Effects of atropine and propranolol on lung inflammation in experimental envenomation: comparison of two buthidae venoms

Background Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam). Results Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury. The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (β adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming. Conclusion These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.

Catecholamine may induce pulmonary edema via both hemodynamic and inflammatory mechanisms, by augmenting the IL-6 level [16]. The autonomic effects on inflammation are not restricted to catecholamine since the use of muscarinic antagonists may prevent some of the underlying cellular inflammatory responses in the lungs in addition to reducing smooth muscle contraction and mucus secretion [17,18]. Furthermore, the muscarinic antagonist, atropine significantly reduces neutrophil influx in lungs [19]. These polynuclear cells migrate into the lungs as a direct response to various proinflammatory stimuli and might contribute to many disorders such as acute respiratory distress syndrome (ARDS) [20,21].The present study is designed to investigate the mechanism by which venoms of two scorpions, found in Algeria and belonging to the same genus Androctonus, lead to lung inflammation by using atropine and propranolol.

Biological materials Venoms
Lyophilized venoms of Androctonus australis hector and Androctonus amoreuxi, with respective LD 50 of 0.85 and 0.75 mg/kg, were obtained from the Pasteur Institute of Algeria.

Animals
Male NMRI mice (20 ± 2 g), provided by the Pasteur Institute of Algeria, were used for all experiments. The animals were kept under controlled environment and received food and water ad libitum. The experimental protocol was in accord with the guidelines for the care of laboratory animals published by the European Union.

Non-biological materials
Chemical products and reagents used in these experiments were purchased from Sigma (USA), Merck (Germany) or Panreac (Spain). Pharmaceutical products were acquired from other firms, the atropine sulfate from Renaudin (France) and the propranolol from AstraZeneca (France).

Effect of Androctonus amoreuxi venom on cytokine levels
Groups of mice were injected by subcutaneous (s.c.) route with a sublethal dose of Aam venom dissolved in saline solution; control mice received 0.2 mL of saline solution alone. Mice were bled at several moments and sera were separated and stored at −20°C.

Effects of venoms on lung tissue
The effects of a sublethal dose of the two venoms, Aam and Aah, in the presence or absence of antagonists on lungs were evaluated by: estimation of myeloperoxydase activity as a marker of neutrophilia and histological study. Atropine sulfate (1 mg/kg) was injected intraperitoneally (i.p. route) 30 minutes before venoms and propranolol (0.1 mg/kg) was injected by the same route at two moments, 15 minutes before and 15 minutes after venom administration.

Myeloperoxydase activity
Three hours after envenomation by Aam or Aah venom, the removed lungs were homogenized in Tris-HCl buffer 50 mM, pH 6.6, then centrifuged at 6000 rpm for 30 minutes. The first supernatant (S1) was conserved at 4°C and the second supernatant (S2) was recovered after three freeze-thaw cycles of the pellet followed by its centrifugation at the above mentioned buffer conditions, rate and duration. One hundred microliters of S1 and 100 μL of S2 were added to 300 μL of chromogene substrate (0.167 mM O-dianisidine prepared in Tris-HCl 50 mM; pH 6.6 and H 2 O 2 8.8 mM) and the resulting mixture was read at the absorbance of 460 nm after one minute of incubation at room temperature.

Histological study
Lungs were fixed in 4% formaldehyde for 48 hours at room temperature, dehydrated in ethanol, cleared in xylen and embedded in paraffin. Histological sections (3-μm thick) were cut and stained with hematoxylineosin (H&E) for microscopic examination (Motic Digital Microscope PAL system).

Statistical analysis
The obtained data were expressed as mean ± SD and analyzed by, ANOVA with the significance level defined as p < 0.05.

Effect of Aam and Aah venoms on cytokine release
In the present study, the sera of mice envenomed by Aam displayed an increase of proinflammatory cytokines (IL-1β and IL-6). The comparison between Aam and Aah showed that the IL-1β level was more important in response to Aah venom (60 ± 12 pg/mL) versus (22.85 ± 2.15 pg/mL) (Figure 1 -A), while the maximum release of IL-6 was detected 60 minutes after Aam injection (237.66 ± 20.5 pg/mL), 180 minutes after administration of Aah venom (56 ± 2.89 pg/mL) followed by a significant elevation at 1440 minutes only in mice envenomed with Aam venom (Figure 1 -B).
In addition to the production of proinflammatory cytokines, the Aam and Aah venoms induced a significant release of an anti-inflammatory cytokine (IL-10). A biphasic profile was observed in response to Aam, the first peak measured at 60 minutes (162 ± 16.35 pg/mL), the second one at 360 minutes (104 ± 10.22 pg/mL), whereas only one peak was observed at 60 minutes (562.5 ± 59.3 pg/mL) in response to Aah venom (Figure 1 -C). Elevation of this cytokine was also reported in mice envenomed with Centruroides noxius venom [22].

Myeloperoxydase activity
Rapid accumulation in lungs of neutrophils in response to any proinflammatory stimulus is one of the first recognizable events in the pathogenesis of many pulmonary diseases [23,24]. Neutrophil recruitment into lungs was evaluated by myeloperoxydase activity in this study, the two venoms induced neutrophil infiltration with more pronounced effect when Aam venom was injected (0.902 ± 0.071) versus (0.474 ± 0.033) (Figure 2).
The increase of pulmonary MPO activity was also observed in mice envenomed with Aah and Tityus serrulatus venoms [25,26].
Antagonists' effectiveness in reducing neutrophil influx depended on the venom injected; in comparison to propranolol, atropine significantly prevented neutrophil recruitment in the presence of Aam venom and showed the same effect in response to Aah venom ( Figure 2).

Lung histology
Lung tissue micrographs from mice envenomed by Aam or Aah showed hemorrhage, thickening of the interalveolar septa with a high accumulation of inflammatory cells (Figure 3 -A1, A'1, B1, B'1). These effects are more pronounced in response to Aah venom which showed, in addition to these anatomopathologic modifications, some edematous area (Figure 3 -B1).
Administration of atropine, a non-selective muscarinic antagonist or propranolol, a β adrenergic antagonist prior to the venom administration showed that atropine is more effective than propranolol at preventing inflammatory cell influx (Figure 3 -A2, B2, A3, B3). The lung edema observed in response to Aah venom was augmented in mice pretreated by the two antagonists ( Figure 3 -B2, B'3).

Discussion
Several studies emphasized the relevance of proinflammatory cytokines in the pathophysiological manifestations of scorpion envenomation and showed their correlation with severity [27][28][29][30].
Cytokine levels differed significantly, with Aam venom inducing an earlier increase of IL-6 compared to Aah venom. This result can be explained by the high early level of anti-inflammatory cytokine (IL-10) in response to Aah venom. Indeed IL10 is known to play a modulatory role, down-regulating multiple aspects of immune and inflammatory responses through the regulation of the proinflammatory cytokines (IL-1β, IL-6 and TNF-α) [34,35].
The discrepancy in neutrophil influx observed in response to the Aam and Aah venoms may be attributable to their different levels of IL-6 release. This result is supported by previous data which showed that proinflammatory cytokines are responsible for leukocyte recruitment by inducing the elevation of chemokines and expression of adhesion molecules such as ICAM-1 and VLA-4 in endothelial cells [36]. The increase of chemokines might result in the binding of acetylcholine to muscarinic receptors [37,38].
Administration of atropine or propranolol prior to envenomation reduced neutrophilia in the lungs. This result suggests an inflammatory effect of both muscarinic and β adrenergic stimulation, as reflected in the ability of each antagonist to reduce neutrophil recruitment; which depends on the venom injected. Atropine was more effective than propranolol in preventing neutrophil influx following Aam injection. This observation is probably related to the amount of neurotransmitter released with a predominance of cholinergic stimulation in the presence of Aam venom.
This cholinergic predominance could explain in part the absence of an edematous area in lungs of mice envenomed by Aam venom, since the binding of Ach to muscarinic receptors decreases cardiac contraction, whereas excessive catecholamine release might give rise to left ventricular dysfunction that, in turn, may form lung edema [14,15,[39][40][41][42][43]. Lung edema observed in pheochromocytoma patients or induced experimentally by catecholamine was prevented by pretreatment with α adrenergic blockers [16,[44][45][46][47]. However our study showed that pulmonary edema subsequent to Aah venom was not reduced by propranolol or atropine. A similar effect was observed following propranolol administration in patients with pheochromcytoma [48]. Ismail [49] explained the effect of atropine by the accentuation of arterial hypertension.
The present comparison of lung micrographs between Aam and Aah also showed that an edematous area is observed only in response to Aah venom which induced a more important leukocyte infiltration in alveolar walls. These data are supported by previous studies which reported that edema formation in scorpion envenomation is attributable in part to activation of the inflammatory cascade and the release of lipid-derived mediators of inflammation, including PAF, leukotrienes and prostaglandins secreted after activation of mast cells by neuropeptides such as substance P [6,12,13].
The influx of inflammatory cells into pulmonary parenchyma was reduced by atropine and propranolol. These results are similar to other studies which ascribed inflammatory effects to β adrenergic stimulation, thus indicating that most inflammatory cells express functional muscarinic receptors and showed that atropine administration inhibits the migration of leucocytes towards the site of inflammation, and blocked increase of leucocytes in splenic venous blood in response to carbacholine [50][51][52][53][54][55][56].

Conclusion
In conclusion, the comparative study of inflammatory response induced by Aam and Aah venoms showed not only the role of the neuroendocrine-immune axis in the development of lung inflammation with more important parasympathetic involvement in envenomed mice by Aam, but also that the role of atropine or propranolol in reducing inflammatory cells influx is independent of their effect on lung edema formation. The variability of venoms must be elucidated before an efficient treatment can be developed.