创缘铁过载的临床样本验证与探讨铁过载对小鼠创面愈合的影响。

(1)收集符合入选标准的2021年1月至2022年2月上海交通大学医学院附属第九人民医院门急诊及住院患者各期创面清创时的创缘组织共36份，按创面形成时间分为3组，分别为<1个月组、1~3个月组、>3个月组，每组12份。观察3组创缘色素沉着情况。取距离创缘前后各5 mm范围内的皮肤全层组织，通过电感耦合等离子体发射光谱检测创缘铁离子含量，苏木精-伊红染色及普鲁士蓝染色观察创缘组织炎症细胞分布与铁沉积部位及铁沉积范围。(2)将48只6~8周龄无特定病原体级雄性C57BL/6小鼠按随机数字表法分为对照组与铁过载组，每组24只。铁过载组小鼠腹腔注射于避光条件下配置而成含铁量为0.1 mg/mL的柠檬酸铁铵溶液，每次注射2 mL/只，每间隔3 d进行1次注射，共注射8次。对照组小鼠腹腔注射0.9%氯化钠溶液，注射含量及注射次数同铁过载组。注射结束后1 d，2组小鼠1%戊巴比妥钠腹腔注射(5 μL/g)麻醉，电动剃毛刀剃除小鼠背部区域毛发，在背部建立1个1.5 cm×1.5 cm的全层创面。以建立创面当日定为造模成功即刻。造模成功后3、7、14 d，观察2组小鼠创缘色素沉着与创面愈合情况，并比较小鼠创面愈合率；2组造模成功即刻，造模成功后3、7、14 d各取6只小鼠脱颈处死并切去小鼠全层皮肤创缘组织，检测铁离子含量；苏木精-伊红染色与普鲁士蓝染色观察创缘组织炎症细胞分布与铁沉积部位及铁沉积范围。数据行单因素方差分析和LSD- t检验。

(1)<1个月组创缘色红伴随局部炎症状态，随着创面形成时间的延长，1~3个月组、>3个月组创缘表现出较为明显的色素沉着；<1个月组、1~3个月组、>3个月组创缘铁离子含量分别为(35.24±2.82)、(70.02±6.16)、(106.90±9.62) μg/g，3组总体比较差异有统计学意义(F＝27.82，P<0.05)。苏木精-伊红染色可见1~3个月组、>3个月组创面细胞核染为深蓝紫色的淋巴细胞聚集明显，局部可见含有红染噬酸性颗粒的巨噬细胞，与<1个月组创面相比，创面炎症细胞积聚明显；普鲁士蓝染色可见，随着创面形成时间的延长，创缘铁沉积加重，局部可见铁沉积带，且铁沉积部位与炎症细胞分布部位重合。(2)造模成功后3 d，铁过载组即出现较对照组更为明显的色素沉着，且随着时间延长色素沉着加深，肉眼观察见铁过载组小鼠创面愈合情况较对照组缓慢；造模成功后3、7、14 d，铁过载组小鼠创面愈合率分别为(40.33±4.55)%、(51.75±4.72)%、(59.75±3.68)%，与对照组[(49.83±3.76)%、(65.25±4.11)%、(90.59±5.25)%]比较，差异均有统计学意义(t＝3.943、4.961、10.570，P<0.05)；造模成功后14 d，对照组创面已基本愈合，而铁过载组小鼠创面仍未完全愈合；造模成功即刻，造模成功后3、7、14 d，铁过载组小鼠创缘铁离子含量分别为(90.35±1.84)、(110.80±3.69)、(125.50±1.85)、(312.10±4.83) μg/g，与对照组[(17.67±0.76)、(24.40±1.08)、(40.37±1.63)、(63.51±2.58) μg/g]比较，差异均有统计学意义(t＝36.53、22.50、34.55、45.42，P<0.05)；造模成功即刻，造模成功后3、7、14 d，对照组无明显淋巴细胞及巨噬细胞聚集，创面表皮及真皮层未见明显的铁沉积带，仅局部血管周围可见少量蓝色铁沉积；铁过载组小鼠创面真皮层可见大量铁沉积，且随创面时间延长出现条状铁沉积带，其周围可见炎症细胞聚集，较对照组明显。

创缘局部铁过载随创面形成时间的延长呈加重趋势，是创面难愈的重要原因之一。

To verify the clinical samples of iron overload at the wound edge and investigate the effect of iron overload on wound healing in mice.

(1) A total of 36 wound marginal tissues from outpatients and inpatients who met the criteria in Shanghai Ninth People′s Hospital, Shanghai Jiaotong University School of Medicine at all stages of wound debridement from January 2021 to February 2022 were collected and divided into 3 groups according to the time of wound formation, <1 month group 1 to 3 months group and >3 months group, with 12 in each group. The pigmentation of the trabecular margin was observed in the three groups. The whole skin tissue within 5 mm from the front and back of the wound edge was taken, the iron ion content of the wound edge was detected by inductively coupled plasma emission spectroscopy, and the distribution of inflammatory cells in the tissue of the lesion was observed by hematoxylin-eosin staining and Prussian blue staining with the site and extent of iron deposition. (2) Forty-eight 6-8 week-old male C57BL/6 mice without specific pathogens were divided into control group and iron overload group according to the random number table method, with 24 mice in each group. The mice in the iron-overloaded group were injected intraperitoneally with ferric ammonium citrate solution at 0.1 mg/mL under light-protected conditions at an interval of every 3 d for 8 injections. The mice in the control group were injected intraperitoneally with 0.9% sodium chloride solution, and the injection content and number of injections were the same as those of the iron overload group. One day after the end of injection, mice in the 2 groups were anesthetized with 1% sodium pentobarbital intraperitoneally (5 μL/g), and the hair in the dorsal region of the mice was shaved with an electric shaver to create a 1.5 cm×1.5 cm full-length wound on the back. The day when the wound was created was defined as the immediately successful modeling. At 3, 7, and 14 d after successful modeling, the pigmentation and healing of the wound margins of the 2 groups were observed, and the healing rates were compared. Six mice from each group were necropsyed and excised from the wound margins immediately after successul modeling and 3, 7, and 14 d after successful modeling, and the iron ion content was measured; hematoxylin-eosin staining and Prussian blue staining were used to observe the distribution of inflammatory cells in the tissue of the lesion and the site and extent of iron deposition. The data were analyzed by one-way analysis of variance and LSD-t test.

(1) In the <1 month group, the red color of the trabecular wound edges was accompanied by a local inflammatory state, and with the prolongation of the trabecular presence, the trabecular wound edges of the 1 to 3 months group and >3 months group showed more obvious pigmentation. The iron ion content of the trabecular wound edges in the <1 month group, 1 to 3 months group and >3 months group was (35.24±2.82), (70.02±6.16) and (106.90±9.62) μg/g, respectively, the difference was statistically significant (F=27.82, P<0.05). Hematoxylin-eosin staining showed significant aggregation of lymphocytes with nuclei stained dark blue-purple on the trabeculae of the 1 to 3 months group and >3 months group, and locally visible macrophages containing red-stained acidophilic granules, and significant accumulation of inflammatory cells on the trabeculae compared with the trabeculae of the <1 month group. Prussian blue staining showed that iron deposition at the wound edge increased with the prolongation of wound existence, and iron deposition bands were visible locally, and the sites of iron deposition overlapped with the distribution of inflammatory cells. (2) At the time of 3 d after successful modeling, pigmentation was more obvious in the iron-overload group than in the control group, and the pigmentation deepened with time. The wounds healing of the mice in the iron overloaded group was slower than that in the control group. At 3, 7, and 14 d after successful modeling, the wound healing rates in the iron overloaded group were (40.33±4.55)%, (51.75±4.72)%, and (59.75±3.68)%, compared with the control group [(49.83±3.76)%, (65.25±4.11)%, and (90.59±5.25)%], the differences were statistically significant (t= 3.943, 4.961, 10.570; P< 0.05); 14 d after successful modeling, the wounds in the control group had basically healed, while the wounds in the iron overloaded group still had not completely healed. Immediately after successful modeling and 3, 7, and 14 d after successful modeling, the iron ion content at the wound edge of the mice in the iron overload group was (90.35±1.84), (110.80±3.69), (125.50±1.85), and (312.10±4.83) μg/g, respectively, compared with the control group [(17.67±0.76), (24.40±1.08), (40.37±1.63), (63.51±2.58) μg/g], the differences were statistically significant (t=36.53, 22.50, 34.55, 45.42; P<0.05). Immediately successful modeling and 3, 7, 14 day after successful modeling, in the control group, there was no obvious lymphocyte and macrophage aggregation, and no obvious iron deposition bands were seen in the epidermis and dermis of the wound, and only a small amount of blue iron deposition was seen around the local blood vessels; in the iron overload group, a large amount of iron was deposited in the dermis of the wound, and stripes of iron deposition bands appeared with the prolongation of the wound, and inflammatory cells aggregation was seen around them, which was obvious compared with the control group.

Local iron overload at the wound edge tends to increase with the duration of wound presence and is an important reason for the difficulty of wound healing.