Recombinantly expressed rhFEB remodeled the skin defect of db/db mice

Abstract

Fibronectin (FN) and collagen are vital components of the extracellular matrix (ECM). These proteins are essential for tissue formation and cell alignment during the wound healing stage. In particular, FN interacts with collagens to activate various intracellular signaling pathways to maintain ECM stability. A novel recombinant extra domain-B fibronectin (EDB-FN)-COL3A1 fusion protein (rhFEB) was designed to mimic the ECM to promote chronic and refractory skin ulcer wound healing. rhFEB significantly enhanced cell adhesion and migration, vascular ring formation, and the production of new collagen I (COL1A1) in vitro. rhFEB decreased M1 macrophages and further modulated the wound microenvironment, which was confirmed by the treatment of db/db mice with rhFEB. Accelerated wound healing was shown during the initial stages in rhFEB-treated db/db mice, as was enhanced follicle regeneration, re-epithelialization, collagen deposition, granulation, inflammation, and angiogenesis. The wound chronicity of diabetic foot ulcers (DFUs) remains the main challenge in current and future treatment. rhFEB may be a candidate molecule for regulating M1 macrophages during DFU healing. KEY POINTS: • A recombinant protein EDB-FN-collagen III (rhFEB) was highly expressed in Escherichia coli • rhFEB protein induces COL1A1 secretion in human skin fibroblasts • rhFEB protein accelerates diabetic wound healing.

Keywords: M1 macrophages; Wound healing; db/db mice; rhFEB.

Fig. 1

Expression and purification of rhFEB protein. a Construction schematic of the recombinant pET20b-rhFEB plasmid. b Nucleic acid electrophoresis of recombinant plasmid rhFEB. M: DNA Ladder 10,000; lane 1: negative control; lane 2: the bands of rhFEB were amplified by PCR. c Electrophoretic expression of rhFEB plasmid in BL21 (DE3) by SDS-PAGE; M: 14.4–116.0 kDa protein marker; lane 1: sediment of rhFEB before induction; lanes 2–6: sediment of different BL21 (DE3)/rhFEB monoclonal after induction. d Electrophoretic diagram of purified rhEGF protein; M: 14.4–116.0 kDa protein marker. e Western blotting analysis of recombinant rhFEB. M: 10–180 kDa protein marker. f HPLC analysis of the purity of rhFEB protein

Fig. 2

Cell biological visibility of rhFEB on proliferation, adhesion, and migration. a Proliferation of rhFEB to HaCaT cells. b Migration and healing rates of HaCaT cells by rhFEB (scale bar = 100 μm). c Adhesion of rhFEB to HaCaT cells (scale bar = 100 μm). d rhFEB-induced tube formation of ECV304-eGFP cells. n = 3, mean ± SD, ** P < 0.01, *** P < 0.001 vs. control group

Fig. 3

Cell biological activity of rhFEB on COL1A1 secretion and inflammatory reaction. a Expression of COL1A1 protein in HSF cells treated with TGFβ1 and rhFEB. b Expression of inflammatory markers, TNF-α, IL-1β, Arg-1, and CD206 in M1 macrophage treated with rhFEB protein. n = 3, * P < 0.05, ** P < 0.01 vs. control group

Fig. 4

Effect of rhFEB on the skin wound healing. a Traces of wound closure were photographed at 0, 7, 10, 14, 21, and 28 days post-surgery. b Quantitative analysis of epithelial thickness at days 3, 7, 10, 14, 21, and 28 using ImageJ software. c H&E staining of wound sections at 28 days post-surgery (40 × , 200 ×). d and e Masson trichrome staining of wound sites at 28 days post-surgery(40 × , 200 ×). Arrows indicate hair follicle structure. n = 3, ** P < 0.01 vs. normal group

Fig. 5

Inflammation and immunolabelling. a Inflammation labeling of iNOS positive cells 28 days after treatment. b The immunohistochemical staining of CD31 within the wounds at 28 days post-surgery. Arrows indicate CD31 expression (scale bar = 100 μm). ** P < 0.01 vs. model group