NGF accelerates cutaneous wound healing by promoting the migration of dermal fibroblasts via the PI3K/Akt-Rac1-JNK and ERK pathways

Abstract

As a well-known neurotrophic factor, nerve growth factor (NGF) has also been extensively recognized for its acceleration of healing in cutaneous wounds in both animal models and randomized clinical trials. However, the underlying mechanisms accounting for the therapeutic effect of NGF on skin wounds are not fully understood. NGF treatment significantly accelerated the rate of wound healing by promoting wound reepithelialization, the formation of granulation tissue, and collagen production. To explore the possible mechanisms of this process, the expression levels of CD68, VEGF, PCNA, and TGF-β1 in wounds were detected by immunohistochemical staining. The levels of these proteins were all significantly raised in NGF-treated wounds compared to untreated controls. NGF also significantly promoted the migration, but not the proliferation, of dermal fibroblasts. NGF induced a remarkable increase in the activity of PI3K/Akt, JNK, ERK, and Rac1, and blockade with their specific inhibitors significantly impaired the NGF-induced migration. In conclusion, NGF significantly accelerated the healing of skin excisional wounds in rats and the fibroblast migration induced by NGF may contribute to this healing process. The activation of PI3K/Akt, Rac1, JNK, and ERK were all involved in the regulation of NGF-induced fibroblast migration.

Figure 1

Wound closure after NGF treatment in rat. (a) Representative photographs of skin full-thickness excisional wounds in rat treated topically every other day with saline control or 10, 20, or 40 μg/mL of NGF for 8, 10, 12, and 14 days after wounding. (b) The rate of wound healing after different concentrations of NGF treatment. *P < 0.05, compared to saline-treated control; ^# P < 0.05, compared to 40 μg/mL NGF group; n = 7.

Figure 2

Histological structure of wounded skin sections. (a) Hematoxylin and eosin stainingof wound healing at 14 days after wounding (×40). As compared with saline-treated control, epithelial crawling (the distance between two black arrows as indicated) in NGF-treated wounds advanced more rapidly, and more granulation tissue was found in the NGF-treated wound. (b) Masson's trichrome staining of the wounded skin sections with saline or NGF treatment for 3 and 21 days after wounding (×200). There is a mild increase of collagen content (green) in NGF-treated wound tissue compared with saline-treated controls at different times after wounding.

Figure 3

NGF administration increases the level of TGF-β1- and PCNA-positive cells in skin wounds. Immunohistochemical staining for (a) TGF-β1 and (b) PCNA was performed at the indicated day after wounding (×200). (c) The optical density of TGF-β1 analyzed with Image J software. (d) The number of cells positive for PCNA staining. *P < 0.05, compared to control group.

Figure 4

The expression of CD68 and VEGF was increased following NGF treatment. Immunohistochemical staining for CD68 (a) and VEGF (b) was performed at the indicated day after wounding (×200). ((c) and (d)) The number of cells positive for CD68 or VEGF in wounds. *P < 0.05, compared to control group.

Figure 5

The effect of NGF on the proliferation and migration of human skin fibroblasts. (a) Cultured human dermal fibroblasts were incubated with different concentrations of NGF protein (0, 10, 100, and 1000 ng/mL) for 24 h, and cell proliferation was assessed by MTT assay. (b) Wound-healing assay of cultured human skin fibroblasts treated with saline or 100 ng/mL NGF for 24 h. (c) The migration rate of cultured fibroblasts after wounding is expressed as migration distance/time (μm/h). *P < 0.05, compared to control group.

Figure 6

The effect on human skin fibroblast migration of blocking JNK, ERK, or PI3K/Akt pathways with specific inhibitors. ((A1), (B1), and (C1)) The levels of Akt, JNK, and ERK activity were all enhanced after treatment with 100 ng/mL of NGF for 5, 15, or 30 min. ((A2), (B2), and (C2)) The activity of Akt, JNK, or ERK following 5 min of NGF treatment was abolished by the corresponding specific inhibitor: LY294002 (10 μM, LY is the abbreviation for LY294002), SP600125 (10 μM, SP is the abbreviation for SP600125), or PD98059 (10 μM, PD is the abbreviation for PD98059). ((A3), (A4), (B3), (B4), (C3), and (C4)) The NGF-induced migration of human skin fibroblasts induced by NG was significantly impaired after incubation with LY294002 (10 μM), SP600125 (10 μM), or PD98059 (10 μM). *P < 0.05, compared to the control group. ((D1) and (D2)) Active Rac1 was pulled down and then detected by Western blotting. *P < 0.05, compared to untreated control group.