Regulatory mechanism of deferoxamine on macrophage polarization and wound healing in mice with deep tissue injury
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摘要:
目的 探讨去铁胺对深部组织损伤（DTI）小鼠巨噬细胞极化和创面愈合的影响及其作用机制。 方法 采用实验研究方法。取54只6~8周龄雄性C57BL/6J小鼠，按随机数字表法分为DTI对照组、2 mg/mL去铁胺组、20 mg/mL去铁胺组，每组18只。采用磁铁压迫法在小鼠背部制造DTI，从伤后1 d开始，每隔1 d在创缘皮下注射100 µL生理盐水或相应质量浓度的去铁胺溶液，直至取材；另取6只不进行任何处理的小鼠为正常对照组。取3组DTI小鼠，每组6只，于伤后3、7、14 d观察创面变化并计算创面愈合率。于其他组伤后3 d取正常对照组小鼠正常皮肤组织（下同）和其余3组小鼠伤后7、14 d创面组织，行苏木精-伊红（HE）染色观察组织形态。取正常对照组小鼠正常皮肤组织和其余3组小鼠伤后7 d创面组织，行免疫组织化学染色观测CD206和CD11c阳性面积百分比，分别采用实时荧光定量反转录PCR法及蛋白质印迹法检测CD206、CD11c和诱导型一氧化氮合酶（iNOS）的mRNA及蛋白表达。取正常对照组小鼠正常皮肤组织和DTI对照组、20 mg/mL去铁胺组小鼠伤后3、7、14 d创面组织，采用蛋白质印迹法检测信号转导及转录活化因子3（STAT3）和白细胞介素10（IL-10）蛋白表达。以上实验各组各时间点样本数均为6。取RAW264.7细胞，分为进行相应处理的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，每组3孔，于培养48 h，采用流式细胞仪检测CD206和CD86阳性细胞百分比。数据分析采用重复测量方差分析、单因素方差分析和LSD检验。 结果 伤后7 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面愈合率分别为（17.7±3.7）%、（21.5±5.0）%，均明显高于DTI对照组的（5.1±2.3）%（P<0.01）；伤后14 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面愈合率分别为（51.1±3.8）%、（57.4±4.4）%，均明显高于DTI对照组的（25.2±3.8）%（P<0.01）。HE染色可见，正常对照组小鼠正常皮肤组织层次清晰，表皮厚度均一，真皮层可见毛囊和汗腺等皮肤附属器。伤后7 d，DTI对照组小鼠创面组织炎症明显，表皮有残缺，血管和皮肤附属器罕见；2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织炎症细胞减少，可见少量血管及皮肤附属器。伤后14 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织炎症明显减轻，血管和皮肤附属器较DTI对照组增多。伤后7 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD206阳性面积百分比均明显高于DTI对照组（P<0.01），DTI对照组小鼠创面组织CD206阳性面积百分比明显低于正常对照组正常皮肤组织（P<0.01），20 mg/mL去铁胺组小鼠创面组织CD206阳性面积百分比明显高于正常对照组正常皮肤组织（P<0.01）。2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD11c阳性面积百分比均明显低于DTI对照组和正常对照组正常皮肤组织（P<0.05或P<0.01），正常对照组小鼠正常皮肤组织CD11c阳性面积百分比明显高于DTI对照组（P<0.05）。伤后7 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD206 mRNA 表达量均明显高于DTI对照组（P<0.01），但均明显低于正常对照组正常皮肤组织（P<0.01）；DTI对照组小鼠创面组织CD206 mRNA表达量明显低于正常对照组正常皮肤组织（P<0.01）。2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD11c和iNOS的mRNA表达量均明显低于DTI对照组（P<0.01）；DTI对照组、2 mg/mL去铁胺组、20 mg/mL去铁胺组小鼠创面组织CD11c mRNA表达量均明显高于正常对照组正常皮肤组织（P<0.01）；与正常对照组正常皮肤组织比较，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织iNOS mRNA 表达量均明显降低（P<0.01），DTI对照组小鼠创面组织iNOS mRNA 表达量明显增多（P<0.01）。伤后7 d，2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD206蛋白表达均明显高于DTI对照组和正常对照组正常皮肤组织（P<0.01），DTI对照组小鼠创面组织CD206蛋白表达明显低于正常对照组正常皮肤组织（P<0.01）。2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD11c和iNOS蛋白表达均明显低于DTI对照组（P<0.01），DTI对照组小鼠创面组织CD11c和iNOS蛋白表达均明显高于正常对照组正常皮肤组织（P<0.01），2 mg/mL去铁胺组和20 mg/mL去铁胺组小鼠创面组织CD11c蛋白表达均明显高于正常对照组正常皮肤组织（P<0.05或P<0.01），2 mg/mL去铁胺组小鼠创面组织iNOS蛋白表达较20 mg/mL去铁胺组和正常对照组正常皮肤组织明显减少（P值均<0.05）。伤后3、7、14 d，20 mg/mL去铁胺组小鼠创面组织STAT3和IL-10蛋白表达均明显高于DTI对照组（P<0.05或P<0.01），STAT3蛋白表达明显高于正常对照组正常皮肤组织（P<0.05或P<0.01）。伤后7、14 d，20 mg/mL去铁胺组小鼠创面组织IL-10蛋白表达均明显高于正常对照组正常皮肤组织（P<0.01）。伤后3、7、14 d，DTI对照组小鼠创面组织IL-10蛋白表达均明显低于正常对照组正常皮肤组织（P<0.05或P<0.01）。培养48 h，与空白对照组比较，100 μmol/L去铁胺组、200 μmol/L去铁胺组CD206阳性细胞百分比均明显升高（P<0.01），100 μmol/L去铁胺组、200 μmol/L去铁胺组CD86阳性细胞百分比均明显降低（P<0.01）。 结论 去铁胺可能通过增强DTI小鼠STAT3/IL-10信号通路，促进巨噬细胞向抗炎M2表型极化，促进创面愈合。 Abstract:Objective To investigate the effects of deferoxamine on macrophage polarization and wound healing in mice with deep tissue injury (DTI) and its mechanism. Methods 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. Results 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 (P<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 (P<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 (P<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 (P<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 (P<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 (P<0.05 or P<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 (P<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 (P<0.01), but significantly lower than that in normal skin tissue in normal control group (P<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 (P<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 (P<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 (P<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 (P<0.01), and the mRNA expression of iNOS in wound tissue of mice in DTI control group was significantly increased (P<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 (P<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 (P<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 (P<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 (P<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 (P<0.05 or P<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 (P<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 (P<0.05 or P<0.01), and the protein expressions of STAT3 were significantly higher than those in normal skin tissue in normal control group (P<0.05 or P<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 (P<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 (P<0.05 or P<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 (P<0.01), while the CD86 positive cell percentages in 100 μmol/L and 200 μmol/L deferoamine groups were significantly decreased (P<0.01). Conclusions 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. -
Key words:
- Wound healing /
- Deferoxamine /
- Macrophage polarization /
- Deep tissue injury
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参考文献
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1 3组深部组织损伤（DTI）小鼠伤后各时间点创面情况。1A、1B、1C.分别是DTI对照组伤后3、7、14 d创面情况；1D、1E、1F.分别是2 mg/mL去铁胺组伤后3、7、14 d创面情况；1G、1H、1I.分别是20 mg/mL去铁胺组伤后3、7、14 d创面情况，图1E、1H创面面积均明显小于图1B，图1F、1I创面面积均明显小于图1C
2 正常对照组小鼠正常皮肤组织和3组深部组织损伤（DTI）小鼠伤后7 d创面组织形态 苏木精-伊红×200。2A.正常对照组组织形态；2B、2C、2D.分别为DTI对照组、2 mg/mL去铁胺组和20 mg/mL去铁胺组组织形态，图2C、2D表皮层扁平，血管和皮肤附属器较图2B增多
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；黑色箭头指示上皮，白色箭头指示炎症细胞，绿色箭头指示皮肤附属器，黄色箭头指示血管
3 正常对照组小鼠正常皮肤组织和3组深部组织损伤（DTI）小鼠伤后7 d创面组织巨噬细胞标志物表达情况 二氨基联苯胺-苏木精×200。3A、3B、3C、3D.分别为DTI对照组、2 mg/mL去铁胺组、20 mg/mL去铁胺组和正常对照组CD206阳性面积，图3B、3C中CD206阳性面积较图3A、3D增加；3E、3F、3G、3H.分别为DTI对照组、2 mg/mL去铁胺组、20 mg/mL去铁胺组和正常对照组CD11c阳性面积，图3F、3G中CD11c阳性面积较图3E、3H减少
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；CD206和CD11c阳性染色均为棕色
4 免疫组织化学法检测的正常对照组小鼠正常皮肤组织和3组深部组织损伤（DTI）小鼠伤后7 d 创面组织中CD206和CD11c阳性面积百分比比较（样本数均为6，
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；4组CD206、CD11c阳性面积百分比总体比较，差异均明显（F值分别为31.60、38.82，P值均<0.001）；与正常对照组比较，a P<0.01，c P<0.05；与DTI对照组比较，b P<0.01，d P<0.05
5 实时荧光定量反转录PCR法检测的正常对照组小鼠正常皮肤组织和3组深部组织损伤（DTI）小鼠伤后7 d 创面组织巨噬细胞标志物的mRNA表达（样本数均为6，
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；iNOS为诱导型一氧化氮合酶；4组CD206、CD11c、iNOS mRNA表达总体比较，差异均明显（F值分别为106.18、98.64、106.44，P值均<0.001）；与正常对照组比较，a P<0.01；与DTI对照组比较，b P<0.01
6 蛋白质印迹法检测正常对照组小鼠正常皮肤组织和3组DTI小鼠伤后7 d创面组织巨噬细胞标志物蛋白表达
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；DTI为深部组织损伤，iNOS为诱导型一氧化氮合酶；条带上方的 1、2、3、4分别为正常对照组、DTI对照组、2 mg/mL去铁胺组、20 mg/mL去铁胺组
7 蛋白质印迹法检测正常对照组小鼠正常皮肤组织和2组DTI小鼠创面组织伤后各时间点STAT3和IL-10蛋白表达。7A.STAT3的蛋白表达条带图；7B.IL-10的蛋白表达条带图；7C.STAT3的蛋白表达条图（样本数均为6，
注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；2组DTI小鼠伤后各时间点均与正常对照组进行比较；DTI为深部组织损伤，STAT3为信号转导及转录活化因子3、IL-10为白细胞介素10 ；条带上方的 1、2、3分别为DTI对照组伤后3、7、14 d，4为正常对照组，5、6、7分别为20 mg/mL去铁胺组伤后3、7、14 d；3组伤后3、7、14 d的STAT3、IL-10蛋白表达总体比较，差异均明显（F值分别为12.15、9.74、31.51，7.94、67.71、141.39，P值分别为0.008、0.013、0.001，0.021、<0.001、<0.001）；与正常对照组比较，a P<0.05，c P<0.01；与DTI对照组比较，b P<0.05，d P<0.01
表1 实时荧光定量反转录PCR法检测小鼠创面组织巨噬细胞特异性标志物基因的引物序列及产物大小
基因名称 引物序列（5'→3'） 产物大小 （bp） CD206 上游：GCAAGTGATTTGGAGGCT 121 下游：ATAGGAAACGGGAGAACC CD11c 上游：GAGCACCAAAGGAAATAAA 183 下游：GCACAGTAGGACCACAAGC iNOS 上游：CACCACCCTCCTCGTTC 132 下游：CAATCCACAACTCGCTCC β肌动蛋白 上游：CTGTGCCCATCTACGAGGGCTAT 155 下游：TTTGATGTCACGCACGATTTCC 注：iNOS为诱导型一氧化氮合酶 
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表2 3组DTI小鼠伤后各时间点创面愈合率比较
表2. （%，x¯±s）
组别 鼠数（只） 7 d 14 d DTI对照组 6 5.1±2.3 25.2±3.8 2 mg/mL去铁胺组 6 17.7±3.7a 51.1±3.8a 20 mg/mL 去铁胺组 6 21.5±5.0a 57.4±4.4a F值 54.22 14.99 P值 <0.001 0.005 注：DTI为深部组织损伤；处理因素主效应，F=63.91，P<0.001；时间因素主效应，F=258.87，P<0.001；两者交互作用，F=6.99，P=0.010；F值、P值为各组各时间点组间总体比较所得；与DTI对照组比较，a P<0.01
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表3 正常对照组小鼠正常皮肤组织和3组DTI小鼠伤后7 d创面组织中巨噬细胞标志物蛋白表达比较（
组别 样本数 CD206 CD11c iNOS 正常对照组 6 0.69±0.04 1.03±0.13 1.02±0.06 DTI对照组 6 0.34±0.07a 7.07±0.36a 1.61±0.04a 2 mg/mL去铁胺组 6 0.95±0.06ab 1.87±0.29ab 0.82±0.07bc 20 mg/mL去铁胺组 6 1.02±0.04ab 1.66±0.08bc 0.99±0.07bd F值 104.90 172.45 96.42 P值 <0.001 <0.001 <0.001 注：正常对照组于其他组伤后3 d取相同部位正常皮肤组织进行检测；DTI为深部组织损伤，iNOS为诱导型一氧化氮合酶；F值、P值为组间各指标总体比较所得；与正常对照组比较，a P<0.01，c P<0.05；与DTI对照组比较，b P<0.01；与2 mg/mL去铁胺组，d P<0.05
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表4 4组RAW264.7巨噬细胞培养48 h的CD206和CD86阳性细胞百分比比较（%，
组别 样本数 CD206 CD86 空白对照组 3 10.3±1.0 29.0±1.5 50 μmol/L去铁胺组 3 13.8±1.6 27.9±1.8 100 μmol/L去铁胺组 3 18.6±0.7a 17.2±1.8a 200 μmol/L去铁胺组 3 23.7±1.1a 13.2±1.1a F值 481.41 309.49 P值 <0.001 <0.001 注：F值、P值为组间各指标总体比较所得；与空白对照组比较，a P<0.01
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