Improvement of diabetic wound healing by topical application of acidified nitrite in type 2 diabetic rats

Improvement of diabetic wound healing by topical application of acidified nitrite in type 2 diabetic rats
Hamideh Afzali,¹ Mohammad Khaksari Hadad,² Reza Norouzirad,³ Sajad Jeddi,⁴ Khosrow Kashfi,⁵ Asghar Ghasemi,^6,*
1. Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2. Department of Physiology and Pharmacology, Afzali Pour Medical Faculty, Kerman University of Medical Sciences, Kerman, Iran
4. Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5. Department of Molecular, Cellular and Biomedical Sciences, Sophie Davis School of Biomedical Education, City University of New York School of Medicine, New York, NY 10031, USA
6. Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Introduction: Impaired nitric oxide (NO) production in the skin is a major contributor to the delayed wound healing in type 2 diabetes (T2D). The present study was designed to determine effects of acidified nitrite on wound closure as well as inflammatory and antioxidants markers in wound tissue of rats with T2D.
Methods: Control and diabetic Wistar rats were divided into four groups: Untreated control (C), acidified nitrite-treated control (CN), untreated diabetes (D), and acidified nitrite-treated diabetes (DN). T2D was induced using a high-fat diet followed by a low-dose of streptozotocin. A full-thickness excisional skin wound was made 28 days after the induction of diabetes on the back of rats. Acidified nitrite (20 mg) was applied once daily from day 3 to day 28 after wounding and wounds were photographed to evaluate CT50% (time taken for 50% closure of a wound), and wound closure percentage. Rats were sacrificed on days 3, 7, 14, 21, and 28 after wounding and wound tissue levels of nitrite + nitrate (NOx), tumor necrosis factor alpha (TNF-), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), total antioxidant capacity (TAC), and reduced glutathione (GSH) as well as activities of superoxide dismutase (SOD) and catalase were measured.
Results: Type 2 diabetic rats had delayed wound healing as demonstrated by ~2–fold higher CT50% and lower wound closure percentage compared to the controls. Compared with untreated diabetic rats, CT50% was significantly lower in acidified nitrite-treated diabetic rats (5.1 vs. 8.0 days, p<0.001). At days 3 and 7 after wounding, diabetic rats had lower levels of TNF-α, iNOS, and PGE2 within the wound tissue, whereas at day 14 after wounding, the parameters peaked in diabetic but not the control rats. These data indicate a delay in the inflammatory response at days 3 and 7 after wounding and persistence of the inflammatory response at day 14 after wounding in the diabetic rats, which is partially restored to control levels following topical application of acidified nitrite. A lower levels of antioxidant markers in diabetic wound, as observed in most time points. Levels of total antioxidant capacity (TAC) and reduced glutathione (GSH) as well as catalase activity (but not SOD activity) were lower in type 2 diabetic rats at day 7 after wounding. Acidified nitrite increased SOD activity, decreased GSH level and had no effect on catalase activity and TAC levels in treated diabetic rats at day 7 after wounding.
Conclusion: Acidified nitrite accelerated wound healing in rats with T2D by restoring delayed inflammatory response and augmentation of antioxidant defense and may be a simple and non–expensive therapy to improve impaired wound healing in T2D.
Keywords: Acidified nitrite; Antioxidants, Diabetic wound; Inflammation; Nitric oxide; Type 2 diabetes.