Beneficial effects of Heparin and l Arginine on dermonecrosis effect induced by Vipera lebetina venom: Involvement of NO in skin regeneration
Introduction
Envenoming by snakes of Viperidae is characterized by multiple deleterious effects, including local and systemic bleeding. Local tissue damage induced by Viperidae venom is characterized by hemorrhage, edema, inflammation, necrosis of skeletal muscle and dermonecrosis. Intramuscular or subcutaneous injection of viperid and some elapid venoms, in human victims results in acute damage of skin, provoking hemorrhage, dermonecrosis and blistering (Mebs et al., 1983, Rucavado et al., 1998).The antivenom neutralizes systemic alterations induced by snake venom but has no effect on local tissue (Lomonte et al., 2009). Tissue damage and necrosis can lead to definitive sequelae to the bitten victims consisting in the loss of tissue mass and organ functions. Thus, alternative treatment such as therapeutic drugs might reduce local tissue damage and improved the tissue regeneration (Lomonte et al., 2009). The pathogenesis of local myonecrosis, hemorrhage and edema induced by VLV has been investigated (Hamza et al., 2010a, Hamza et al., 2010b, Sebia-Amrane and Laraba-Djebari, 2013). VLV is a complex mixture of pharmacologically active peptides and proteins both phospholipases A2, proteases, coagulant and anticoagulant enzymes (Amel and Fatima, 2015, Bennacef-Heffar and Laraba-Djebari, 2003, Boumaiza et al., 2016, Trummal et al., 2005). Hemorrhagic metalloproteinases play a relevant role in the pathogenesis of local damage after envenoming, they induce myonecrosis (Gutiérrez and Rucavado, 2000), hemorrhage (Baldo et al., 2010) and skin damage (Rucavado et al., 2000), SVMP induced hemorrhage in the dermis of mouse ear skin (Jiménez et al., 2008). The pathogenesis of necrosis is characterized by an inflammatory response, an increase in the release of cytokines TNF-α, IL-1, IL-6, IL-10 and IFN-γ and nitric oxide (Petricevich et al., 2000).
Heparin is known to be able to sequestrate growth factors and cytokines that are involved in cell proliferation and angiogenesis (Landucci et al., 2000). It was reported that the use of heparin can reduced the effects of a myotoxic PLA2 and inhibited activation of mast cells induced by venom toxins (Landucci et al., 1998). Skin tissue regeneration was also rescue by exogenous administration of l-arginine, the precursor of endogenous synthesis of NO (Oussedik-Oumehdi and Laraba-Djebari, 2012). The l-arginine administered prior to Bothrops jararacussu venom enhanced the muscle regeneration (Santo Neto et al., 2006). Numerous studies have demonstrated that supplemental l-arginine improves endothelial NO in several pathologies enhances collagen synthesis and improves wound repair (Schwentker et al., 2002).Wound healing is a dynamic and complex process of tissue repair, which involves various cellular and molecular events.
The aim of this study was, therefore, to characterize the pathogenesis of dermonecrosis induced by VLV in mice. The contribution of heparin and l-arginine to reduce the dermonecrosis and the evaluation of the role of NO in the re-epithelialization after dermonecrosis was investigated.
Section snippets
Venom and animals
Lyophilized Vipera lebetina venom (VLV) was obtained from Pasteur Institute of Algeria. Crude venom was lyophilized and kept frozen at −20 °C. When required, the venom was diluted with saline solution.
Male Balb/c mice (18 ± 2 g of body mass) were obtained from the animal breeding of the Pasteur Institute of Algeria. They were housed in controlled temperature rooms and received water and food ad libitum. All animal experiments were conducted in accordance with the actual guidelines for the care of
Dermonecrotic activity of Vipera lebetina venom: macroscopic analysis
Vipera lebetina venom induced dermonecrotic activity after intradermal injection in mice. A necrotic black areas and hemorrhage were macroscopically observed in dorsal skin (Fig. 1A). The diameters of necrotic spots were evaluated 3 days after injection. This dermonecrotic effect was dose-response and Minimal Necrotic Dose (MND) was estimated at 19 μg/20 g body mass (Fig. 1B).
Inhibition of dermonecrotic activity
The dermonecrotic response to the venom was totally abolished after preincubation with chelating agent EDTA, EGTA and 1–10
Discussion
We report in this study, several alterations induced by Vipera lebetina venom on skin characterized by edema, inflammation and dermonecrosis mainly due to snake venom metalloproteinases (Rucavado et al., 2000). Dermonecrosis was significantly reduced when the venom was incubated with pBPB and totally abolished by 1,10-phenanthroline, EDTA, and EGTA but not by PMSF indicating the requirement of zinc and calcium for SVMPs (Fox and Serrano, 2009). It was reported that the envenomation with Naja
Conclusion
Our results indicate that VLV induces hemorrhage, edema, massive inflammation and dermonecrosis. Treatment with l-arginine and heparin can promote a significant reduction of dermonecrosis; these pro-angiogenic drugs could improve skin regeneration. Then, re-epithelization process occurred, and the skin regained its normal structure by 2 weeks.
Thus, these treatments could be used in therapeutic approach for dermal lesions after viper bite. Obtained data indicate that endogenous production of NO
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