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Effect of blocking receptor for advanced glycation end product pathway on inflammation of diabetic wound healing in mice

Objective 
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. 
 
 
Methods 
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. 
 
 
Results 
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. 
 
 
Conclusions 
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. 
 
 
Key words: 
Mice; Diabetes mellitus; Ulcer; Inflammation; Wound healing; Receptor for advanced glycation end product