Oral administration of linoleic acid induces new vessel formation and improves skin wound healing in diabetic rats

Introduction Impaired wound healing has been widely reported in diabetes. Linoleic acid (LA) accelerates the skin wound healing process in non-diabetic rats. However, LA has not been tested in diabetic animals. Objectives We investigated whether oral administration of pure LA improves wound healing in streptozotocin- induced diabetic rats. Methods Dorsal wounds were induced in streptozotocin-induced type-1 diabetic rats treated or not with LA (0.22 g/kg b.w.) for 10 days. Wound closure was daily assessed for two weeks. Wound tissues were collected at specific time-points and used to measure fatty acid composition, and contents of cytokines, growth factors and eicosanoids. Histological and qPCR analyses were employed to examine the dynamics of cell migration during the healing process. Results LA reduced the wound area 14 days after wound induction. LA also increased the concentrations of cytokine-induced neutrophil chemotaxis (CINC-2 alpha beta), tumor necrosis factor-alpha (TNF-alpha) and leukotriene B-4 (LTB4), and reduced the expression of macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1). These results together with the histological analysis, which showed accumulation of leukocytes in the wound early in the healing process, indicate that LA brought forward the inflammatory phase and improved wound healing in diabetic rats. Angiogenesis was induced by LA through elevation in tissue content of key mediators of this process: vascular-endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2). Conclusions Oral administration of LA hastened wound closure in diabetic rats by improving the inflammatory phase and angiogenesis1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP446562/2014-9sem informação2012/10653-9; 2013/06810-

Oral Administration of Linoleic Acid Induces New Vessel Formation and Improves Skin Wound Healing in Diabetic Rats.

Impaired wound healing has been widely reported in diabetes. Linoleic acid (LA) accelerates the skin wound healing process in non-diabetic rats. However, LA has not been tested in diabetic animals.We investigated whether oral administration of pure LA improves wound healing in streptozotocin-induced diabetic rats.Dorsal wounds were induced in streptozotocin-induced type-1 diabetic rats treated or not with LA (0.22 g/kg b.w.) for 10 days. Wound closure was daily assessed for two weeks. Wound tissues were collected at specific time-points and used to measure fatty acid composition, and contents of cytokines, growth factors and eicosanoids. Histological and qPCR analyses were employed to examine the dynamics of cell migration during the healing process.LA reduced the wound area 14 days after wound induction. LA also increased the concentrations of cytokine-induced neutrophil chemotaxis (CINC-2αβ), tumor necrosis factor-α (TNF-α) and leukotriene B4 (LTB4), and reduced the expression of macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1). These results together with the histological analysis, which showed accumulation of leukocytes in the wound early in the healing process, indicate that LA brought forward the inflammatory phase and improved wound healing in diabetic rats. Angiogenesis was induced by LA through elevation in tissue content of key mediators of this process: vascular-endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2).Oral administration of LA hastened wound closure in diabetic rats by improving the inflammatory phase and angiogenesis

Experimental protocol.

<p>Experimental protocol.</p

Transcription factors activation.

<p>NF-KB and AP-1 activation in wound tissues from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA). Results are presented as mean ± SD. D (5 animals) and DLA (8 animals). (*) Indicates significant difference between D and DLA rats (AP-1 1 h–p = 0.02; AP-1 24hs–p = 0.001)</p

Myeloperoxidase and CINC-2αβ contents.

<p>Myeloperoxidase (MPO) activity (1 hour), mRNA expression (1 h, 1, 3 and 7 days) and CINC-2αβ concentration (1 h) in wound tissue. Results are presented as mean ± SD. D (6 rats) and DLA (6 rats). (*) Indicates significant differences between D and DLA rats (MPO activity–p = 0.02; mRNA expression 1h 0.006; mRNA 1 day–p = 0.03; CINC-2αβ –p = 0.04).</p

Time course of wound healing and glycemia.

<p><b>(a)</b> Macroscopic and time course of wound closure in control (C) and diabetic rats (D). (*) Indicates significant differences among C versus D (p = 0.006) <b>(b)</b> Macroscopic and time course of wound closure in diabetic (D) and diabetic rats treated with LA (DLA) (*) Indicates significant differences between D and DLA (p = 0.02). Representative photos of the wound tissue obtained during the time-course of 18 days. Results are presented as mean ± SD. D (5 rats) and DLA (9 rats). <b>(c)</b> Glycemia of rats during the wound healing process: (D) diabetic; (DLA) diabetic rats treated with LA. Dashed line indicates the mean of glycemia in control rats.</p

Macrophages cell markers expression.

<p><b>(a)</b> mRNA expression of F4/80 in wound tissue 1 hour and 1–7 days after wounding. Results are presented as mean ± SD. D (5 rats) and DLA (10 rats). <b>(b)</b> mRNA expression of MIP-1, MCP-1 and iNOS in wound tissue from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA). Results are presented mean ± SD. D (5 animals) and DLA (9 animals). (*) Indicates significant differences between D and DLA rats (p<0.001)</p

Cytokines production during wound healing.

<p>CINC-2α, IL-6, IL-1β and TNF-α concentrations in wound tissue from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA). Results are presented mean ± SD. D (5 animals) and DLA (6 animals). (*) Indicates significant differences between D and DLA rats (TNF-α 3d –p<0,05; TNF-α 7d –p<0.01)</p

Histological analysis and angiogenic growth factors expression in wound tissue.

<p><b>(a)</b> Samples were isolated from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA) at the 1^st, 3^rd, 7^th and 14^th days after wounding. <b>(b)</b> Representative new vessel formation in wound tissue from the D and DLA groups. Samples were collected on the 7^th day after wounding. <b>(c)</b> Vessels quantification. Results are presented as mean ± SD. D (4 rats) and DLA (5 rats). (*) Indicates significant difference between D and DLA (p = 0.0001). <b>(d)</b> TGF-β mRNA expression and VEGF concentration in wound tissues from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA). Results are presented as mean ± SD. D (9 rats) and DLA (4 rats). (*) Indicates significant difference between D and DLA (VEGF–p<0.01). <b>(e)</b> eNOS and ANGPT-2 mRNA expression in wound tissues from diabetic rats (D) and diabetic rats treated with linoleic acid (DLA). Results are presented as mean ± SD. D (9 rats) and DLA (4 rats). (*) Indicates significant difference between D and DLA (ANGPT-2 –p = 0.01). V: vessel. DE: derm. Objective 10X.</p