Intradermal Application of Crotamine Induces Inflammatory and Immunological Changes In Vivo.

Ana Silvestrini,Maíra Mendes,Maurício Mazzi,Luana De Macedo,Acácio Pigoso,Thiago De Andrade

doi:10.3390/toxins11010039

Ana Silvestrini, Maíra Mendes + Show 4 more

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https://doi.org/10.3390/toxins11010039

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Abstract

Crotamine is a single-chain polypeptide with cell-penetrating properties, which is considered a promising molecule for clinical use. Nevertheless, its biosafety data are still scarce. Herein, we assessed the in vivo proinflammatory properties of crotamine, including its local effect and systemic serum parameters. Sixty male Wistar rats were intradermically injected with 200, 400 and 800 µg crotamine and analyzed after 1, 3 and 7 days. Local effect of crotamine was assessed by determination of MPO and NAG activities, NO levels and angiogenesis. Systemic inflammatory response was assessed by determination of IL-10, TNF-α, CRP, NO, TBARS and SH groups. Crotamine induced macrophages and neutrophils chemotaxis as evidenced by the upregulation of both NAG (0.5–0.6 OD/mg) and MPO (0.1–0.2 OD/mg) activities, on the first and third day of analysis, respectively. High levels of NO were observed for all concentrations and time-points. Moreover, 800 μg crotamine resulted in serum NO (64.7 μM) and local tissue NO (58.5 μM) levels higher or equivalent to those recorded for their respective histamine controls (55.7 μM and 59.0 μM). Crotamine also induced a significant angiogenic response compared to histamine. Systemically, crotamine induced a progressive increase in serum CRP levels up to the third day of analysis (22.4–45.8 mg/mL), which was significantly greater than control values. Crotamine (400 μg) also caused an increase in serum TNF-α, in the first day of analysis (1095.4 pg/mL), however a significant increase in IL-10 (122.2 pg/mL) was also recorded for the same time-point, suggesting the induction of an anti-inflammatory effect. Finally, crotamine changed the systemic redox state by inducing gradual increase in serum levels of TBARS (1.0–1.8 μM/mL) and decrease in SH levels (124.7–19.5 μM/mL) throughout the experimental period of analysis. In summary, rats intradermally injected with crotamine presented local and systemic acute inflammatory responses similarly to histamine, which limits crotamine therapeutic use on its original form.

Highlights

Crotamine is a basic polypeptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus and a member of the α-myotoxin family
C-reactive protein (CRP) is an important marker of acute inflammation in response to various stimuli caused by infectious agents or tissue damage and it was used to evaluate the inflammatory effect of crotamine
All doses of crotamine resulted in average serum levels of CRP equal to or greater than those induced by 1000 μg histamine on the respective days of analysis

Summary

Introduction

Crotamine is a basic polypeptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus and a member of the α-myotoxin family. Positively charged regions distributed throughout the structure and a small area of negative charge optimize electrostatic interactions between crotamine and diverse cell membranes [1,2,3,4,5]. This toxin displays different cellular and molecular targets as well as several activities, including neurotoxicity and myotoxicity. Its myotoxic potential is related to the electrophysiological changes in sodium and potassium channels, changes in mitochondrial calcium homeostasis and degeneration of myofibrils, with consequent structural damage to muscle fibers [6,7,8,9]. Studies have shown that the mechanism of action of crotamine is not restricted to the muscle tissue, involving other tissues, mainly liver and kidneys or involving other cells such as fibroblasts, neural and embryonic stem cells [2,10]

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