In vivo and in vitro antileishmanial activity of Bungarus caeruleus snake venom through alteration of immunomodulatory activity
Graphical abstract
Introduction
Leishmaniasis (kala-azar) is a group of disease prevalent in tropical and sub-tropical countries caused by protozoan parasites belonging to the genus leishmania. The disease is of different types (cutaneous, mucocutaneous and visceral leishmaniasis), among which visceral leishmaniasis is fatal. More than 350 million people are affected by leishmaniasis over 88 countries (Banerjee et al., 2008). WHO estimated 500,000 new cases of visceral leishmaniasis globally each year and one tenth of this is fatal (WHO, 2005). The recommended drugs for leishmaniasis are pentavalent antimonials among which sodium stibogluconate (Pentosam®) and meglumine antimoniate (Glucantime®) that have been used over the last 50 years (Kolodziej and Kiderlen, 2005). These drugs possess several side effects including renal toxicity and cardiotoxicity. The problem is aggravated by the emergence of drug resistant strains. The new line of drugs like amphotericin B, miltefosine and paromomycin are expensive and having low bioavailability (Haldar et al., 2009). Search for low cost, less toxic antileishmanial drugs with better efficacy are ventured throughout the world especially from natural products.
It has been reported that snake/scorpion venom possessed antileishmanial activity. Scorpion venom Tityus discrepans showed in vitro leishmanicidal activity (Borges et al., 2006). Bothrops moojeni snake venom killed Leishmania sp (Tempone et al., 2001). Venoms from Cerastes, Vipera and Naja snake species had inhibitory effect on Leishmania donovani infantum (Fernandez-Gomez et al., 1994). Antileishmanial activity of three species of Crotalus durissus venom (Passero et al., 2007) and five South American rear-fanged snake venoms on Leishmania major was reported (Peichoto et al., 2011).
Macrophages play an important role in leishmaniasis not only as host cells, but activated macrophages show immunomodulatory activity against pathogens. Activated macrophages produce toxic molecules during innate and adaptive immune responses (Kolodziej and Kiderlen, 2005), that involve morphological and functional changes like cell spreading, secretion of cytokines and augmented phagocytosis (Binker et al., 2007). Nitric oxide and reactive oxygen species produced from activated macrophages show important biological functions like tumoricidal effects, cytotoxic activity against several pathogens. Presence of pathogen or their secreted products increase the production of IFN-γ by the host that later interacts with macrophage through IFN-γ receptors and thereby activates macrophages (Binker et al., 2007). IFN-γ production by leishmania infected macrophages is important for leishmanicidal activity. Increased production of TNF-α is a hallmark of macrophage activation (Kolodziej and Kiderlen, 2005).
Venom of Bungarus caereleus, a common venomous snake found in the Indian subcontinent, North Asia, Sri Lanka caused neuro-muscular blockade, resulting in paralysis. A neurotoxin purified from B. caereleus caused blockade in isolated frog rectus abdominis muscle preparation. It can be used as a therapeutic agent in the treatment of neuromuscular disorder (Mirajkar et al., 2006). There is no report on the anti parasitic activity of Indian krait venom. In this study, we have evaluated the effect of Indian common krait venom (BCV) on leishmanial parasite L. donovani promastigote, amastigote in in vitro and in vivo experimental models.
Section snippets
Snake venom
Lypholized Bungarus caeruleus venom was purchased from Calcutta Snake Park, Kolkata, India and was kept in desiccator for further use.
Animals used
BALB/c mice (20 ± 2 g) and Golden hamster (75 ± 5 g) of either sex were procured from Institute’s animal house. Animals were maintained at 25 ± 2 °C, 12 h day/night cycle, fed a standard pellet diet and provided water ad libitum. Experiments using animals conform to the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH
Effect of BCV on promastigote of L. donovani
BCV showed a dose dependent cytotoxic effect on L. donovani promastigotes. BCV (1–50 μg/ml) treatment increased cell death of promastigotes by 15 ± 0.91–60 ± 1.95% (Fig. 1). IC50 value of BCV on L. donovani promastigote was calculated to be 14.5 μg/ml for 48 h.
Effect of BCV on macrophage
BCV (1–20 μg/ml) did not produce any cytotoxicity on murine peritoneal macrophages. However BCV at a dose of 50 μg/ml showed 10 ± 1.2% cytotoxic effect which was not significant. BCV (10 μg/ml and 15 μg/ml) produced macrophage spreading (8–30%) as
Discussion
It had been reported that C. cerastes and Naja haje snake venoms had inhibitory activity on L. donovani promastigotes multiplication in a dose dependent manner (Fernandez-Gomez et al., 1994). It had been reported that T. discrepans scorpion venom killed 50% of Leishmania mexicana mexicana promastigotes at 0.4 μg/ml dose (Borges et al., 2006). Three species of C. durissus venom (Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus) showed cytotoxicity on
Conflict of interest
The authors declared that there is no conflict of interest among the authors.
Acknowledgments
We are thankful to CSIR, Govt. of India for funding the work and providing infrastructural facilities. We are thankful to Dr. Syamal Roy, Scientist, Infectious Disease and Immunology Division, Indian Institute of Chemical Biology, Kolkata for his kind support and help.
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