Venom of the giant ant Dinoponera quadriceps attenuates inflammatory pain in mouse cutaneous wound healing model

. Arthropod venoms are potential sources of bioactive substances, providing tools for the validation of popular use and new drugs design. Ants belonging to the genus Dinoponera are used in the folk medicine to treat inflammatory conditions. It was previously demonstrated that the venom of the giant ant Dinoponera quadriceps (DqV), containing a mixture of polypeptides, elicit antinociceptive effect in mice models of chemical, mechanical and thermal nociception. The aim of this study was to evaluate DqV antiinflammatory and antihypernociceptive effects in a mice model of traumatic cutaneous wound. Colonies of D. quadriceps were collected in the ‘ Serra de Maranguape ’ (State of Ceará, northeastern Brazil), a small mountain range located on the coastal zone, and the venom secreted by the ant glands was extracted with capillary tubes, further lyophilized and maintained at -20 ± 1ºC until use. Wounds were performed in the dorsum of Swiss mice. Animals received intravenous (i.v.) injection of DqV (50 µg kg -1 day -1 ) during 3 days for evaluation of inflammatory parameters present in the wounds: hypernociception, leukocyte infiltrate, myeloperoxidase activity, nitrite/nitrate content. Data was tested by two-way ANOVA and Bonferroni’s post-hoc test. DqV reduced (2.7 folds) hypernociception at 48 hours, leukocyte infiltration by 65% at 6 hours and myeloperoxidase activity by 60% at 0.5 hour after wound induction. In conclusion, the venom extracted from D. quadriceps glands attenuates inflammation and hypernociception in mice cutaneous wounds. Based on the both popular use of Dinoponera ants as analgesic and antiinflammatory and experimental results with DqV in mice models of nociception, the aim of this study was to evaluate its antiinflammatory and antinociceptive effects in a model of traumatic cutaneous wound.

Based on the both popular use of Dinoponera ants as analgesic and antiinflammatory and experimental results with DqV in mice models of nociception, the aim of this study was to evaluate its antiinflammatory and antinociceptive effects in a model of traumatic cutaneous wound.

Material
Colonies of D. quadriceps Santschi, 1921 were collected (SISBIO authorization 28794-1) in the 'Serra de Maranguape' (State of Ceará, northeastern Brazil), a small mountain range located on the coastal zone, and maintained at the Laboratory of Entomology (Universidade Estadual do Ceará). The venom secreted by the ant glands was extracted with capillary tubes, lyophilized and maintained at -20 ± 1ºC until use (Sousa et al., 2012).
Female Swiss mice (22-35 g), kept at 26 ± 1ºC under 12/12 hours light/dark cycle and receiving food and water ad libitum, were brought to the laboratory 1h before experiments conducted according to protocols approved by our institutional Animal Care and Use Committee (CEUA/UECE 1776865/2015) and international principles (NIH publication #85-23, revised in 2011).

Leukocyte infiltrate
Skin fragments were fixed for 24 hours in 10% formaldehyde, subjected to dehydration in an increasing alcohol series, cleared in xylol, embedded in paraffin (60°C), sectioned in microtome (5 µm thickness) and stained with hematoxylin and eosin (H&E). Five fields were selected per slide in areas close to the ulcer presenting high cell density. Polymorphonuclear cells (segmented nucleus cells with multilobulated nucleus morphology/field) were quantified by optical microscopy (1000x) (Andrade et al., 2011). The sample unit for each animal was the sum of 5 fields per slide.

Hypernociception
Hypernociception was measured in grams (g) using the analgesiometer (Insight Equipamentos, Brasil), composed of a pressure transducer connected to a digital counter. The mechanical pressure, using a polypropylene tip -0.5 mm diameter, was applied to the ulcer edges as nociceptive stimuli, being the frequency of the behavioral responses (squeaking and writhing) measured. The nociceptive threshold (average of three values) was measured prior to ulceration and after DqV treatment at 0.5, 1, 6, 12, 24, 48 and 72 hours) (Pereira et al., 2016), and expressed as the area under the curve (AUC).

Statistical analysis
Results were expressed as mean ± SEM and tested by two-way Analysis of Variance (ANOVA) followed by Bonferroni's post-hoc test. Statistical significance was set at α = 0.05.

Results and discussion
The present study demonstrated that DqV inhibits the nociceptive behavior and the number of polymorphonuclear leukocytes at the inflammatory stage of the healing process, corroborating the popular use in inflammatory and painful conditions (Haddad et al., 2015).
The three-day treatment with DqV (50 µg kg -1 day -1 ; i.v) brought the nociceptive threshold at the wound margins to control values. The antinociceptive effect of DqV (AUC: 300) started 30 min after injection and was maintained until the 2 nd day of treatment (48 hours) compared to saline (AUC: 100). The reference drug celecoxib was also antinociceptive (AUC: 200) ( Figure 1A/B). Although DqV had inhibited nociception in the wound area at several stages of healing (0.5 until 48 hours), it only decreased by 65% the number of polymorphonuclear cells at 0.5 and at 6h post-treatment ( Figure 1C and Figure 2). These data suggest that DqV antinociceptive effect could be associated with the inhibition of inflammatory cells and nociceptive mediators. Our data are in line with the literature, since cutaneous wounds, such as those caused by pressure, venous insufficiency, neuropathy, or arterial disease, may evoke considerable pain (Young & McNaught, 2011). Besides, during the healing inflammatory phase, several cellular mediators are released, primarily by neutrophils and macrophages, in order to clean the wound site of cellular debris and pathogens and to attract other cells accounted for closing the wound (Patel, Maheshwari, & Chandra, 2016). These mediators, such as cytokines (interleukins, tumor necrosis factor, etc.), arachidonic acid by-products (prostaglandins), reactive species (hydrogen peroxide, superoxide anion, nitrate, etc.) and enzymes (MPO, nitric oxide synthase, etc.) are also involved in the genesis of pain, signaling to the nociceptor (Singh, Kumar, & Vinayak, 2018). Accordingly, Dqv inhibition on leukocyte infiltrate was confirmed by the inhibition of MPO activity at 0.5 (12.8 µg mg -1 tissue) and 6h (30 µg mg -1 tissue), by 60 and 48%, respectively ( Figure 1D). However, DqV did not alter the nitrite/nitrate content (0.5h: 5.17 ± 0.28 vs. saline: 5.14 ± 0.29 µM; 6h: 5.05 ± 0.2 vs. saline: 5.49 ± 0.08 µM). This parameter is a feature of acute inflammation, being involved in vascular alterations, such as vasodilatation and augment of vascular permeability (Patel et al., 2016). The lack of DqV effect on the nitrate content could be seen as beneficial, considering the dual role of NO in the inflammatory response, being toxic to infectious organisms and/or to host tissues or acting as antiinflammatory agent (Bogdan, 2001).
The present study is in accordance with the literature that show antinociceptive and antiinflammatory activities of other Hymenoptera venoms, such as bee venom constituents (Bogdan, 2001;Lee & Bae, 2016) and ant venoms (Altman et al., 1984;Yoon at al., 2015), including those of D. quadriceps (Sousa et al., 2012). In this line, the novel data concerning the antiinflammatory and antinociceptive effects of DqV on traumatic wounds in mice open new and better possibilities for wound care.

Conclusion
The venom extract of D. quadriceps glands attenuates inflammation and hypernociception in traumatic cutaneous wounds of mice.