Effects of advanced glycation end products on neutrophil migration and aggregation in diabetic wounds

Abstract

Increased accumulation of advanced glycation end products (AGEs) in diabetic skin is closely related to delayed wound healing. Studies have shown that the concentration of AGEs is elevated in the skin tissues and not subcutaneous tissues in refractory diabetic wounds, which suggests there may be a causal relationship between the two. In the present study, in vitro experiments revealed that AGEs activated neutrophils, and the migratory and adhesive functions of neutrophils decreased once AGE levels reached a certain threshold. Different levels of AGE expression differentially affected the function of neutrophils. Messenger RNA (mRNA) sequencing analysis combined with real-time polymerase chain reaction (PCR) showed that poliovirus receptor (PVR/CD155) and CTNND1, which play a role in migration- and adhesion-related signaling pathways, were decreased following AGE stimulation. Consequently, neutrophils cannot effectively stimulate the formation of the inflammatory belt needed to remove necrotic tissues and defend against foreign microorganisms within diabetic chronic wounds. In addition, this phenomenon may be related to the differential accumulation of AGEs in different layers of the skin.

Keywords: AGEs; CTNND1; diabetes; neutrophil migration; wound repair.

Figure 1

Hematoxylin and eosin (H&E) staining, advanced glycation end products (AGEs) immunohistochemical staining. (A) H&E staining. Scale bar as 0.1mm. (B) AGEs immunohistochemical staining (Red arrows indicate positive expressions). Scale bar as 0.025mm.

Figure 2

Protein expression of advanced glycation end products (AGEs) and interleukin-8 receptor A (IL8-RA) in wound skin edge by western blotting. (A) Western blotting images of advanced glycation end products (AGEs) and interleukin-8 receptor A (IL8-RA) expression. (B) Results of AGEs and IL8-RA expression in diabetic and control rat skin edge. *: P<0.05, **: P<0.01, ***: P<0.001. N = normal, D = diabetic, d = days, h = hours.

Figure 3

Interleukin-8 receptor A (IL8-RA) immunohistochemical staining to determine the formation and width of the inflammatory belt and density of neutrophils. (A) IL8-RA immunohistochemical staining of skin tissues. (B) IL8-RA immunohistochemical staining of subcutaneous tissues. (C) Comparation of inflammatory belt formation, inflammatory belt width, and density of neutrophils. The blue circle indicates abnormal neutrophil aggregation under the dermis of diabetic rat. *: P<0.05, **: P<0.01, ***: P<0.001.

Figure 4

Enzyme-linked immunosorbent assay (ELISA) experiment of rat skin and CCK-8 test of advanced glycation end product (AGE)-stimulated neutrophils. (A) Leukotriene B4 (LTB4) expression in diabetic and control rat skin at different time points after injury. (B) Myeloperoxidase (MPO) expression in diabetic and control rat skin. (C) The results of MPO activity in diabetic and control rat skin. (D) CCK-8 test for neutrophils that were stimulated by AGEs and bovine serum albumin (BSA) for 24 h. *: P<0.05, **: P<0.01, ***: P<0.001. D = days, H = hours.

Figure 5

U-slide chemotaxis experiments to observe neutrophil viability, migration, and clustering ability that was affected by advanced glycation end products (AGEs). Scale bar as 1 mm. (A) Measurement of cell migration and aggregation. The blue and red squares represent middle-range and near-range areas, respectively. The blue circle represents neutrophil aggregation (clustering). (B, G) Neutrophil clustering ability that was affected by AGEs stimulation and suspension. (C, E) Neutrophil migration ability that was affected by AGEs stimulation at different time points. (D, F) Neutrophil migration ability that was affected by AGEs resuspension. *: P<0.05, **: P<0.01, ***: P<0.001.

Figure 6

Heat map of the differentially expressed mRNA between neutrophils that were incubated with advanced glycation end products (AGEs) and neutrophils that were incubated with bovine serum albumin (BSA).

Figure 7

Results of RNA-SEQ for the differential expression of mRNA between neutrophils that were incubated with advanced glycation end products (AGEs) and neutrophils that were incubated with bovine serum albumin (BSA). (A) Volcano and scatter map of the differentially expressed genes between the two groups. (B) Significantly up- and down-regulated genes between the two groups (P < 0.05). (C) Results of Gene Ontology (GO) analysis. Red bars represent BP (biological process), green bars represent CC (cellular component) and blue bars represent MF (molecular function). (D) Results of Gene Set Enrichment Analysis (GSEA) (partial).

Figure 8

RT-PCR results for the expression of CTNND1 and poliovirus receptor (PVR) between neutrophils that were incubated with AGEs and BSA. *: P<0.05. (A) The expression of CTNND1 between the two groups. (B) The expression of PVR between the two groups.