Curcuminoids from Curcuma longa L. reduced intestinal mucositis induced by 5-fluorouracil in mice: Bioadhesive, proliferative, anti-inflammatory and antioxidant effects

Abstract

Introduction: Intestinal mucositis is a frequent limiting factor in anticancer therapy and there is currently no broadly effective treatment targeted to cure this side effect.

Objective: This study aimed to evaluate the effects of a mucoadhesive formulation containing curcuminoids (MFC) from Curcuma longa L. on the pathogenesis of 5-fluorouracil (5-FU)-induced intestinal mucositis.

Methods: Three intraperitoneal 5-FU injections (200 mg/kg) were used to induce intestinal mucositis in adult Swiss male mice. Treatment was provided orally (MFC 3.75, 7.5 and 15 mg/kg), thirty minutes before 5-FU injections, daily until euthanasia. Duodenal samples were collected to perform morphometric and histopathological analysis, to investigate the expression of Ki-67, p53, Bax and Bcl-2 by immunohistochemistry, to evaluate neutrophil activity myeloperoxidase (MPO)-mediated and oxidative stress by malondialdehyde (MDA) determination. Mice body weight was assessed as well.

Results: As expected, 5-FU induced a significant weight loss (∼17%, P < 0.001), shortening in villi height (∼55.4%) and crypts depth (∼47%), and increased (∼64%) the histological severity score when compared to other groups (P < 0.05). These pathological changes were markedly alleviated by the three MFC treatment doses (P < 0.05), in special with the dose MFC 15 mg/kg. This dose also stimulated cell proliferation by ∼90% in the epithelial cells lining from villi and crypts (P < 0.05), reduced MPO levels and MDA formation by 60% and 44%, respectively (P < 0.05).

Conclusions: Our data suggest the therapeutic potential of the formulation for treating intestinal mucositis in mice. Supplementary studies are underway searching for the elucidation of mechanisms involved in the protective effects of MFC in order to make this formulation a clinical tool for mucositis treatment.

Keywords: 5-Fluorouracil; Curcuma longa L; Curcuminoids; Intestinal mucositis; Mucoadhesive formulation.

Fig. 1

Schematic representation of MFC treatment and intestinal mucositis induction by 5-FU.

Fig. 2

Body weight variation in mice subjected to intestinal mucositis and treated with MFC in relation to initial mass. Experiments performed in triplicate with 5 animals per group. Mice were daily weighed throughout the experimental period (7 days). Data ​​expressed as mean ± SEM. ^***P < 0.001 comparing 5-FU vs Control; ^#P < 0.05 comparing 5-FU vs MFC 3.75 mg/kg + 5-FU; ^##P < 0.001 comparing 5-FU vs MFC 7.5 mg/kg + 5-FU; and ^###P < 0.001 comparing 5-FU vs MFC 15 mg/kg + 5-FU. For statistical analysis, one-way ANOVA and Bonferroni post-tests were applied.

Fig. 3

Morphometric analysis of mice duodenum subjected to intestinal mucositis and treated with MFC. On day 7, duodenum segments were removed to measure villus height (panel A) and crypt depth (panel B). Data expressed as mean ± SEM. ^*P < 0.05 comparing 5-FU vs Control and ^#P < 0.05 comparing 5-FU vs MFC 3.75 mg/kg + 5-FU, MFC 7.5 mg/kg + 5-FU, MFC 15 mg/kg + 5-FU. For statistical analysis, two-way ANOVA and Bonferroni post-tests were applied.

Fig. 4

Histological sections of mice duodenum subjected to intestinal mucositis and treated with MFC. Control (panels A and B) showing normal villi and crypts. 5-FU (panels C and D) showing villi shortening (arrows), crypts deepening, intense inflammatory infiltration, vacuolization and mucosal edema (arrowheads). MFC 3.75 mg/kg + 5-FU (panels E and F), MFC 7.5 mg/kg + 5-FU (panels G and H), MFC 15 mg/kg + 5-FU (panels I and J) showing villi and crypts preserved – both in size and integrity –, however, inflammatory infiltrate was observed. H&E staining (panels A, C, E, G, I; 20X and B, D, F, H, J; 40X).

Fig. 5

Expression of Ki-67 (cell proliferation), p53 (pro-apoptotic), Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) in mice bearing intestinal mucositis and treated with MFC. See black arrows. Immunohistochemical staining, original magnification, 40X. Data expressed as mean ± SEM. ^*P < 0.05 comparing 5-FU vs Control, ^#P < 0.05 comparing 5-FU vs MFC 15 mg/kg. For statistical analysis, two-way ANOVA and Bonferroni post-tests were applied.

Fig. 6

Effect of MFC treatment on MPO activity and MDA determination in duodenal portions of mice bearing intestinal mucositis. Experiments performed in triplicate with 5 animals per group. Data expressed as mean ± SEM. ^*P < 0.05 comparing 5-FU vs Control, ^#P < 0.05 comparing 5-FU vs MFC 15 mg/kg. For statistical analysis, one-way ANOVA and Bonferroni post-tests were applied.