Crotoxin from <i>Crotalus durissus terrificus</i> Is Able to Down-Modulate the Acute Intestinal Inflammation in Mice

<div><p>Inflammatory bowel diseases (IBD) is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX) is the main component of <i>Crotalus durissus terrificus</i> snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI), macroscopic tissue damage, histopathological score and myeloperoxidase (MPO) activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1β and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3) and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4⁺Tbet⁺ T cells induced by TNBS instillation in mice. In contrast, increased CD4⁺FoxP3⁺ cell population as well as secretion of TGF-β, prostaglandin E₂ (PGE₂) and lipoxin A₄ (LXA₄) was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the induction of a local anti-inflammatory profile in mice.</p></div

Effect of CTX treatment on the colitis induced by TNBS instillation in BALB/c mice.

<p><b>(A)</b> Body weight changes (%) of BALB/c mice during four days after the intrarectal instillation of TNBS (2.5 mg/animal) in 45% ethanol solution. The control mice received the 45% of ethanol solution. CTX (0.035 mg/kg) was administered i.p. 18 h after TNBS-induced colitis, and saline solution was administered as control. # <i>(p</i><0.05) ETOH versus TNBS and TNBS+CTX; o <i>(p</i><0.05) ETOH+CTX versus TNBS and TNBS+CTX; α <i>(p</i><0.05) TNBS versus TNBS+CTX (n = 4–6 mice/group). <b>(B)</b> Disease activity index calculated as described in material and methods. The results were expressed as mean ± SEM (n = 4–6 mice/group); <b>(C)</b> Macroscopic appearance of colonic portion (4 cm) obtained from each mice/group at 4 days after TNBS-induced colitis; <b>(D)</b> Histological analysis of perirectal segment from mice of distinct experimental groups stained with H&E (Structures: (e) epithelial damage, (i) inflammatory infiltrate and (s) submucosa edema) obtained after 4 days of TNBS-colitis; <b>(E)</b> Histological score of inflammatory reaction perirectal segment of each experimental group of mice (n = 4–5 mice/group); <b>(F)</b> MPO activity of colonic tissue of each experimental mice-group. Groups: ETOH (control- 45% ETOH); ETOH+CTX (45% ethanol group treated with CTX); TNBS (TNBS instillation in 45% ETOH- inflammatory bowel disease) and TNBS+CTX (TNBS-instillation in 45% ETOH that received the CTX) (n = 4–5 animals/group). * <i>p</i><0.05; *** <i>p</i><0.001.</p

Analysis of CD4⁺FoxP3⁺ cells and anti-inflammatory mediators in TNBS-mice treated or not with CTX.

<p>Cell suspensions were prepared from lamina propria of distinct experimental groups after 4 days of TNBS-induced colitis for the analysis of CD4⁺FoxP3⁺ cells by flow cytometry. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2 independent experiments. <b>(A)</b> Representative dot plots of CD4⁺FoxP3⁺ cells in the lamina propria obtained from distinct experimental groups. <b>(B)</b> Results of CD4⁺FoxP3⁺ cells expressed as a mean of the absolute number of cells in duplicate of 2 independent experiments ± SEM. Secretion of TGF-β <b>(C)</b> and IL-10 <b>(D)</b> in colonic tissue homogenates determined by ELISA. Production of PGE₂<b>(E)</b> and LXA₄<b>(F)</b> was performed by ELISA in colonic tissue homogenates. The results represent the mean obtained in individual mice/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001; (n = 4–5 animals/group).</p

CD4⁺Tbet⁺ cell population and IFN-γ secretion of mice with acute colitis induced by TNBS treated or not with CTX.

<p><b>(A)</b> Representative dot plots of CD4⁺Tbet⁺ cells in the lamina propria of distinct group of mice. <b>(B)</b> CD4⁺Tbet⁺ cells were expressed as a mean of the absolute number of cells ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 3 independent experiments. <b>(C)</b> Secretion of IFN-γ in colonic tissue homogenates determined by ELISA. The results represent the mean obtained in the individual samples/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001; (n = 4–5 animals/group).</p

Secretion of pro-inflammatory cytokines in colonic tissue homogenates of TNBS-mice treated or not with CTX.

<p>Production of IL-1β (A), IL-6 (B) and TNF-α (C) was measured in homogenates of colonic segments by ELISA. The results represent the mean of the cytokine secretion in individual mice/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 (n = 4–5 animals/group).</p

Effect of CTX on Th17 cells, ILC3 and IL-17 secretion of mice with acute colitis induced by TNBS.

<p>Cell suspensions were prepared from lamina propria of distinct experimental groups after 4 days of TNBS-induced colitis and that received or not the CTX. The TCRß⁺CD4⁺RORγt⁺, TCRß⁺CD4⁺IL-17⁺ and Lin^-CD90⁺IL-17⁺ cells were analyzed by flow cytometry. <b>(A, B and C)</b> Strategy for the analysis of TCRß⁺CD4⁺RORγt⁺ and TCRß⁺CD4⁺IL-17⁺ cells in the lamina propria obtained from each group of mice. The results of TCRß⁺CD4⁺RORγt⁺<b>(D)</b> and TCRß⁺CD4⁺IL-17⁺ cells <b>(E)</b> expressed as the mean of the absolute number ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2–3 independent experiments. <b>(F)</b> Strategy for the analysis of the Lin^-CD90⁺IL-17⁺ cells. <b>(G)</b> The results of Lin^-CD90⁺IL-17⁺ cells were expressed as a mean of the absolute number ± SEM. The samples were prepared from a pool of cells from 4–5 animals/group performed in duplicate. The results are from 2 independent experiments. <b>(H)</b> Secretion of IL-17 in colonic tissue homogenates determined by ELISA. The results represent the mean obtained in the individual samples/group ± SEM. * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 (n = 4–5 animals/group).</p

Chronic training.

<p>*Cisplatin administration.</p><p>Chronic training.</p

IL-6 contributes to kidney protection induced by exercise.

<p>The expression of the cytokine IL-6 was determined by qPCR in the muscle (<b>A</b>) and the kidney (<b>B</b>). The CIS group showed increased levels of IL-6 in both tissues compared to sedentary groups (CT and EX). However, CIS-EX showed increased IL-6 expression in the kidney, which indicates the protective role of IL-6 in this model. *P<0.05; **P<0.01.</p

Aerobic exercise minimizes cisplatin-induced cachexia and renal damage.

<p>Exercise efficacy was confirmed by an increase in muscle angiogenesis in the exercised (EX) and exercised with cisplatin injection (CIS-EX) groups (<b>A</b> and <b>B</b>). Decreased weight loss (<b>C</b>) and lower levels of serum urea (<b>D</b>) in the CIS-EX group compared to the sedentary goup injected with cisplatin (CIS) indicate the protective effect of exercise. The sedentary group without cisplatin injection was used as the control (CT). *P<0.05; **P<0.01; ***P<0.001.</p

Aerobic exercise reduces cell death in the kidney.

<p>Representative TUNEL staining (<b>A</b>) and quantification (<b>B</b>) showed a significantly reduced percentage of TUNEL-positive cells (dead cells) in the CIS-EX group. The expression of Bcl2 and Bax genes was evaluated by qPCR and is represented by the ratio of the pro- to the anti-apoptotic molecule (Bax/Bcl2) (<b>C</b>) the CIS group was more likely to exhibit a pro-apoptotic profile, confirming the protective role of exercise. ***P<0.001.</p