Fig 1.
A schematic outline of the experimental design for the six study groups (n = 6 in each group).
In the first group, saline was given once at time point 0 h (control). In 4 groups, gastrointestinal (GI) toxicity was induced by a single intraperitoneal (IP) injection of 5-Fluorouracil (5-FU, 200 mg/kg) at time point 0 h. In 3 of these 5-FU groups, a treatment with melatonin (10 mg/kg) and/or misoprostol (1 mg/kg) was given once a day (OD) for 4 days (-24 to 48 h). In the last group, the combination of melatonin and misoprostol was given once daily for 4 days but without any induction of GI toxicity with 5-FU. At 72 h, the rats were sedated and samples were collected.
Fig 2.
Preventive and treatment effects of melatonin (Mel, IP 10 mg/kg daily starting 24 h before 5-FU) and/or misoprostol (Mis, IP 1 mg/kg daily starting 24 h before 5-FU) on jejunal villus height following systemic exposure to 5-Fluorouracil (5-FU, IP 200 mg/kg).
Each point represents the mean value from a single animal based on ten separate determinations, the short horizontal line signifies the group mean. The statistical data analysis was performed with an ordinary one-way ANOVA test with Dunnett’s multiple comparisons test, and significant comparisons were indicated by one (p < 0.05), three (p 0.001), or four (p < 0.0001) stars.
Fig 3.
a-f. Apoptosis (number of apoptotic cells per crypt determined with TUNEL-positive staining) in jejunum samples 72 h after IP dosing of 5-Fluorouracil (5-FU, 200 mg/kg) and melatonin (Mel, IP 10 mg/kg daily) and/or misoprostol (Mis, IP 1 mg/kg daily). Representative tissue images for each study group are shown in Fig 3A-3E for saline (1 ml/kg Control), 5-FU only, and 5-FU with daily dosing (starting 24 h before 5-FU dosing) of melatonin, misoprostol, or melatonin and misoprostol. The results from the staining analysis are shown in Fig 3E, where each point represents the value from a single animal and the horizontal line signifies the group mean. The statistical data analysis was performed with a Kruskal-Wallis test with Dunn’s multiple comparison test. The different treatments were not significantly different from each other. Scale bars represent 100 μm.
Fig 4.
a-f. Proliferation (percentage of sample area stained for Ki67) in jejunum samples 72 h after IP dosing of 5-Fluorouracil (5-FU, 200 mg/kg) and melatonin (Mel, IP 10 mg/kg daily) and/or misoprostol (Mis, IP 1 mg/kg daily). Representative tissue images for each study group are shown in Fig 4A-4E for saline (1 ml/kg Control), 5-FU only, and 5-FU with daily dosing (starting 24 h before 5-FU dosing) of melatonin, misoprostol, or melatonin and misoprostol. The results from the staining analysis are shown in Fig 4E, where each point represents the value from a single animal and the horizontal line signifies the group mean. The statistical data analysis was performed with a Kruskal-Wallis test with Dunn’s multiple comparison test. The different treatments were not significantly different from each other. Scale bars represent 100 μm.
Fig 5.
Colonic fecal water content (%) reflects diarrhea 72 h after dosing of 5-Fluorouracil (5-FU, 200 mg/kg) and melatonin (Mel, 10 mg/kg daily starting 24 h before 5-FU) and/or misoprostol (Mis, 1 mg/kg daily starting 24 h before 5-FU).
Each point represents the percent water content from a single animal, the horizontal line signifies the group mean. The dotted line at 80% represents a threshold from which diarrhea can be assumed. The statistical analysis was performed with a Brown-Forsythe and Welch ANOVA test with Dunnett’s T3 multiple comparisons test. Comparisons with p < 0.05 were considered significant, indicated by one star.
Fig 6.
Body weight loss 72 h after dosing of 5-Fluorouracil (5-FU, 200 mg/kg) and melatonin (Mel, 10 mg/kg daily starting 24 h before 5-FU) and/or misoprostol (Mis, 1 mg/kg daily starting 24 h before 5-FU).
Each point represents the percent water content from a single animal, the horizontal line signifies the group mean. The statistical data analysis was performed with a Kruskal-Wallis test with Dunn’s multiple comparison test. Comparisons with p < 0.05 were considered significant and significant comparisons were indicated with one (p < 0.05) or three (p < 0.001) stars.
Fig 7.
Expression of the MT2 melatonin receptor in rat jejunum.
Animals administered 5-FU and melatonin (a-c) or 5-FU alone (d-f). Original microscopy pictures are shown in a and d. Color deconvolution was performed to split the different color channels and show only the DAB portion (b, e). Finally, a threshold was set to highlight the areas stained with a higher intensity (in red) from unspecific background staining and (c, f).
Fig 8.
Murine intestinal organoids experiments.
(A) In vitro growth of healthy murine intestinal organoids. Representative images showing early (a-c) and late (d-f) stages of development. Morphology and viability were assessed by light microscopy (a, d), haematoxylin and eosin staining (b, e) and fluorescent microscopy (c, f). (B) Cell viability curves of murine intestinal organoids treated with either 5-FU (0–100,000 μM) or 5-FU + melatonin (0–100,000 μM and 0.1 mM respectively). Data points represent the median value ± 95% CI (n = 2–4 organoids/treatment) from 3 independent experiments. (C) 3-D confocal immunofluorescence imaging of murine intestinal organoids embedded in BME. Representative images of organoids treated with either 5-FU (10 μM), 5-FU (10 μM) + melatonin (0.1 mM) or control. Blue = DAPI (nuclei), green = calcein (live cells), red = ethidium (dead cells). Scale bars = 100 μm.