To better understand the inflammatory effects of Pelagia noctiluca crude venom in injected rats, we preliminary monitored blood pressure within 3 h and mortality within 90 min, at three different doses of crude venom (6, 30 and 60 µg/kg). A significant reduction of blood pressure and increase of mortality percent at 30 and 60 µg/kg compared to 6 µg/kg were observed (Figure 1a,b). No significant difference was found between the dose of 30 and 60 µg/kg (Figure 1a,b). No blood pressure alterations and no mortality percent was found in the sham group (Figure 1a,b). Based on these preliminary results, 30 µg/kg crude venom dose was considered for experiments. Thus, for the next step, to investigate the mechanisms related to the inflammatory process and oxidative stress possibly induced by Pelagia noctiluca crude venom, venom-injected rats were treated with tempol, as a potent antioxidant.

Hepatocellular injury. Crude venom injected rats significantly exhibited higher ALT (Figure 2b), AST (Figure 2a), bilirubin (Figure 2c) and alkaline phosphatase (Figure 2d) plasma levels, compared to the control group. All these findings are consistent with the development of hepatocellular injury. Treatment with tempol significantly decreased the liver injury caused by crude venom injection.

Pancreatic injury. Crude venom injected rats exhibited significantly higher lipase and amylase levels than the control group: such findings are consistent with the development of pancreatic injury (Figure 2e,f). Treatment with tempol significantly decreased the pancreatic injury caused by crude venom injection (Figure 2e,f).

Renal dysfunction. Crude venom injected rats exhibited a significant increase in plasmatic creatinine concentration compared to the control group, predictive of renal dysfunction development (Figure 2g). Treatment reduced the crude venom-induced renal dysfunction (Figure 2g).

No histological alteration was observed in tissues from control group rats (Figure 3a,d). Histological evaluation of lung (Figure 3b) and intestine sections (Figure 3e) 6 h after crude venom injection (30 µg/kg) revealed marked pathological changes. In particular, lung and intestine sections revealed inflammatory infiltration by neutrophils, macrophages and plasma cells (Figure 3b,e). In that regard, Pelagia noctiluca crude venom caused a significant histological damage in injected rats, the treatment with tempol caused a substantial reduction in the extent of histological damage in both lung and intestine (Figure 3c,f).

Inhibitor of kappa B (IκB-α) degradation and nuclear factor kappa B (NF-κB p65) expression were evaluated by Western blot analysis to investigate the cellular mechanisms underlying the development of acute lung and intestine injury after injection with crude venom. Basal expression of IκB-α was detected in lung (Figure 4a) and intestine samples (Figure 4d) from control group animals, whereas IκB-α levels were substantially reduced in both lung and intestine tissues obtained from venom-injected animals. Treatment with tempol prevented crude venom-induced IκB-α degradation, with increased IκB-α levels in treated rats (Figure 4a,d). Moreover, NF-κB p65 levels in lung (Figure 4b) and intestine (Figure 4e) nuclear fractions were also significantly increased at 6 h after crude venom injection, compared to control group rats. In that regard, we demonstrated that tempol treatment significantly reduced NF-κB p65 levels in both lung and intestine inflamed tissues.

The expression of cyclooxygenase-2 (COX2) was also assessed by Western blot analysis, to better understand the effects of Pelagia noctiluca crude extract on lipid degradation processes and on the subsequent production of leukotrienes and prostaglandins. As seen in the figure, the expression of COX2 is increased in lung (Figure 4c) as well as in intestine (Figure 4f), compared to the control group. On the other hand, rats treated with tempol showed a significant reduction in COX2 levels in both organs (Figure 4c,f).

Six hours after crude venom injection, expression of the adhesion molecules ICAM-1 and P-Selectin were evaluated to assess neutrophil infiltration. In crude venom-injected rats, an increase in immunohistochemical staining for ICAM-1 and P-Selectin was demonstrated on the surface of endothelial cells in both inflamed lung (Figure 5a; see particles a; Figure 5c; see particles c1) and intestine (data not shown), while the immunostainings for ICAM-1 and P-Selectin were markedly reduced in lung (Figure 5b,d; see particles b1,d1) and intestine (data not shown) tissues of tempol-treated rats. No staining for either ICAM-1 or P-Selectin was found in tissue sections obtained from the control group (data not shown). Six hours after crude venom injection, MPO activity, accounting for PMN infiltration, was measured in lung (Figure 5e) and intestine (Figure 5f). As shown in figure, MPO activity was significantly increased in both organs 6 h after crude venom injection. MPO activity was also significantly reduced by tempol treatment (Figure 5e,f).

To determine the role of nitric oxide (NO) produced by the injection of crude venom, iNOS expression was evaluated by Western blot and immunohistochemistry. Samples of lung tissue, taken 6 h after injection, were processed for immunohistological staining for iNOS. Lung sections from control group rats did not stain for iNOS (data not shown), whereas lung sections obtained from crude venom-treated rats exhibited a positive staining for iNOS (Figure 6a; see particles a1). Here, we also showed a reduction in NO production and iNOS activity in lung (Figure 6b; see particles b1) and intestine (data not shown) by tempol treatment. Western blot analysis revealed a markedly reduced iNOS expression in lung (Figure 6d) and intestine (Figure 6e) tissues from tempol-treated rats. Both groups showed a significant increase of expression compared to the control group. In addition, plasma analysis showed that the injection of crude extract of Pelagia noctiluca induced a significant concentration of NOx compared to sham rats. The increase in plasma NO₂/NO₃ levels was significantly reduced in tempol-treated animals (Figure 6f).

Six hours after crude venom injection, the presence of nitrotyrosine was investigated in both lung and intestine sections. Immunohistochemical analysis, using a specific anti-nitrotyrosine antibody, revealed a positive staining in lung (Figure 7a; see particles a1) and intestine (data not shown) of crude venom-injected rats. Treatment with tempol reduced nitrotyrosine staining in both organs from crude venom-challenged rats (Figure 7b; see particles b1). Immunohistochemical analysis of lung (Figure 7c; see particles c1) and intestine (data not shown) sections, obtained from crude venom-challenged rats, also revealed a positive staining for Poly ADP-ribose (PAR). In contrast, staining for PAR was absent in sections of lung (Figure 7d; see particles d1) and intestine (data not shown) from tempol-treated crude venom-challenged rats. There was no staining for either nitrotyrosine or PAR in sections of lung (data not shown) or intestine (data not shown) from control group rats. In addition, 6 h after crude venom injection, MDA concentration was measured as an indicator of the degree of lipid peroxidation. As shown in figure, MDA levels were significantly increased in lung (Figure 7f) and intestine (Figure 7g), while MDA levels were significantly reduced by tempol treatment (Figure 7f,g).

Six hours after crude venom injection, the appearance of proapoptic protein Bax in lung (Figure 8a) and intestine (Figure 8c) homogenates was investigated by Western blot, showing that Bax levels were appreciably increased (Figure 8a,c). In addition, Bcl-2 expression was analyzed by Western blot analysis in homogenates from lung (Figure 8b) and intestine (Figure 8d) tissues. A basal level of Bcl-2 expression was detected in tissues of the control group (Figure 8b,d), while 6 h after crude venom injection, Bcl-2 expression was significantly reduced in lung and intestine (Figure 8b,d). Treatment with tempol induced a decrease of Bax (Figure 8a,c) and an increase in Bcl-2 expression (Figure 8b,d) in both lung and intestine tissues from injected animals, attenuating the effect of Pelagia noctiluca crude venom on the apoptotic pathway.

The study was carried out on Sprague-Dawley male rats (200–230 g, Harlan, Nossan, Italy). Food and water were available ad libitum. The study was approved by the University of Messina Review Board for the care of animals. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

Briefly, specimens of Pelagia noctiluca were collected in the Strait of Messina. The oral arms were excised and submerged in distilled water for 2 h at 4 °C. After a complete detachment of the epidermis the tissue was removed from the suspension containing both epidermis and undischarged nematocysts deriving from the osmotic rupture of nematocytes. The nematocysts suspension was repeatedly washed in cold distilled water and filtered through plankton nets (100, 60, and 40 μm mesh, respectively) to remove most of the tissue debris, and then centrifuged at 4 °C (ALC PK 120R, 4000× g for 5 min). The isolated nematocysts were classified as holotrichous isorhizas according to [55]. Then, the suspension was frozen at −20 °C until use. Before each experiment, the nematocysts were defrosted, filtered, and washed again in distilled water. This clean suspension of nematocysts was re-suspended in 0.01 M phosphate buffer containing 0.9% NaCl (pH 7.4; osmotic pressure = 300 mOsm/kg_H2O). Crude venom was extracted by sonication on ice (Sonoplus, 70 mHz, 30 times, 20 s) of a population of isolated nematocysts (90 nematocysts/μL) and the crude extract was then separated from crushed capsules by refrigerated centrifugation at 4000× g for 10 min. The obtained venom was kept at 4 °C until use or stored at −20 °C or −80 °C, unless otherwise stated. Aliquots of crude venom were employed to measure the protein content by Bio-Rad Protein Assay (BioRad, Richmond, CA, USA), in agreement with previous studies [18].

The experiment was divided into two steps: in the first one, we preliminary evaluated the toxic effect of crude venom on blood pressure and mortality. For this purpose, rats (n = 25 rats/group) were randomly allocated into the following groups:

As a second step, to confirm the inflammatory effects caused by crude venom injection, we planned to treat crude venom-injected rats with an excellent antioxidant such as tempol. With this aim, the dose of 30 μg/kg of Pelagia noctiluca crude venom was used. Rats were randomly allocated into the following groups:

The choice of this dose of tempol was based on our previous studies [56].

Six hours after administration of crude venom, rats were sacrificed and the following organs were taken: intestine, kidney, liver, lung. Blood samples, were centrifuged (1610× g for 3 min at room temperature) to separate plasma successively used for analysis of nitrites and nitrates, amylase, lipase, creatinine, bilirubin, AST, ALT and alkaline phosphatase levels.

Rats were anaesthetized by sodium pentobarbital (45 mg/kg i.p.). Following anaesthesia, catheters were placed in the carotid artery and jugular vein. The mean arterial blood pressure (MABP) was continuously recorded by a Maclab A/D converter (Ugo Basile Biological Research Apparatus, Varese, Italy), displayed and stored on a Macintosh personal computer. A rectal thermometer was inserted, and the rats were kept at a body temperature of 37–38 °C by a homeothermic blanket. The left jugular vein was cannulated to allow administration of further anesthetic and crude venom at three different doses 6, 30 and 60 µg/kg.

Myeloperoxidase activity, which is an index of polymorphonuclear (PMN) leukocyte accumulation, was determined as previously described [57]. Intestine and lung tissues, collected 6 h after crude venom injection, were homogenized in a solution containing 0.5% hexa decyl-trimethylammoniumbromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000× g at 4 °C. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H₂O₂. The rate of change in absorbance was measured with a spectrophotometer at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 mmol of peroxide 1 min at 37 °C, and was expressed in units per gram weight of wet tissue.

Levels of MDA in the intestine and lung tissue were determined as an index of lipid peroxidation. Six hours after injection of crude venom, the animals were euthanized and the organs were removed with a scalpel. Each piece of tissue (100 mg) was homogenized in 1.15% KCl solution (1 mL per 250 mg of tissue). An aliquot (100 mL) of the homogenate was added to a reaction mixture containing 200 mL of 8.1% SDS, 1.5 mL of 20% acetic acid (pH 3.5), 1.5 mL of 0.8% thiobarbituric acid and 700 mL distilled water. Samples were then boiled for 1 h at 95 °C and centrifuged at 3000× g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 650 nm.

Nitrite/nitrate (NO₂⁻/NO₃⁻) production, an indicator of NO synthesis, was measured in plasma collected 6 h after vehicle or crude venom administration. Plasma was incubated with nitrate reductase (0.1 U·mL⁻¹), nicotinamide adenine dinucleotide phosphate (1 mM) and flavin adenine dinucleotide (50 mM) at 37 °C for 15 min, followed by further incubation with lactate dehydrogenase (100 U·mL⁻¹) and sodium pyruvate (10 mM) for 5 min. Nitrite concentration in the samples was measured by Griess reaction, by adding 100 mL of Griess reagent [0.1% (w/v) naphthylethylenediamide dihydrochloride in H2O and 1% (w/v) sulphanilamide in 5% (v/v) H₂PO₄; vol. 1:1 to the 100 µL sample]. The optical density at 550 nm (OD550) was measured using an ELISA microplate reader (SLT-Lab Instruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution.

For histopathological examination, the animals were euthanized 6 h following the intravenously injection of crude venom and tissues (intestine and lung) were removed with a scalpel. The tissue slices were fixed in Dietric solution (14.25% ethanol, 1.85% formaldehyde, 1% acetic acid) for 1 week at room-temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical, Sherwood, OR, USA). Section (thickness 7 μm) were deparaffinized with xylene, stained with Haematoxylin/Eosin (H&E), studied using light microscopy (Dialux 22 Leitz; Leica Microsystems SpA, Milan, Italy). Sections of each type of tissue were evaluated by an experienced histopathologist, without knowledge of the treatments. The following morphological criteria were used for scoring lung injury: 0, normal lung; grade 1, minimal oedema or infiltration of alveolar or bronchiolar walls; grade 2, moderate oedema and inflammatory cell infiltration without obvious damage to lung architecture; and grade 3, severe inflammatory cell infiltration with obvious damage to lung architecture. The following morphological criteria were used for intestine injury: 0, no damage; 1 (mild), focal epithelial oedema and necrosis; 2 (moderate), diffuse swelling and necrosis of the villi; 3 (severe), necrosis with the presence of neutrophil infiltrate in the submucosa; and 4 (highly severe), widespread necrosis with massive neutrophil infiltrate and haemorrhage.

Tissues, taken 6 h after crude venom injection, were fixed in 10% (w/v) PBS-buffered formaldehyde, and 8 mm sections were prepared from paraffin-embedded tissues. After de-paraffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (Santa Cruz, Milan, Italy) respectively. Sections were incubated overnight with the following antibodies: rabbit antinitrotyrosine (1:500 in PBS, w/v); anti-iNOS (1:500 in PBS, w/v); anti-PAR (1:500 in PBS, v/v); anti-ICAM-1 antibody (1:500 in PBS, w/v); anti-P-selectin (1:500 in PBS, v/v). Sections were washed with PBS, and incubated with secondary antibody. Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex. The counter stain was developed with diaminobenzidine (brown) and nuclear fast red (red background). To confirm that the immunoreactions for the nitrotyrosine were specific, sections were incubated with primary antibody (anti-nitrotyrosine) in presence of excess nitrotyrosine (10 mM). Similarly, to confirm the binding specificity for iNOS, P-Selectin, ICAM-1 or PAR, some sections were incubated, alternatively, with primary or secondary antibody alone. Under these conditions, there was no positive staining indicating an effective immunoreaction. Immunocyto-chemistry photographs (N = 5) were assessed by densitometry by using Imaging Densitometer (AxioVision, Zeiss, Milan, Italy) and a computer program (AxioVision).

Cytosolic and nuclear extracts were prepared as described previously [58] with slight modifications. Briefly, tissues from each rat were suspended in extraction buffer A (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 3 μg/mL pepstatin A, 2 μg/mL leupeptin, 15 μg/mL Trypsin inhibitor, 40 μM Benzamidine), homogenized at the highest setting for 2 min, and centrifuged at 13,000× g for 3 min at 4 °C. Supernatants were collected as the cytosolic fraction. The pellets containing nuclei were resuspended in buffer B (20 mM Hepes, 1.5 mM MgCl₂, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 3 μg/mL pepstatin A, 2 μg/mL leupeptin, 15 μg/mL Trypsin inhibitor, 40 μM Benzamidine, NONIDET P40 1%, Glycerol 20%). After centrifugation 10 min at 13,000× g at 4 °C, the supernatants were collected as nuclear extracts and then stored at −80 °C for further analysis. The levels of IκB-α, iNOS, COX2, Bax and Bcl-2 were quantified in cytosolic fraction from the tissue collected 6 h after crude venom administration, while NF-κB p65 levels were quantified in nuclear fraction. The blotting membranes were blocked with 1 PBS, 5% (w/v) nonfat dried milk (PM) for 1 h at room temperature and subsequently probed with specific Abs IκB-α (1:1000; Santa Cruz Biotechnology, DBA, Milan, Italy), or anti-Bax (1:500; Santa Cruz Biotechnology), or anti-Bcl-2 (1:500; Santa Cruz Biotechnology), or anti-iNOS (1:1000; Transduction) or anti-NF-κB p65 (1:1000; Santa Cruz Biotechnology), or anti-COX2 (1:1000; Santa Cruz Biotechnology) in 1 PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 (PMT) at 4 °C, overnight. Membranes were incubated with peroxidase-conjugated bovine antimouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of proteic lysates, they were also incubated in the presence of the antibody against β-actin protein (1:10,000 Sigma-Aldrich Corp., Milan, Italy). The relative expression of the protein bands of IκB-α (37 kDa), NF-κB p65 (65 kDa), Bax (23 kDa), Bcl-2 (29 kDa) iNOS (130 kDa), COX2 (72 kDa) was quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (GS-700, Bio-Rad Laboratories, Milan, Italy) and a computer program (Molecular Analyst, IBM), and standardized for densitometric analysis to β-actin protein levels.

Blood samples, taken 6 h after crude venom or vehicle injection, were centrifuged (1610× g for 3 min at room temperature) to separate plasma. All plasma samples were analyzed within 24 h in a veterinary clinical laboratory using standard laboratory techniques. The following enzymes were measured in plasma as indicators of multiple organ injury/dysfunction: (i) amylase and lipase as indicators of pancreatic injury; (ii) alkaline phosphatase, aspartate aminotransferase (AST, a non-specific marker for hepatic injury), alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury) and bilirubin as indicators of liver injury; (iii) creatinine, as an indicator of reduced glomerular filtration rate, and hence, renal failure.

Unless otherwise stated, all compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK). All solutions used for in vivo infusions were prepared using nonpyrogenic saline (0.9% w/v NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).

All values in the figures and text are expressed as mean values ± standard error of the mean (SEM) of n observations. For in vivo studies, n represents the number of animals studied. In histology or immunohistochemistry experiments, figures are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value less than 0.05 was considered significant.