Losartan improves intestinal mucositis induced by 5-fluorouracil in mice

Abstract

Intestinal mucositis (IM) is a common side effect of 5-fluorouracil (5-FU)-based chemotherapy, which negatively impacts therapeutic outcomes and delays subsequent cycles of chemotherapy resulting in dose reductions and treatment discontinuation. In search of new pharmacological alternatives that minimize your symptoms, this work set out to study the effect of losartan (LOS), a receptor type I (AT1) angiotensin II antagonist, on intestinal mucositis induced by 5-FU. Intestinal mucositis was induced by a single intraperitoneal administration of 5-FU (450 mg/kg) in Swiss mice. Losartan (5, 25 or 50 mg/kg) or saline was orally administered 30 min before 5-FU and daily for 4 days. On 4th day, the animals were euthanized and segments of small intestine were collected to evaluate histopathological alterations (morphometric analysis), concentration of inflammatory cytokines, oxidative stress markers and genic expression of NF-κB p65, Fn-14 and TWEAK. Weight evaluation and changes in leukogram were also analyzed. 5-FU induced intense weight loss, leukopenia and reduction in villus height compared to saline group. Losartan (50 mg/kg) prevented 5-FU-induced inflammation by decreasing in the analyzed parameters compared to the 5-FU group. Our findings suggest that 50 mg/kg of losartan prevents the effects of 5-FU on intestinal mucosa in mice.

Figure 1

Histopathological analysis of intestinal segments of the duodenum, jejunum, ileum and colon of animals submitted to intestinal mucositis (IM) induced by 5-fluorouracil. Photomicrographs on a 100 μm scale demonstrated that the animals in the saline group maintained the integrity of the intestinal mucosa, with villi and crypts without damage, as well as the absence of inflammatory infiltrate. The 5-FU caused the loss of integrity, structure and shortening of the villi (black arrow), injury and disruption of the crypts with the presence of vacuolization (green arrow) and the presence of inflammatory infiltrate (red arrow). Losartan treatment at a dose of 50 mg/kg decreased the effects caused by 5-FU, improving the appearance of villi (red arrow) and crypts (black arrow).

Figure 2

(A) Images of histological slides stained in H&E, of fragments of the jejunum of mice submitted to intestinal mucositis. Animals saline without IM (a), with intestinal mucositis induced by 5-FU, without treatment (b) and animals with IM, treated with losartan at a dose of 50 mg/kg. The measurement of villus integrity is represented by dashed lines in yellow and the size of crypts is represented by dashed lines in blue. Segments of the duodenum, jejunum and ileum were collected to measure the villus height (10 villi/lamina). Scale bars = 100 µm (B) and crypt depth measurements (C). The bars represent the mean ± SEM of 5 mice in each group. ^#p < 0.001 vs. saline group, **p < 0.001 vs. 5-FU group. One way ANOVA with Bonferroni post-test.

Figure 3

(A) Effect of losartan on weight loss and (B) total leukocyte count in mice with 5-FU-induced intestinal mucositis (IM). (A) Weight assessment is shown as a percentage. The bars represent the mean ± SEM (n = 5) in each group. ^#p < 0.01 versus Saline group; *p < 0.001 versus group 5-FU (Two-way ANOVA analysis of variance followed by the Tukey test). (B) Animals with IM, group 5-FU, reduced the number of leukocytes compared to the saline group (**p < 0.0001). LOS 50 mg/kg significantly increased the number of cells when compared to the 5-FU group. *p < 0.0001 vs. group 5-FU; **p < 0.001 versus 5-FU group and ***p < 0.05 versus 5-FU group. ANOVA one-way test followed by the Tukey test.

Figure 4

Cytokines tumor necrosis factor alpha (TNF-α) (A) and interleukin 1 beta (IL-1β) (B) in the jejunal portion of mice submitted to the experimental model of intestinal mucositis (IM). The saline group comprised animals without IM; In the 5-FU group, the animals received 5-FU and without treatment. The LOS groups received 5-FU, underwent IM and were treated with losartan (orally), in doses (5, 25 or 50 mg/kg) (n = 5/group). The results are presented as mean ± standard error of the mean (n = 5/group). *p < 0.001 (compared to saline); **p < 0.0001 (compared to 5-FU) (ANOVA analysis of variance with the Tukey post-test).

Figure 5

Malondialdehyde (MDA) (A) and glutathione (GSH) (B) levels in the IM model. The experimental groups saline, 5-FU and losartan are presented in doses of 5, 25 or 50 mg/kg, the values are shown as mean ± SEM. For statistical analysis, ANOVA was used, followed by the Tukey test. *p < 0.01 vs. saline group and **p < 0.01 vs. group 5-FU.

Figure 6

Polymerase chain reaction in real time for TWEAK, Weak apoptosis inducer like cytokine tumor necrosis factor (A); FN-14, inducible fibroblast growth factor 14 (B); NF-κB p65, the nuclear transcription factor kappa B (C). 5-Fluorouracil (5-FU) increased the expression of these genes compared to the Saline group. Losartan decreased the expression of TWEAK, FN-14 and NF-κB p65, compared to the 5-FU group (n = 5); *p < 0.05 compared with the Saline group; **p < 0.05 compared with the group 5-FU; analysis of variance with Tukey's post-test).

Figure 7

Representation of pharmacological modulation of the intestinal mucositis induced by 5-fluorouracil (5-FU) treated with the losartan. DNA damage due to 5-FU promotes the release of reactive oxygen species (ROS) and activates NF-κB that induces the expression of proinflammatory cytokines such as IL-1β and TNF-α, in addition, occurs the oxidative stress, which that together promote inflammation in the intestinal mucosa. Losartan was reducing NF-κB p65 in response the decreased of gene expression TWEAK/Fn14, reducing IL-1β and TNF-α and decreasing oxidative stress, therefore improving intestinal mucositis. TWEAK, Tumor necrosis factor (TNF)-like weak inducer of apoptosis; Fn14, Fibroblast growth factor-inducible 14; NF-κB p65, nuclear transcription factor kappa B.