通过观察阻断糖基化终末产物受体(RAGE)效应后对小鼠糖尿病创面炎性细胞浸润及炎症因子分泌情况的影响，推测RAGE对创面炎症反应的可能影响和效应环节。

96只雄性8周龄C57BL/6J小鼠随机分成糖尿病组(n＝72)和正常对照组(N组)(n＝24)。糖尿病组小鼠多次小剂量链脲佐菌素腹腔注射，诱导糖尿病小鼠模型，并运用灭菌9 mm直径环钻于所有小鼠背部，制造全层皮肤缺损创面模型，伤后N组小鼠创面局部运用0.9%氯化钠溶液20 μL，糖尿病组随机分成3组，糖尿病空白对照组(C组)局部外用0.9%氯化钠溶液20 μL、糖尿病IgG对照组(I组)局部外用兔IgG 20 μL、糖尿病RAGE干预组(R组)创面局部外用兔抗鼠RAGE多克隆抗体20 μL。隔日分别给予上述对应的药物，直至伤后7 d。致伤当日及伤后1、3、7 d取材，制备标本待用，苏木精-伊红(HE)染色观察各组创面愈合的情况及愈合过程中中性粒细胞的浸润变化，免疫组织化学染色观察RAGE表达和巨噬细胞的浸润，酶联免疫吸附测定(ELISA)检测创周组织炎症因子肿瘤坏死因子-α(TNF-α)、γ干扰素、白细胞介素-1α(IL-1α)、白细胞介素-6(IL-6)、髓过氧化物酶(MPO)含量；透射电子显微镜下观察创面巨噬细胞吞噬现象的情况。对数据行方差分析、t检验。

N组和C组小鼠皮肤组织中均可见RAGE阳性表达，且C组小鼠创面表达明显强于N组，RAGE主要分布于细胞表面。伤后14 d，R组创面愈合率为(89.65±1.49)%，明显高于C组(76.21±0.69)%与Ⅰ组(78.81±1.01)%，差异均有统计学意义(t＝18.771、14.026，P值均小于0.05)。伤后1 d，N组TNF-α、IL-1α、IL-6水平均明显高于C组，差异均有统计学意义(t＝5.871、34.005、17.698，P值均小于0.05)；R组TNF-α、IL-1α、IL-6水平均明显高于C组，差异均有统计学意义(t＝10.275、27.295、18.591，P值均小于0.05)。伤后3 d，N组TNF-α、IL-1α、IL-6水平均明显低于C组，差异均有统计学意义(t＝24.758、26.674、27.275，P值均小于0.05)；R组TNF-α、IL-1α、IL-6水平均明显低于C组，差异均有统计学意义(t＝17.819、15.584、29.360，P值均小于0.05)。伤后1 d，MPO含量N组(444.71±28.27) pg/mL和R组(476.65±38.76) pg/mL均明显高于C组(173.73±28.27) pg/mL，差异均有统计学意义(t＝11.759、10.937，P值均小于0.05)；相反，伤后3 d，MPO含量N组(214.05±46.55) pg/mL和R组(267.07±52.49) pg/mL均明显低于C组(491.90±38.21) pg/mL，差异均有统计学意义(t＝7.991、5.998，P值均小于0.05)。伤后3 d，中性粒细胞个数，相比于N组(35.67±5.03)个和R组(36.67±4.16)个，C组创面创周中性粒细胞数量(83.00±4.58)个明显增多，差异均有统计学意义(t＝10.833、5.376，P值均小于0.05)。伤后1、3 d，CD68^＋细胞数量计数N组(76.60±9.07)、(121.40±9.96)个和R组(51.00±8.92)、(89.60±7.89)个均明显多于C组(15.00±4.93)、(50.00±8.40)个，差异均有统计学意义(P值均小于0.05)。伤后3 d，N组和R组均可探测到典型巨噬细胞吞噬中性粒细胞现象存在，而C组和R组未及此现象。

糖尿病创面环境中，中性粒细胞的消退、早期巨噬细胞的浸润及吞噬均受到了抑制，伤后炎症因子分泌表现为早期不足和消退迟滞的现象，且均呈现RAGE途径依赖性，提示糖尿病创面早期炎症反应失调，创面迁延难愈与RAGE介导效应相关。

To investigate the influence of blocking receptor for advanced glycation end product(RAGE) pathway on inflammatory cells infiltration and inflammatory cytokines secretion on diabetic wound in mice, and to explore the effects of RAGE on inflammatory response of wound healing and the possible action pathway.

Ninty-six C57BL/6J mice (male, 8 weeks old) were divided into diabetic groups (n=72) and normal group (group N) (n=24) randomly. Diabetic mice were induced by streptozocin multiple intraperitoneal injection. One full-thickness excisional wound (9 mm diameter) was created by a sterilized punch for all the mice. Diabetic mice were divided into 3 groups in which different topical treatments were applied to the wounds: anti-RAGE antibody for 20 μL applied topically (group R), rabbit IgG for 20 μL applied topically (group I) and saline for 20 μL applied topically (group C), while normal mice group N were applied with saline for 20 μL topically. All treatments were applied every 2 days from days 0-7 after wounding. The wound and surrounding tissues (a margin of approximately 5 mm into the unwounded skin) from animals in each group were excised on 1, 3, and 7 days after wounding. Hematoxylin and eosin (HE) staining was used to observe granulation tissues and neutrophils infiltration. Immunohistochemistry was utilized to investigate expression of RAGE and macrophage infiltrations. Inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ, interleukin-1α(IL-1α), interleukin-6(IL-6) and myeloperoxidase deficiency(MPO) were analyzed by enzyme-linked immunosorbent assay (ELISA). Macrophage phagocytosis was observed through transmission electron microscopy. All data were analyzed with one-way ANOVA and the Student′s t-test.

RAGE were expressed in both normal and diabetic mice skin tissue which had a higher level in diabetic mice. On day 14, the rate of wound healing of group R was (89.65±1.49)%, which was higher than group C(78.81±1.01)%, group I (78.81±1.01)%, (t=18.771, 14.026, with P values below 0.05). On day 1, the contents of TNF-α, IL-1α, IL-6 in group N and R [(444.71±28.27) pg/mL, (476.65±38.76)pg/mL] were higher than group C (173.73±28.27) pg/mL, (t=5.871 and 10.275, 34.005 and 27.295, 17.698 and 18.591, with P values below 0.05). On day 3, the contents of TNF-α, IL-1α, IL-6 in group N and R [(1214.05±46.55) pg/mL, (267.07±52.49) pg/mL] were lower than group C (t=24.758 and 17.819, 26.674 and 15.584, 27.275 and 29.360, with P values below 0.05). On day 1, the contents of MPO in group N and R [(444.71±28.27) pg/mL, (476.65±38.76) pg/mL] were higher than group C (173.73±28.27) pg/mL (t=11.759, 10.937, with P values below 0.05); while on day 3, the contents of MPO in group N and R [(214.05±46.55) pg/mL, (267.07±52.49) pg/mL] were lower than group C (491.90±38.21) pg/mL (t=7.991, 5.998, with P values below 0.05). On day 3, neutrophils infiltration in group N and R were 35.67±5.03 and 36.67±4.16, when group C 83.00±4.58 was obvious higher (t=10.833, 5.376, with P values below 0.05). The number of macrophages infiltration on day 1 and 3, group N and R were 76.60±9.07, 121.40±9.96 and 51.00±8.92, 89.60±7.89, while group C 15.00±4.93, 50.00±8.40 was obvious lower (with P values below 0.05). Phagocytosis on the wound edge was observed in day 3 in group N and R, while not in group C and I.

Subsiding of neutrophils, macrophage infiltration and phagocytosis in the inflammatory stage are all been inhibited on diabetic wound. Secretion of pro-inflammatory factors are insufficient in early stage and subsided lingeringly. All of the phenomenon showed RAGE pathway depending manner. It suggests inflammatory disorders and impaired healing of diabetic wound are both closely related to RAGE pathway.