Effects and mechanism of cerium-myricetin nanosystem on wound healing in rats with full-thickness burns
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摘要:
目的 探讨铈-杨梅素纳米体系（Ce-MYR）对Ⅲ度烧伤大鼠创面愈合的作用及其机制。 方法 该研究为成组设计与重复测量设计实验研究。采用配位自组装法制备Ce-MYR并表征。取小鼠RAW264.7细胞,分为常规培养的对照组,用过氧化氢处理的过氧化氢组,以及用过氧化氢处理后加入终质量浓度分别为5、10、20 μg/mL Ce-MYR的低Ce-MYR组、中Ce-MYR组、高Ce-MYR组,培养24 h后,采用荧光探针法检测细胞内活性氧水平。取小鼠骨髓来源巨噬细胞（BMDM）,分为常规培养的对照组、用内毒素/脂多糖（LPS）处理的LPS组及用LPS联合Ce-MYR处理的Ce-MYR组,培养24 h后,行免疫荧光染色后检测并计算CD86和CD206阳性面积百分比。另取小鼠BMDM,分为同前处理的LPS组和Ce-MYR组,培养24 h后,采用蛋白质印迹法检测血红素加氧酶1（HO-1）、精氨酸酶1（Arg-1）、诱导型一氧化氮合酶（iNOS）和NOD样受体热蛋白结构域相关蛋白3（NLRP3）的蛋白表达。上述实验样本数均为4。取10只6周龄雄性SD大鼠,于每只大鼠背部建立4个Ⅲ度烧伤创面,按随机数字表法将大鼠分为磷酸盐缓冲液（PBS）组和Ce-MYR组（每组5只）,于伤后0（即刻）、3、6 d分别在创缘皮下注射PBS或Ce-MYR,计算伤后3、7、14、21 d剩余创面面积百分比；伤后21 d,取大鼠创面组织,行免疫组织化学染色后计数CD31阳性血管数,行免疫荧光染色后计算CD86和CD206阳性面积百分比,采用酶联免疫吸附测定法检测白细胞介素6（IL-6）、IL-1β、肿瘤坏死因子α（TNF-α）的水平。 结果 培养24 h后,过氧化氢组RAW264.7细胞内活性氧水平明显高于对照组（P<0.05）；中Ce-MYR组RAW264.7细胞内活性氧水平明显低于低Ce-MYR组（P<0.05）,明显高于高Ce-MYR组（P<0.05）。培养24 h后,Ce-MYR组BMDM中CD86阳性面积百分比明显低于LPS组（P<0.05）,明显高于对照组（P<0.05）；Ce-MYR组BMDM中CD206阳性面积百分比明显高于对照组和LPS组（P值均<0.05）。与LPS组比较,Ce-MYR组BMDM中HO-1和Arg-1的蛋白表达均明显增加（P<0.05）,iNOS和NLRP3的蛋白表达均明显减少（P<0.05）。伤后3、7、14、21 d,Ce-MYR组大鼠剩余创面面积百分比分别为（87.1±2.4）%、（65.1±2.2）%、（16.6±1.9）%、（0.5±0.4）%,均明显低于PBS组的（101.5±3.5）%、（79.9±3.2）%、（36.2±3.9）%、（14.5±1.9）%（t值分别为7.604、8.478、10.193、16.166,P<0.05）。伤后21 d,与PBS组比较,Ce-MYR组大鼠创面组织中阳性血管数明显增加（t=3.395,P<0.05）；创面组织中CD86阳性面积百分比明显降低（t=4.474,P<0.05）,CD206阳性面积百分比明显升高（t=4.713,P<0.05）；创面组织中IL-1β、IL-6和TNF-α的水平均明显降低（t值分别为3.999、5.040、6.023,P<0.05）。 结论 Ce-MYR可通过降低巨噬细胞内活性氧水平、增强HO-1相关抗氧化应答,促进巨噬细胞由M1型向M2型转化,改善Ⅲ度烧伤大鼠创面氧化应激与炎症失衡状态,从而促进创面愈合。 Abstract:Objective To investigate the effects and mechanism of cerium-myricetin nanosystem (Ce-MYR) on wound healing in rats with full-thickness burns. Methods This study was an experimental study with a grouped design. Ce-MYR was prepared by coordination self-assembly and characterized. Mouse RAW264.7 cells were divided into control group cultured normally, hydrogen peroxide group treated with hydrogen peroxide, and low Ce-MYR group, medium Ce-MYR group, and high Ce-MYR group treated with hydrogen peroxide followed by Ce-MYR at final mass concentrations of 5, 10, and 20 μg/mL, respectively. After 24 h of culture, intracellular reactive oxygen species (ROS) levels were detected using the fluorescent probe method. Mouse bone marrow-derived macrophages (BMDMs) were divided into control group cultured normally, lipopolysaccharide (LPS) group treated with LPS, and Ce-MYR group treated with LPS combined with Ce-MYR. After 24 h of culture, immunofluorescence staining was performed to detect and calculate the percentages of CD86- and CD206-positive areas. Additional mouse BMDMs were divided into LPS group and Ce-MYR group treated as the described above. After 24 h of culture, the protein expressions of heme oxygenase-1 (HO-1), arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) were detected by Western blotting. The sample size in all the above experiments was 4. Ten 6-week-old male Sprague-Dawley rats were used to establish four full-thickness burn wounds on the back of each rat, which were divided into phosphate-buffered saline (PBS) group and Ce-MYR group according to the random number table method, with 5 rats in each group. PBS or Ce-MYR was subcutaneously injected at the wound margin on post-injury days 0 (immediately), 3, and 6, respectively. The percentages of residual wound area were calculated on post-injury days 3, 7, 14, and 21. On post-injury day 21, the wound tissue of rats was collected to evaluate granulation tissue thickness after hematoxylin-eosin staining, to calculate the type Ⅰ/type Ⅲ collagen ratio after Sirius red-acid fuchsin staining, to count CD31-positive blood vessels after immunohistochemical staining, to determine the percentages of CD86-and CD206-positive areas after immunofluorescence staining, and to detect the levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) after enzyme-linked immunosrbent assay. Results After 24 h of culture, the ROS level in RAW264.7 cells in hydrogen peroxide group was significantly higher than that in control group (P<0.05); the ROS level in RAW264.7 cells in medium Ce-MYR group was significantly lower than that in low Ce-MYR group (P<0.05) and significantly higher than that in high Ce-MYR group (P<0.05). After 24 h of culture, the percentage of CD86-positive area in BMDMs in LPS group was significantly higher than that in control group and Ce-MYR group (with both P values <0.05), while the percentage of CD206-positive area in BMDMs in Ce-MYR group was significantly higher than that in control group and LPS group (with both P values <0.001). Compared with those in LPS group, the protein expressions of HO-1 and Arg-1 in BMDMs in Ce-MYR group were significantly increased (P<0.05), whereas the protein expressions of iNOS and NLRP3 were significantly decreased (P<0.05). On post-injury days 3, 7, 14, and 21, the percentages of residual wound area in rats in Ce-MYR group were (87.1±2.4)%, (65.1±2.2)%, (16.6±1.9)%, and (0.5±0.4)%, respectively, which were significantly lower than (101.5±3.5)%, (79.9±3.2)%, (36.2±3.9)%, and (14.5±1.9)% in PBS group (with t values of 7.604, 8.478, 10.193, and 16.166, respectively; P<0.001). On post-injury day 21, compared with those in PBS group, the granulation tissue thickness, type Ⅰ/type Ⅲ collagen ratio, and number of CD31-positive blood vessels in wound tissue of rats in Ce-MYR group were significantly increased (with t values of 5.179, 6.212, and 3.395, respectively, P<0.05). The percentage of CD86-positive area in wound tissue was significantly decreased (t=4.474, P<0.05), whereas the percentage of CD206-positive area was significantly increased (t=4.713, P<0.05). The levels of IL-1β, IL-6, and TNF-α in wound tissue were significantly decreased (with t values of 3.999, 5.040, and 6.023, respectively, P<0.05). Conclusions Ce-MYR may promote macrophage polarization from the M1 phenotype toward the M2 phenotype by reducing ROS levels and enhancing HO-1-related antioxidant responses, in macrophages thereby improving oxidative stress and inflammatory imbalance in rats with full-thickness burn wounds and promoting wound repair. -
参考文献
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图 1 Ce-MYR的表征。1A.Ce-MYR为分散、较均一的近球形纳米颗粒 透射电子显微镜 ×100 000；1B.Ce-MYR的水合粒径分布；1C.Ce-MYR在PBS和DMEM培养基中21 d内的水合粒径变化；1D.Ce-MYR的X射线光电子能谱全谱；1E.Ce-MYR的铈元素3d能级高分辨X射线光电子能谱及分峰拟合结果
注：Ce-MYR为铈-杨梅素纳米体系,PBS为磷酸盐缓冲液；图1B为横坐标经log2处理的图
图 2 5组小鼠RAW264.7细胞培养24 h后活性氧水平 2′,7′-二氯二氢荧光素二乙酸酯 ×400。2A、2B、2C、2D、2E.分别为对照组、过氧化氢组、低Ce-MYR组、中Ce-MYR组和高Ce-MYR组,图2A荧光强度最低,图2B~2E荧光强度依次降低
注：对照组细胞行常规培养,过氧化氢组细胞用过氧化氢处理,低Ce-MYR组、中Ce-MYR组、高Ce-MYR组细胞用过氧化氢处理后分别加入终质量浓度5、10、20 μg/mL的Ce-MYR；绿色荧光强度反映细胞内活性氧水平；Ce-MYR为铈-杨梅素纳米体系
图 3 3组小鼠BMDM培养24 h后细胞中CD86、CD206的表达 Alexa Fluor 594-Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚 ×400。3A、3B、3C.分别为对照组、LPS组和Ce-MYR组细胞中CD86阳性染色情况,图3B阳性面积较图3A和图3C明显增加；3D、3E、3F.分别为对照组、LPS组和Ce-MYR组细胞中CD206阳性染色情况,图3F阳性面积较图3D和3E明显增加
注：对照组细胞行常规培养,LPS组细胞用LPS处理,Ce-MYR组细胞用LPS和Ce-MYR处理；CD86阳性染色为红色,CD206阳性染色为绿色,细胞核阳性染色为蓝色；BMDM为骨髓来源巨噬细胞,LPS为内毒素/脂多糖,Ce-MYR为铈-杨梅素纳米体系
图 4 蛋白质印迹法检测的2组BMDM培养24 h后细胞中HO-1、Arg-1、iNOS和NLRP3的蛋白表达
注：BMDM为骨髓来源巨噬细胞,HO-1为血红素加氧酶1,Arg-1为精氨酸酶1,iNOS为诱导型一氧化氮合酶,NLRP3为NOD样受体热蛋白结构域相关蛋白3,LPS为内毒素/脂多糖,Ce-MYR为铈-杨梅素纳米体系；条带上方1、2分别表示用LPS处理的LPS组、用LPS和Ce-MYR处理的Ce-MYR组
图 5 2组Ⅲ度烧伤大鼠伤后各时间点创面愈合情况。5A、5B、5C.分别为PBS组伤后3、14、21 d创面情况；5D、5E、5F.分别为Ce-MYR组伤后3、14、21 d创面情况,图5D、5E、5F剩余创面面积分别小于图5A、5B、5C
注：PBS组和Ce-MYR组大鼠创面分别采用PBS和Ce-MYR处理；PBS为磷酸盐缓冲液,Ce-MYR为铈-杨梅素纳米体系；图上方刻度尺分度值为1 mm
图 6 2组Ⅲ度烧伤大鼠伤后21 d创面组织中肉芽组织、胶原沉积与重塑及血管生成情况。6A~6D及6E~6H.分别为PBS组和Ce-MYR组,6A和6E为肉芽组织情况 苏木精-伊红 ×200,6B和6F为胶原纤维沉积和排列情况 Masson ×400,6C和6G为Ⅰ/Ⅲ型胶原形成情况 天狼星红-苦味酸 ×400,6D和6H为血管生成情况 二氨基联苯胺-苏木精 ×400,与图6A、6B、6C、6D比较,图6E、6F、6G、6H中肉芽组织厚度增加,胶原沉积增多且排列更规则,Ⅰ/Ⅲ型胶原比值升高,CD31阳性血管增多
注：PBS组和Ce-MYR组大鼠创面分别采用PBS和Ce-MYR处理；PBS为磷酸盐缓冲液,Ce-MYR为铈-杨梅素纳米体系；图6A和6E中双向箭头长度表示肉芽组织厚度；图6C和6G中Ⅰ型胶原阳性染色为黄色,Ⅲ型胶原阳性染色为绿色；图6D和6H中CD31阳性染色为棕黄色
图 7 2组Ⅲ度烧伤大鼠伤后21 d创面组织中CD86、CD206的表达 Alexa Fluor 594-Alexa Fluor 488-4′,6-二脒基-2-苯基吲哚 ×400。7A、7B.分别为PBS组、Ce-MYR组CD86阳性染色情况,图7B中CD86阳性面积较图7A明显减少；7C、7D.分别为PBS组、Ce-MYR组CD206阳性染色情况,图7D中CD206阳性面积较图7C明显增加
注：PBS组和Ce-MYR组大鼠创面分别采用PBS和Ce-MYR处理；PBS为磷酸盐缓冲液,Ce-MYR为铈-杨梅素纳米体系；CD86阳性染色为红色,CD206阳性染色为绿色,细胞核阳性染色为蓝色
Table 1. 2组Ⅲ度烧伤大鼠伤后各时间点剩余创面面积百分比比较（%,
组别 鼠数（只） 3 d 7 d 14 d 21 d PBS组 5 101.5±3.5 79.9±3.2 36.2±3.9 14.5±1.9 Ce-MYR组 5 87.1±2.4 65.1±2.2 16.6±1.9 0.5±0.4 t值 7.604 8.478 10.193 16.166 P值 <0.001 <0.001 <0.001 <0.001 注：PBS组和Ce-MYR组大鼠创面分别采用PBS和Ce-MYR处理；PBS为磷酸盐缓冲液,Ce-MYR为铈-杨梅素纳米体系；时间因素主效应,F=3 079.282,P<0.001；分组因素主效应,F=204.695,P<0.001；两者交互效应,F=3.265,P=0.039 
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