检测试剂盒检测IL-4-AuNP的过氧化氢清除率和超氧阴离子清除率。取小鼠成纤维细胞系3T3细胞，采用随机数字表法（下同）将其分为空白对照组、仅使用过氧化氢处理的单纯过氧化氢组、先使用IL-4-AuNP处理0.5 h再使用过氧化氢处理的过氧化氢+IL-4-AuNP组，培养24 h后，采用免疫荧光法检测细胞活性氧水平，采用细胞计数试剂盒8检测细胞相对存活率。取Raw264.7小鼠巨噬细胞，将其分为空白对照组和用IL-4-AuNP处理的IL-4-AuNP组，培养24 h后，采用免疫荧光法观测细胞中精氨酸酶1（Arg-1）的表达。取12只8~10周龄雄性BALB/c小鼠（小鼠周龄、性别、品系下同），分为IL-4-AuNP组和空白对照组，分别作相应处理。在分组处理第16天，采集小鼠全血分析全血中白细胞计数（WBC）、红细胞计数（RBC）、血红蛋白水平与血小板计数和天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）、尿素与肌酐水平；采用苏木精-伊红（HE）染色观察小鼠心、肝、脾、肺和肾组织的炎症、出血或坏死情况。另取36只鼠，制作糖尿病模型后，在其背部制作全层皮肤缺损创面，将创面分为空白对照组、单纯AuNP组和IL-4-AuNP组，每组12只鼠，分别进行相应处理。于分组处理第0（即刻）、4、9、15天，观察创面情况并计算创面面积。分组处理第9天，采用HE染色检测创面中新生上皮长度和肉芽组织厚度。分组处理第15天，采用免疫荧光法检测创面组织中活性氧水平及Arg-1阳性细胞数。样本数均为6。对数据行独立样本t检验、校正t检验、Tukey检验或Dunnett T3检验。 结果 AuNP及IL-4-AuNP大小均匀，其粒径、表面电位、水合粒径分别为（13.0±2.1）、（13.9±2.5）nm及（-45.8±3.2）、（-20.3±2.2）mV与（14±3）、（16±4）nm。IL-4-AuNP的过氧化氢清除率和超氧阴离子清除率分别为（69±4）%和（52±5）%。分组培养24 h后，单纯过氧化氢组3T3细胞活性氧水平明显高于空白对照组（q=26.12，P<0.05）；过氧化氢+IL-4-AuNP组细胞活性氧水平明显低于单纯过氧化氢组（q=25.12，P<0.05），而与空白对照组接近（P>0.05）。分组培养24 h后，过氧化氢+IL-4-AuNP组3T3细胞相对存活率明显高于单纯过氧化氢组（t=51.44，P<0.05）。分组培养24 h后，IL-4-AuNP组Raw264.7细胞Arg-1的表达明显高于空白对照组（t'=8.83，P<0.05）。分组处理第16天，空白对照组和IL-4-AuNP组小鼠的WBC、RBC、血红蛋白水平与血小板计数和AST、ALT、尿素与肌酐水平比较差异均无统计学意义（P>0.05）；IL-4-AuNP组小鼠的心、肝、脾、肺、肾等重要脏器中均未观察到明显的炎症、出血或坏死，与空白对照组相比，无明显变化。分组处理第0、4天，空白对照组、单纯AuNP组和IL-4-AuNP组糖尿病小鼠创面面积比较差异均无统计学意义（P>0.05）。分组处理第9天，单纯AuNP组和IL-4-AuNP组创面面积均明显小于空白对照组（q值分别为9.45、14.87，P<0.05），IL-4-AuNP组创面面积显著小于单纯AuNP组（q=5.42，P<0.05）。分组处理第15天，单纯AuNP组和IL-4-AuNP组创面面积均明显小于空白对照组（q值分别为4.84、20.64，P<0.05），IL-4-AuNP组创面面积显著小于单纯AuNP组（q=15.80，P<0.05）；且IL-4-AuNP组创面部位红、肿等炎症反应较其他2组明显减轻。分组处理第9天，相比于空白对照组和单纯AuNP组，IL-4-AuNP组糖尿病小鼠创面中新生上皮长度明显更长（P值均<0.05），创面中的肉芽组织厚度显著增厚（q值分别为11.33、9.65，P值均<0.05）。分组处理第15天，相比于空白对照组，单纯AuNP组和IL-4-AuNP组糖尿病小鼠创面组织的活性氧水平均明显降低（P<0.05）。分组处理第15天，IL-4-AuNP组糖尿病小鼠创面中Arg-1阳性的细胞数显著多于空白对照组和单纯AuNP组（P值均<0.05）。 结论 IL-4-AuNP在体使用安全，可以通过清除活性氧改善氧化微环境和诱导巨噬细胞向M2表型极化，促进糖尿病小鼠全层皮肤缺损创面高效愈合与修复再生。 Abstract:Objective To investigate the effects and mechanism of interleukin-4-modified gold nanoparticle (IL-4-AuNP) on the wound healing of full-thickness skin defects in diabetic mice. Methods Experimental research methods were adopted. Gold nanoparticle (AuNP) and IL-4-AuNP were synthesized by improving the methods described in published literature. The morphology of those two particles were photographed by transmission electron microscopy, and their particle sizes were calculated. The surface potential and hydration particle size of the two particles were detected by nanoparticle potentiometer and particle size analyzer, respectively. The clearance rate of IL-4-AuNP to hydrogen peroxide and superoxide anion was measured by hydrogen peroxide and superoxide anion kits, respectively. Mouse fibroblast line 3T3 cells were used and divided into the following groups by the random number table (the same below): blank control group, hydrogen peroxide alone group treated with hydrogen peroxide only, hydrogen peroxide+IL-4-AuNP group treated with IL-4-AuNP for 0.5 h and then treated with hydrogen peroxide. After 24 h of culture, the reactive oxygen species (ROS) levels of cells were detected by immunofluorescence method; cell count kit 8 was used to detect relative cell survival rate. The macrophage Raw264.7 mouse cells were then used and divided into blank control group and IL-4-AuNP group that treated with IL-4-AuNP. After 24 h of culture, the expression of arginase 1 (Arg-1) in cells was observed by immunofluorescence method. Twelve male BALB/c mice (mouse age, sex, and strain, the same below) aged 8 to 10 weeks were divided into IL-4-AuNP group and blank control group, treated accordingly. On the 16th day of treatment, whole blood samples were collected from mice for analysis of white blood cell count (WBC), red blood cell count (RBC), hemoglobin level, or platelet count and the level of aspartate aminotransferase (AST), alanine transaminase (ALT), urea, or creatinine. The inflammation, bleeding, or necrosis in the heart, liver, spleen, lung, and kidney tissue of mice were detected by hematoxylin-eosin (HE). Another 36 mice were selected to make diabetic model, and the full-thickness skin defect wounds were made on the back of these mice. The wounds were divided into blank control group, AuNP alone group, and IL-4-AuNP group, with 12 mice in each group, and treated accordingly. On the 0 (immediately), 4th, 9th, and 15th day of treatment, the wound condition was observed and the wound area was calculated. On the 9th day of treatment, HE staining was used to detect the length of neonatal epithelium and the thickness of granulation tissue in the wound. On the 15th day of treatment, immunofluorescence method was used to detect ROS level and the number of Arg-1 positive cells in the wound tissue. The number of samples was 6 in all cases. Data were statistically analyzed with independent sample t test, corrected t test, Tukey test, or Dunnett T3 test. Results The size of prepared AuNP and IL-4-AuNP were uniform. The particle size, surface potential, and hydration particle size of AuNP and IL-4-AuNP were (13.0±2.1) and (13.9±2.5) nm, (-45.8±3.2) and (-20.3±2.2) mV, (14±3) and (16±4) nm, respectively. For IL-4-AuNP, the clearance rate to hydrogen peroxide and superoxide anion were (69±4)% and (52±5)%, respectively. After 24 h of culture, the ROS level of 3T3 in hydrogen peroxide alone group was significantly higher than that in blank control group (q=26.12, P<0.05); the ROS level of hydrogen peroxide+IL-4-AuNP group was significantly lower than that in hydrogen peroxide alone group (q=25.12, P<0.05) and close to that in blank control group (P>0.05). After 24 h of culture, the relative survival rate of 3T3 cells in hydrogen peroxide+IL-4-AuNP group was significantly higher than that in hydrogen peroxide alone group (t=51.44, P<0.05). After 24 h of culture, Arg-1 expression of Raw264.7 cells in IL-4-AuNP group was significantly higher than that in blank control group (t'=8.83, P<0.05).On the 16th day of treatment, there were no significant statistically differences in WBC, RBC, hemoglobin level, or platelet count and the level of AST, ALT, urea, or creatinine of mice between blank control group and IL-4-AuNP group (P>0.05). No obvious inflammation, bleeding or necrosis was observed in the heart, liver, spleen, lung, and kidney of important organs in IL-4-AuNP group, and no significant changes were observed compared with blank control group. On the 0 and 4th day of treatment, the wound area of diabetic mice in blank control group, AuNP alone group, and IL-4-AuNP group had no significant difference (P>0.05). On the 9th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 9.45 and 14.87, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=5.42, P<0.05). On the 15th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 4.84 and 20.64, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=15.80, P<0.05); moreover, inflammations such as redness and swelling were significantly reduced in IL-4-AuNP group compared with the other two groups. On the 9th day of treatment, compared with blank control group and AuNP alone group, the length of neonatal epithelium in the wound of diabetic mice in IL-4-AuNP group was significantly longer (all P<0.05), and the thickness of the granulation tissue in the wound was significantly increased (with q values of 11.33 and 9.65, respectively, all P<0.05). On the 15th day of treatment, compared with blank control group, ROS levels in wound tissue of diabetic mice in AuNP alone group and IL-4-AuNP group were significantly decreased (P<0.05). On the 15th day of treatment, the number of Arg-1 positive cells in the wounds of diabetic mice in IL-4-AuNP group was significantly more than that in blank control group and AuNP alone group, respectively (all P<0.05). Conclusions IL-4-AuNP is safe in vivo, and can improve the oxidative microenvironment by removing ROS and induce macrophage polarization towards M2 phenotype, thus promote efficient diabetic wound healing and regeneration of full-thickness skin defects in diabetic mice. -
Key words:
- Diabetes mellitus, experimental /
- Interleukin-4 /
- Reactive oxygen species /
- Macrophages /
- Skin /
- Gold nanozymes /
- Wound repair
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姚梦云：酝酿和设计实验，起草文章，实验研究，采集数据，分析、解释数据，获取研究经费；张宁、张庆、卢毅飞：实验研究，分析、解释数据，对文章的知识性内容作批评性审阅；黄勇、贺登峰、陈云霞：实验研究，采集数据；罗高兴：酝酿和设计实验，对文章的知识性内容作批评性审阅，获取研究经费，行政、技术或材料支持，指导性贡献所有作者均声明不存在利益冲突
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参考文献
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1 金纳米酶颗粒和白细胞介素4修饰的金纳米酶颗粒（IL-4-AuNP）形貌 透射电子显微镜×25 000。1A.金纳米酶颗粒大小均匀；1B.IL-4-AuNP大小均匀，其粒径略大于图1A
2 3组小鼠成纤维细胞系3T3细胞培养24 h后活性氧水平 2,7-二氯荧光素二乙酸酯×200。2A.空白对照组细胞活性氧水平低；2B.单纯过氧化氢组细胞活性氧水平较图2A明显升高；2C.过氧化氢+白细胞介素4修饰的金纳米酶颗粒组细胞活性氧水平较低，与图2A相当
注：绿色荧光反映细胞活性氧水平，且呈正相关
3 2组Raw264.7小鼠巨噬细胞培养24 h后精氨酸酶1的定位与表达 Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚×600。3A、3B、3C.分别空白对照组细胞精氨酸酶1、细胞核及复合染色情况，图3A红色荧光弱；3D、3E、3F.分别为白细胞介素4修饰的金纳米酶颗粒组细胞精氨酸酶1、细胞核及复合染色情况，图3D红色荧光强度明显高于图3A
注：精氨酸酶1阳性染色为红色，细胞核阳性染色为蓝色
4 2组小鼠分组处理第16天主要脏器组织学分析 苏木精-伊红×200。4A、4B、4C、4D、4E.依次为空白对照组小鼠的心、肝、脾、肺、肾组织切片染色情况，显示无明显炎症、出血或坏死；4F、4G、4H、4I、4J.依次为白细胞介素4修饰的金纳米酶颗粒组小鼠的心、肝、脾、肺、肾组织切片染色情况，分别与图4A、4B、4C、4D、4E相似
5 3组糖尿病小鼠构建全层皮肤缺损创面模型后不同时间点愈合情况观察。5A、5B、5C、5D.依次为空白对照组小鼠分组处理第0（即刻）、4、9、15天的创面，周围长期有红肿渗液，愈合速度缓慢；5E、5F、5G、5H.依次为单纯金纳米酶颗粒组小鼠分组处理第0、4、9、15天的创面，其中图5G、5H创面面积分别较图5C、5D有所减小；5I、5J、5K、5L.依次为白细胞介素4修饰的金纳米酶颗粒组小鼠分组处理第0、4、9、15天的创面，其中图5K、5L红肿渗液减少，愈合速度均明显分别较图5G、5H加快
注：图中黑色卡片为参照圆片，直径为6 mm
6 3组糖尿病小鼠全层皮肤缺损分组处理第9天创面组织新生上皮长度和肉芽组织厚度 苏木精-伊红×40。6A.空白对照组创面中新生上皮长度短，肉芽组织的厚度薄；6B.单纯金纳米酶颗粒组创面中新生上皮较长，肉芽组织较厚；6C.白细胞介素4修饰的金纳米酶颗粒组创面中新生上皮长度较图6A、6B显著增加，肉芽组织厚度较图6A、6B明显增厚
注：图中黑色双向箭头长度指示新生上皮长度，绿色双向箭头长度指示肉芽组织厚度
7 3组糖尿病小鼠全层皮肤缺损分组处理第15天创面组织中活性氧水平 二氢乙锭-4′,6-二脒基-2-苯基吲哚×400。7A、7B、7C.分别为空白对照组创面组织中活性氧、细胞核及复合染色情况，图7A红色荧光强度高；7D、7E、7F.分别为单纯金纳米酶颗粒组创面组织中活性氧、细胞核及复合染色情况，图7D红色荧光强度明显低于图7A；7G、7H、7I.分别为白细胞介素4修饰的金纳米酶颗粒组创面中活性氧、细胞核及复合染色情况，图7G红色荧光强度较图7A弱
注：二氢乙锭荧光探针阳性染色为红色，反映活性氧水平，且呈正相关；细胞核阳性染色为蓝色
8 3组糖尿病小鼠全层皮肤缺损分组处理第15天创面组织中精氨酸酶1表达情况 Alexa Fluor 594-4′,6-二脒基-2-苯基吲哚×200。8A、8B、8C.分别为空白对照组、单纯金纳米酶颗粒组、白细胞介素4修饰的金纳米酶颗粒组创面，图8C红色荧光细胞数明显较图8A、8B增多
注：精氨酸酶1阳性染色为红色，细胞核阳性染色为蓝色
表1 空白对照组和IL-4-AuNP组小鼠分组处理第16天血常规及血生化主要指标比较（
组别 鼠数（只） WBC（×109/L） RBC（×1012/L） 血红蛋白水平（×102 g/L） PLT（×1011/L） ALT（U/L） AST（U/L） 尿素（mmol/L） 肌酐（μmol/L） 空白对照组 6 8.6±1.2 8.5±0.4 1.45±0.12 8.4±0.6 37±12 101±15 8.3±0.7 13.7±1.9 IL-4-AuNP组 6 8.9±1.0 8.7±0.5 1.49±0.18 8.6±0.7 33±7 103±11 8.4±0.9 13.3±2.1 t值 0.41 0.62 0.63 0.35 0.70 0.22 0.06 0.30 P值 0.688 0.551 0.543 0.736 0.506 0.834 0.952 0.775 注：IL-4-AuNP为白细胞介素4修饰的金纳米酶颗粒；WBC为白细胞计数，RBC为红细胞计数，PLT为血小板计数，ALT为丙氨酸转氨酶，AST为天冬氨酸转氨酶 
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表2 3组糖尿病小鼠分组处理不同时间点全层皮肤缺损未愈合面积比较（mm2，
组别 鼠数（只） 0 d 4 d 9 d 15 d 空白对照组 6 43.3±3.5 41.2±2.8 39.3±3.2 25.3±2.5 单纯AuNP组 6 44.1±3.9 41.1±2.8 29.9±2.0a 21.3±2.1a IL-4-AuNP组 6 44.7±3.4 40.9±2.3 24.4±2.0ab 8.3±1.2ab F值 0.23 0.02 56.61 116.48 P值 0.800 0.983 <0.001 <0.001 注：AuNP为金纳米酶颗粒；IL-4为白细胞介素4；处理因素主效应，F=15.03，P<0.001；时间因素主效应，F=1 143.93，P<0.001；二者交互作用，F=72.16，P<0.001；与空白对照组相比，aP<0.05；与单纯AuNP组相比，bP<0.05
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