Improvement of diabetic wound healing by topical application of Vicenin-2 hydrocolloid film on Sprague Dawley rats

Abstract

Background: Impaired wound healing is a debilitating complication of diabetes that leads to significant morbidity, particularly foot ulcers. The risk of developing diabetic foot ulcers for diabetic patients is 15% over their lifetime and approximately 85% of limb amputations is caused by non-healing ulcers. Unhealed, gangrenous wounds destroy the structural integrity of the skin, which acts as a protective barrier that prevents the invasion of external noxious agents into the body. Vicenin-2 (VCN-2) has been reported to contain prospective anti-oxidant and anti-inflammatory properties that enhance cell proliferation and migration. Sodium Alginate (SA) is a natural polysaccharide that possesses gel forming properties and has biodegradable and biocompatible characteristics. Therefore, the objective of this study is to evaluate the effect of SA wound dressings containing VCN-2 on diabetic wounds.

Methods: Wounds were inflicted in type-1 diabetic-streptozotocin (STZ) induced male Sprague Dawley rats. Subsequently, relevant groups were topically treated with the indicated concentrations (12.5, 25 and 50 μM) of VCN-2 hydrocolloid film over the study duration (14 days). The control group was treated with vehicle dressing (blank or allantoin). Wounded tissues and blood serum were collected on 0, 7 and 14 days prior to sacrifice. Appropriate wound assessments such as histological tests, nitric oxide assays, enzyme-linked immunosorbent assays (ELISA) and immunoblotting assays were conducted to confirm wound healing efficacy in the in vivo model. One-way Analysis of Variance (ANOVA) was used for statistical analysis.

Results: Results showed that hydrocolloid film was recapitulated with VCN-2 enhanced diabetic wound healing in a dose-dependent manner. VCN-2 reduced pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), mediators (iNOS and COX-2), and nitric oxide (NO) via the NF-κB pathway. Data suggests that the VCN-2 film facilitated healing in hyperglycemic conditions by releasing growth factors such as (VEGF and TGF-β) to enhance cell proliferation, migration, and wound contraction via the VEGF and TGF-β mechanism pathways.

Conclusions: This study's findings suggest that VCN-2 may possess wound healing potential since topical treatment with VCN-2 hydrocolloid films effectively enhanced wound healing in hyperglycemic conditions.

Keywords: Diabetic wound; Hydrocolloid film; Sodium alginate; Vicenin-2.

Fig. 1

Representative image of wounds of the normal control, diabetic control, 12.5, 25, 50 μM VCN-2 and 316 μM allantoin treated groups on day 0, 7 and 14. Result shows treatment with VCN-2 enhanced wound healing in diabetic rats as compared to diabetic rats treated with blank film (n = 6)

Fig. 2

a Images of healed wound site from a normal rat and diabetic rat with blank, VCN-2 and allantoin dressing treatment on day 7 and 14. Normal rat demonstrating complete re-epithelialization and well-form granulation tissue. Diabetic rat treated with VCN-2 12.5, 25 and 50 μM film consequence re-epithelialization and well-formed granulation tissues on day 7 and 14, (100x with H&E stain). Abbreviation (br): boundary between unhealed and healed tissue; (bv): blood vessels; (h): healed area with multiple layers of fibrous connective tissue; (e): epithelium; ie): immature epidermis; (ig): immature granulation tissue and (u): ulcer. b Histologic scoring for epidermal regeneration. c Histologic scoring for granulation tissues thickness of the rats on day 7 and 14 from 10 randomly chosen fields. Data expressed as means ± SEM (n = 6). ### p < 0.001 is diabetic group compared with normal group; *** p < 0.001 is treated group compared with diabetic group on the same day

Fig. 3

a Normal rat and diabetic rat treated with blank, VCN-2 and allantoin dressing on day 7 and 14. Diabetic rats treated with VCN-2 showed more neovascularization on day 14 compared to day 7. Wound of rat on day 14 contained more blood vessels and showed well connective tissue deposition, n = 6, 400x with H&E staining. Abbreviations: (I): Inflammatory cell and (C): Connective tissue. Representation: angiogenesis and : fibroblast cell. b Histological finding score of fibroblast proliferation; (c) angiogenesis and (d) inflammatory cells infiltrate (score from 0 to 4) from 10 randomly chosen high-power fields (× 400) from three sections in wound treated with difference films on day 7 and 14. Data are means ± SEM histological score (n = 6). ###p < 0.001, diabetes versus normal; *p < 0.05, **p < 0.01 and ***p < 0.001, treatment groups versus diabetic

Fig. 4

Nitrite levels. On 0, 7, and 14 postoperative days, animals were sacrificed and samples of excisional wounds were removed to determine nitrite levels by Griess reaction. Data are mean of three independent experiments and are expressed as mean ± SEM of nitric oxide levels (μM). ###p < 0.001 indicated statistical difference diabetic group compared to normal group; * p < 0.05, ** p < 0.01 and *** p < 0.001 indicated treatments groups with respect to diabetic group. (n = 6 animals/group/time point/experiment, ANOVA-Tukey test)

Fig. 5

a Effect of VCN-2 hydrocolloid film which was topically applied on normal and diabetic rats inflicted with excision wound for 14 days. Production of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α after different concentration of VCN-2 treatments. b Secretions of growth factors like VEGF and TGF-1β on day 14 with treatment of VCN-2 in concentration 12.5, 25, 50 μM. All values are expressed as mean ± SEM. Each group consists of six rats. ### p < 0.001, diabetic group compared to normal group; ***p < 0.001, treatments groups compared to diabetic group

Fig. 6

a Effect of VCN-2 hydrocolloid film on expression of mediators after 14 days treatment was detected by Western blotting. Down-regulation of various anti-inflammatory and up-regulation of expression of several wound healing markers in skin tissues were observed. β-actin acted as control marker. b Effect of VCN-2 film on expression of mediators after 14 days topically treatment was detected by immunoblotting. A graph represented densitometry analysis results of the effect of VCN-2 on proteins expression proinflammatory mediators (c) anti-HIF1α, (d) MMP-9, and (e) VEGF and TGF-β. All data are expressed as mean ± SEM. ### p < 0.001 represented statistical difference diabetic group compared to normal group; *** p < 0.001 represented statistical difference treatment groups compared to the diabetic group

Fig. 7

Mechanism pathways of VCN-2 to enhance excision wound healing in diabetic rats. Abbreviation: Cyclooxygenase2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of κB (IκB-α), nitric oxide (NO), nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin (IL), metalloproteinase (MMP), hypoxia inducible transcription factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β)