Effect of Ac2-26 treatment on the mesenteric inflammation induced by CV and MjTX-II.

<p>Intact mast cells (arrows) in the control mesentery (A). Inflamed mesentery of CV—(B) and MjTX-II-induced peritonitis (C-D) with extravasated neutrophils in the tissue (arrowheads) as observed at 4 and 24 h. Reduced neutrophil influx (arrowheads) after Ac2-26 post-treatment at 4 (E) and 24 h (F) of MjTX-II-induced peritonitis. Vessels (V). Stain: Toluidine blue. Scale bars: 10 μm. Quantitative analysis of extravasated neutrophils in the mesentery after CV- (G) and MjTX-II–induced peritonitis (H). The data represent the mean ± SEM of cell numbers/mm² (n = 5 animals/group). **P < 0.01 vs control; ^§P < 0.05 vs MjTX-II-4 h.</p

Expression of AnxA1 in the mesenteric neutrophils.

<p>Strong AnxA1 immunoreactivity in neutrophils (arrows) from CV (B, C) and MjTX-II (E, F) groups, after 4 and 24 h, compared with cells of control group (A). The absence of immunoreactivity in the negative control (D). Scale bars: 5 μm. Counterstain: hematoxylin. Densitometric analysis of mesenteric neutrophils immunostained for AnxA1 in the CV (G) and MjTX-II (H) groups. The values (arbitrary units) are expressed as mean ± SEM of the sections analyzed from 5 rats /group. **P < 0.01 and ***P < 0.001 vs control.</p

Effect of Ac2-26 treatment on proinflammatory cytokine secretion.

<p>Peritonitis was induced in rats by i.p. injection of CV (100 μg) or MjTX-II (100 μg) in 0.5 mL of PBS. Control group received i.p. only PBS. Another set of animals were treated i.p. with 1 mg/kg of Ac2-26 peptide after 15 minutes of CV or MjTX-II injection. Peritoneal exudate was collected 4 and 24 hours after peritonitis inductions and cytokine dosages were performed by ELISA, as described in Methods. Values are expressed as the mean ± SEM of cytokine levels (n = 5 rats/group). ND < 31.25 pg/mL (not detected).</p><p>* P < 0.05 and</p><p>*** P < 0.001 vs control</p><p>^##P < 0.01 vs CV 4 h.</p><p>Effect of Ac2-26 treatment on proinflammatory cytokine secretion.</p

Model to summarize the effects of Ac2-26 treatment after either CV or MjTX-II induced peritonitis.

<p>Local inflammation in the peritoneal exudate augments the number of neutrophils and the pro-inflammatory cytokines, IL-1β and IL-6 (A). This inflammation is decreased after Ac2-26 treatment (B). In the mesentery, the inflammatory response promotes the influx of macrophages, and increases the number of neutrophils, degranulated mast cells and the levels of AnxA1 expression (C). The Ac2-26 treatment decreased the numbers of all the inflammatory cells (D). The systemic inflammation results in an increase of neutrophils in the bloodstream (E); these levels are restored after Ac2-26 post-treatment (F). In the kidney (G), MjTX-II augmented the infiltration by macrophages (H) and Ac2-26 prevented this influx (I). Histopathological analysis showed direct (casts) and indirect (pyknotic nuclei/karyolysis) effects of the envenoming (J), which were restored by the Ac2-26 treatment (K). Higher levels of AnxA1 were observed in the distal tubules and glomerular cells after the administration of either CV or MjTX-II (L), and these levels decreased with the anti-inflammatory treatment (M).</p

Presence of renal macrophages after MjTX-II-induced peritonitis.

<p>An increased number of cells was observed in the glomeruli (arrows) and interstice (arrowheads) of the juxtamedullary region 24 h after peritonitis induced by MjTX-II (B), compared to the control group (A). Ac2-26 peptide post-treatment prevented the macrophages influx in renal tissue (C). Scale bars: 20 μm. Counterstain: Hematoxylin. The data represent the mean ± SEM of the number of interstitial (D) and glomerular (E) macrophages per 20 fields in the renal tissue (n = 5 animals/group). **P < 0.01 and ***P < 0.001 vs control; ^§P < 0.05 and ^§§P < 0.01 vs MjTX-II 24 h.</p

Histopathological analysis of kidneys after CV- and MjTX-II-induced peritonitis.

<p>(A) Normal histological condition of proximal (PT) and distal convuluted tubules (DT) in the renal control. (B-E) CV- and MjTX-II PLA₂-induced peritonitis provoked renal damage characterized by the presence of pyknotic nuclei (arrowheads), karyolisis (white arrow) and hyaline casts (asterisks) in proximal tubules. (F) Ac2-26 peptide restores the normal morphological aspect of juxtamedullary structures. Stain: Hematoxylin-Eosin. Scale bars: 20 μm.</p

Analysis of AnxA1 expression in the juxtamedullary renal structures.

<p>AnxA1 immunostaining was detected in the epithelial cells under all of the experimental conditions (A, C, E, G, I), concentrated in the glomerular capsule (arrows) and distal convuluted tubules (asterisks). Ac2-26 post-treatment decreased the immunostaining for endogenous AnxA1 in the distal tubules and glomerular capsule (D, F, H, J) 4 and 24 h after CV- and MjTX-II-induced peritonitis. Negative control of the reaction (B). Counterstain: hematoxylin. Scale bars: 20 μm. Densitometric analysis of the epithelial cells from distal convuluted tubules (K-L) and glomerular capsule (M-N) immunostained for AnxA1. Values (arbitrary units) are expressed as the mean ± SEM of sections analyzed from 5 rats /group. **P < 0.01 and ***P < 0.001 vs control; ^###P < 0.001 vs CV groups; ^§§P < 0.01 and ^§§§P < 0.001 vs MjTX-II groups at the corresponding experimental time.</p

Blood and peritoneal neutrophils after CV- and MjTX-II-induced peritonitis.

<p>Blood (A) and peritoneal (B) neutrophil counts. Peritonitis was induced in rats by i.p. injection of either CV or MjTX-II (100 μg) in 0.5 mL of PBS. Control animals were injected with PBS alone. Another set of animals from CV and MjTX-II groups were treated i.p. with 1 mg/kg of Ac2-26 peptide 15 minutes after the induction of peritonitis. The data represent the mean ± SEM of the cell numbers x 10⁵ per mL. *P < 0.05 and ***P < 0.001 vs control; ^###P < 0.001 vs CV 4h; ^§P < 0.05 and ^§§§P < 0.001 vs MjTX-II 24h.</p

Quantitative analysis of degranulated mast cell and macrophages into mesentery.

<p>Peritonitis was induced in rats by i.p. injection of CV (100 μg) or MjTX-II (100 μg) in 0.5 mL of PBS. Control group received i.p. only PBS. Another set of animals were treated i.p. with 1 mg/kg of Ac2-26 peptide after 15 minutes of CV or MjTX-II injection. Mesentery was collected after 4 and 24 hours after peritonitis induction and cellular counts performed as described in Methods. Values are expressed as the mean ± SEM of the number of cells per mm² (n = 5 rats/group).</p><p>** P < 0.01 and</p><p>*** P < 0.001 vs control</p><p>^#P < 0.05 and</p><p>^###P < 0.001 vs CV 4 h.</p><p>Quantitative analysis of degranulated mast cell and macrophages into mesentery.</p