Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats Mahendra Ram, Vishakha Singh, Sanjay Kumawat, Vinay Kant, Surendra Kumar Tandan, Dinesh Kumar ⁎ Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122 (U.P.), India abstract article info Article history: Received 25 May 2015 Received in revised form 29 November 2015 Accepted 30 November 2015 Available online 8 December 2015 Bilirubin has shown cutaneous wound healing potential in some preliminary studies. Here we hypothesize that bilirubin facilitates wound healing in diabetic rats by modulating important healing factors/candidates and anti- oxidant parameters in a time-dependent manner. Diabetes was induced in male Wistar rats by streptozotocin. In all diabetic rats wounds were created under pentobarbitone anesthesia. All the rats were divided into two groups, of which one (control) was treated with ointment base and other with bilirubin ointment (0.3%). Wound closer measurement and tissue collection were done on days 3, 7, 14 and 19 post-wounding. The relative expressions of hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 alpha (SDF-1α), transforming growth factor- beta 1 (TGF-β 1 ) , tumor necrosis factor-α (TNF-α) and interlukin-10 (IL-10) mRNA and proteins and the mRNA of interlukin-1 beta (IL-1β) and matrix metalloprteinase-9 (MMP-9) were determined in the wound tissues. CD-31 staining and collagen content were evaluated by immunohistochemistry and picrosirius red staining, respectively. Histopathological changes were assessed by H&E staining. The per cent wound closer was significantly higher from day 7 onwards in bilirubin-treated rats. HIF-1α, VEGF, SDF-1α, TGF-β 1 , IL-10 mRNA and protein levels were significantly higher on days 3, 7 and 14 in bilirubin-treated rats. The mRNA expression and protein level of TNF-α and the mRNA of IL-1β and MMP-9 were progressively and markedly reduced in bilirubin-treated rats. The collagen deposition and formation of blood vessels were greater in bilirubin-treated rats. Bilirubin markedly facilitated cutaneous wound healing in diabetic rats by modulating growth factors, cytokines, neovasculogenesis and collagen contents to the wound site. Topical application of bilirubin ointment might be of great use in cutaneous wound healing in diabetic patients. © 2015 Elsevier B.V. All rights reserved. Keywords: Bilirubin Cytokines Growth factors Angiogenesis Diabetic rats Wound healing 1. Introduction The primary function of skin is to serve as a vindicatory barrier against any harmful environmental insult that may result in loss of the integrity of skin (such as wound) and ultimately lead to morbidity or even death [1]. Healing is an integral part of recovery of damaged skin [2]. Optimum healing of a cutaneous wound requires a well- orchestrated integration of complex cellular and molecular events of cell migration, proliferation, extracellular matrix deposition and remod- eling [3]. Several cellular and molecular biological studies have demon- strated that many cytokines, growth factors, enzymes and cells are closely involved in the wound-healing process to complete normal tis- sue repair after damage [1]. The cytokines and growth factors are useful candidates and markers for determination of wound vitality and age [4]. There are a number of factors that adversely affect wound healing process, among them diabetes mellitus is one of the most inextricable and complicated impediment, wherein development of chronic non- healing foot ulcerations is one of the most serious and debilitating com- plications. The most common complications associated with delayed wound healing in diabetes are: reduction in chemotactic and phagocytic activities of neutrophils [5,6], decreased angiogenesis [7], decreased vasculogenesis due to decreased number of endothelial progenitor cells (EPC) [8,9], decreased endothelial nitric oxide synthase (eNOS) ac- tivity [10], increased oxidative stress [11], reduced number of growth factors like platelet derived growth factor (PDGF) [12], vascular endo- thelial growth factor (VEGF) [13], etc. The impairment in the healing of wounds in diabetic mice has been attributed to reduced angiogenesis, granulation tissue formation, decreased collagen formation and delay in transformation of fibroblasts to myofibroblasts [14]. Further chronic wounds seen in diabetic patients get stuck in the inflammatory phase featured by continuing influx of neutrophils that release cytotoxic en- zymes, free radicals and inflammatory mediators that cause extensive collateral damage to surrounding tissue. These destructive processes International Immunopharmacology 30 (2016) 137–149 ⁎ Corresponding author at: Division of Pharmacology & Toxicology, Indian Veterinary Research Institute, Izatnagar (U.P.) Pin 243 122, India. E-mail addresses: dineshks17@gmail.com, dineshks17@ivri.res.in (D. Kumar). http://dx.doi.org/10.1016/j.intimp.2015.11.037 1567-5769/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp