Shan H, Zhang Z R, Wang X Y, Hou J Y, Zhang J
School of Nursing, Qingdao University, Qingdao 266071, China.
Department of Intensive Care Medicine, Affiliated Hospital of Qingdao University, Qingdao 266555, China.
Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi. 2022 Aug 20;38(8):767-777. doi: 10.3760/cma.j.cn501225-20220114-00007.
To investigate the effects of deferoxamine on macrophage polarization and wound healing in mice with deep tissue injury (DTI) and its mechanism. The experimental research methods were adopted. Fifty-four male C57BL/6J mice of 6-8 weeks old were divided into DTI control group, 2 mg/mL deferoxamine group, and 20 mg/mL deferoxamine group according to random number table, with 18 mice in each group. DTI was established on the back of mice by magnet compression method. From post injury day (PID) 1, mice were injected subcutaneously with 100 µL normal saline or the corresponding mass concentration of deferoxamine solution every other day at the wound edge until the samples were collected. Another 6 mice without any treatment were selected as normal control group. Six mice in each of the three DTI groups were collected on PID 3, 7, and 14 to observe the wound changes and calculate the wound healing rate. Normal skin tissue of mice in normal control group was collected on PID 3 in other groups (the same below) and wound tissue of mice in the other three groups on PID 7 and 14 was collected for hematoxylin-eosin (HE) staining to observe the tissue morphology. Normal skin tissue of mice in normal control group and wound tissue of mice in the other three groups on PID 7 were collected, and the percentages of CD206 and CD11c positive area were observed and measured by immunohistochemical staining, and the mRNA and protein expressions of CD206, CD11c, and inducible nitric oxide synthase (iNOS) were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Normal skin tissue of mice in normal control group and wound tissue of mice in DTI control group and 20 mg/mL deferoxamine group were collected on PID 3, 7, and 14, and the protein expressions of signal transducer and activator of transcription 3 (STAT3) and interleukin-10 (IL-10) were detected by Western blotting. The sample number in each group at each time point in the above experiments. The RAW264.7 cells were divided into 50 μmol/L deferoxamine group, 100 μmol/L deferoxamine group, 200 μmol/L deferoxamine group, and blank control group, which were treated correspondingly, with 3 wells in each group. The positive cell percentages of CD206 and CD86 after 48 h of culture were detected by flow cytometry. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. On PID 7, the wound healing rates of mice in 2 mg/mL and 20 mg/mL deferoamine groups were (17.7±3.7)% and (21.5±5.0)%, respectively, which were significantly higher than (5.1±2.3)% in DTI control group (<0.01). On PID 14, the wound healing rates of mice in 2 mg/mL and 20 mg/mL deferoamine groups were (51.1±3.8)% and (57.4±4.4)%, respectively, which were significantly higher than (25.2±3.8)% in DTI control group (<0.01). HE staining showed that the normal skin tissue layer of mice in normal control group was clear, the epidermis thickness was uniform, and skin appendages such as hair follicles and sweat glands were visible in the dermis. On PID 7, inflammation in wound tissue was obvious, the epidermis was incomplete, and blood vessels and skin appendages were rare in mice in DTI control group; inflammatory cells in wound tissue were reduced in mice in 2 mg/mL and 20 mg/mL deferoxamine groups, and a few of blood vessels and skin appendages could be seen. On PID 14, inflammation was significantly alleviated and blood vessels and skin appendages were increased in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups compared with those in DTI control group. On PID 7, the percentages of CD206 positive area in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly higher than that in DTI control group (<0.01), the percentage of CD206 positive area in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue of mice in normal control group (<0.01), the percentage of CD206 positive area in wound tissue of mice in 20 mg/mL deferoxamine group was significantly higher than that in normal skin tissue of mice in normal control group (<0.01). The percentages of CD11c positive area in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly lower than those in DTI control group and normal skin tissue in normal control group (<0.05 or <0.01), and the percentage of CD11c positive area in normal skin tissue of mice in normal control group was significantly higher than that in DTI control group (<0.05). On PID 7, the CD206 mRNA expressions in the wound tissue of mice in 2 mg/mL and 20 mg/mL deferoxamine groups were significantly higher than that in DTI control group (<0.01), but significantly lower than that in normal skin tissue in normal control group (<0.01); the CD206 mRNA expression in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue in normal control group (<0.01). The mRNA expressions of CD11c and iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly lower than those in DTI control group (<0.01). The mRNA expressions of CD11c in the wound tissue of mice in DTI control group, 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than that in normal skin tissue in normal control group (<0.01). Compared with that in normal skin tissue in normal control group, the mRNA expressions of iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly decreased (<0.01), and the mRNA expression of iNOS in wound tissue of mice in DTI control group was significantly increased (<0.01). On PID 7, the protein expressions of CD206 in the wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than those in DTI control group and normal skin tissue in normal control group (<0.01), and the protein expression of CD206 in wound tissue of mice in DTI control group was significantly lower than that in normal skin tissue in normal control group (<0.01). The protein expressions of CD11c and iNOS in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly lower than those in DTI control group (<0.01). The protein expressions of CD11c and iNOS in wound tissue of mice in DTI control group were significantly higher than those in normal skin tissue in normal control group (<0.01). The CD11c protein expressions in wound tissue of mice in 2 mg/mL and 20 mg/mL deferoamine groups were significantly higher than those in normal skin tissue in normal control group (<0.05 or <0.01). The protein expression of iNOS in wound tissue of mice in 2 mg/mL deferoamine group was significantly lower than that in 20 mg/mL deferoamine group and normal skin tissue in normal control group (<0.05). On PID 3, 7, and 14, the protein expressions of STAT3 and IL-10 in wound tissue of mice in 20 mg/mL deferoxamine group were significantly higher than those in DTI control group (<0.05 or <0.01), and the protein expressions of STAT3 were significantly higher than those in normal skin tissue in normal control group (<0.05 or <0.01). On PID 7 and 14, the protein expressions of IL-10 in wound tissue of mice in 20 mg/mL deferoxamine group were significantly higher than those in normal skin tissue in normal control group (<0.01). On PID 3, 7, and 14, the protein expressions of IL-10 in wound tissue of mice in DTI control group were significantly lower than those in normal skin tissue in normal control group (<0.05 or <0.01). After 48 h of culture, compared with those in blank control group, the CD206 positive cell percentages in 100 μmol/L and 200 μmol/L deferoamine groups were significantly increased (<0.01), while the CD86 positive cell percentages in 100 μmol/L and 200 μmol/L deferoamine groups were significantly decreased (<0.01). Deferoxamine can promote the polarization of macrophages toward the anti-inflammatory M2 phenotype and improve wound healing by enhancing the STAT3/IL-10 signaling pathway in DTI mice.
探讨去铁胺对深部组织损伤（DTI）小鼠巨噬细胞极化及伤口愈合的影响及其机制。采用实验研究方法。将54只6 - 8周龄的雄性C57BL/6J小鼠按随机数字表法分为DTI对照组、2 mg/mL去铁胺组和20 mg/mL去铁胺组，每组18只。采用磁压缩法在小鼠背部建立DTI模型。从损伤后第1天（PID 1）起，每隔一天在伤口边缘皮下注射100 μL生理盐水或相应质量浓度的去铁胺溶液，直至采集样本。另选6只未作任何处理的小鼠作为正常对照组。在PID 3、7和14时，每组采集6只DTI组小鼠，观察伤口变化并计算伤口愈合率。在PID 3时采集正常对照组小鼠的正常皮肤组织（其他组同），在PID 7和14时采集其他三组小鼠的伤口组织，进行苏木精-伊红（HE）染色，观察组织形态。在PID 7时采集正常对照组小鼠的正常皮肤组织和其他三组小鼠的伤口组织，通过免疫组织化学染色观察并测量CD206和CD11c阳性面积百分比，分别采用实时荧光定量逆转录聚合酶链反应和蛋白质免疫印迹法检测CD206、CD11c和诱导型一氧化氮合酶（iNOS）的mRNA和蛋白质表达。在PID 3、7和14时采集正常对照组小鼠的正常皮肤组织、DTI对照组和20 mg/mL去铁胺组小鼠的伤口组织，采用蛋白质免疫印迹法检测信号转导子和转录激活子3（STAT3）和白细胞介素-10（IL-10）的蛋白质表达。上述实验中每组各时间点的样本数。将RAW264.7细胞分为50 μmol/L去铁胺组、100 μmol/L去铁胺组、200 μmol/L去铁胺组和空白对照组，进行相应处理，每组设3个复孔。培养48 h后，采用流式细胞术检测CD206和CD86的阳性细胞百分比。数据采用重复测量方差分析、单因素方差分析和最小显著差法进行统计学分析。在PID 7时，2 mg/mL和20 mg/mL去铁胺组小鼠的伤口愈合率分别为（17.7±3.7）%和（21.5±5.0）%，显著高于DTI对照组的（5.1±2.3）%（P<0.01）。在PID 14时，2 mg/mL和20 mg/mL去铁胺组小鼠的伤口愈合率分别为（51.1±3.8）%和（57.4±4.4）%，显著高于DTI对照组的（25.2±3.8）%（P<0.01）。HE染色显示，正常对照组小鼠的正常皮肤组织层次清晰，表皮厚度均匀，真皮内可见毛囊、汗腺等皮肤附属器。在PID 7时，DTI对照组小鼠伤口组织炎症明显，表皮不完整，血管和皮肤附属器少见；2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中的炎症细胞减少，可见少量血管和皮肤附属器。在PID 14时，与DTI对照组相比，2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织炎症明显减轻，血管和皮肤附属器增多。在PID 7时，2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD206阳性面积百分比显著高于DTI对照组（P<0.01），DTI对照组小鼠伤口组织中CD206阳性面积百分比显著低于正常对照组小鼠的正常皮肤组织（P<0.01），20 mg/mL去铁胺组小鼠伤口组织中CD206阳性面积百分比显著高于正常对照组小鼠的正常皮肤组织（P<0.01）。2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD11c阳性面积百分比显著低于DTI对照组和正常对照组的正常皮肤组织（P<0.05或P<0.01），正常对照组小鼠的正常皮肤组织中CD11c阳性面积百分比显著高于DTI对照组（P<0.05）。在PID 7时，2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD206 mRNA表达显著高于DTI对照组（P<0.01），但显著低于正常对照组的正常皮肤组织（P<0.01）；DTI对照组小鼠伤口组织中CD206 mRNA表达显著低于正常对照组的正常皮肤组织（P<0.01）。2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD11c和iNOS的mRNA表达显著低于DTI对照组（P<0.01）。DTI对照组、2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD11c的mRNA表达显著高于正常对照组的正常皮肤组织（P<0.01）。与正常对照组的正常皮肤组织相比，2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中iNOS的mRNA表达显著降低（P<0.01），DTI对照组小鼠伤口组织中iNOS的mRNA表达显著升高（P<0.01）。在PID 7时，2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD206的蛋白质表达显著高于DTI对照组和正常对照组的正常皮肤组织（P<0.01），DTI对照组小鼠伤口组织中CD206的蛋白质表达显著低于正常对照组的正常皮肤组织（P<0.01）。2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD11c和iNOS的蛋白质表达显著低于DTI对照组（P<0.01）。DTI对照组小鼠伤口组织中CD11c和iNOS的蛋白质表达显著高于正常对照组的正常皮肤组织（P<0.01）。2 mg/mL和20 mg/mL去铁胺组小鼠伤口组织中CD11c的蛋白质表达显著高于正常对照组的正常皮肤组织（P<0.05或P<0.01）。2 mg/mL去铁胺组小鼠伤口组织中iNOS的蛋白质表达显著低于20 mg/mL去铁胺组和正常对照组的正常皮肤组织（P<0.05）。在PID 3、7和14时，20 mg/mL去铁胺组小鼠伤口组织中STAT3和IL-10的蛋白质表达显著高于DTI对照组（P<0.05或P<0.01），STAT3的蛋白质表达显著高于正常对照组的正常皮肤组织（P<0.05或P<0.01）。在PID 7和14时，20 mg/mL去铁胺组小鼠伤口组织中IL-10的蛋白质表达显著高于正常对照组的正常皮肤组织（P<0.01）。在PID 3、7和14时，DTI对照组小鼠伤口组织中IL-10的蛋白质表达显著低于正常对照组的正常皮肤组织（P<0.05或P<0.01）。培养48 h后，与空白对照组相比，100 μmol/L和200 μmol/L去铁胺组的CD206阳性细胞百分比显著升高（P<0.01），而100 μmol/L和200 μmol/L去铁胺组的CD86阳性细胞百分比显著降低（P<0.01）。去铁胺可促进DTI小鼠巨噬细胞向抗炎M2表型极化，并通过增强STAT3/IL-10信号通路改善伤口愈合。
