2. Methods
Chemicals were obtained from the following manufacturers: Methotrexate (MTX; Immunex Corporation; Seatle, WA); Camellia sinensis (laboratório Brasmed, Brazil); Vitamins E (Laboratório Sundow). Diets included total nitrogen content around 10% - 13%. All groups consumed commercial chow (Fri-Ribe—SP, Brazil).
Animal experiments were performed with permission of the Experimental Ethical Committee of Federal University of Ceará-Brazil; and the protocol was complied with the Occupational Health and Safety in the Care and Use of Research Animal, National Research Council (National Academic Press, 1977). Male adults Swiss Mus musculus (n = 32, 35 - 40 g) from Pharmacology and Physiology Department, Federal University of Ceará, were used throughout the experiments. Animals were housed in individual metabolic cages, maintained under a 12 hours light-dark cycle, and controlled access to a standard palletized diet and water. Mice were monitored daily, for signs of diarrhea, metabolic control and mortality. An experimental model of intestinal mucositis in rats as described previously [12], was modified and adapted for mice.
Control (PBS, phosphate buffer solution, via gavage), methotrexate (2.75 mg/kg/24 h sc) and Camellia sinensis decoction and vitamin E (10 IU/animal/24 h via gavage) groups (n = 8/group) were treated during three days. Tea decoction (20%) was prepared by soaking 20 g of green tea in 100 ml boiled water separately, soaked for 2 minutes and thereafter filtered. This filtrate was designated as “green tea decoction”. The dose of this decoction orally administered to each mice was 0.1 ml/10g of body weight. Initial pilot study suggested that 20% of green tea preparation had given significant results, than 10% green tea. Therefore, we have decided to set forward our study with 20% green tea decoction. Since the maximum mucosal damage caused by MTX occurs after 72 hours after the MTX administration, and MTX+CS (methotrexate plus Camellia sininsis) or vitamin E (methotrexate plus vitamin E), all the mice were sacrificed (at 4th day), under general anesthesia (50 mg/kg ketamine + 10 mg/kg xilazine im). Intestines of the mice were removed for histological examination just after they have been sacrificed, and put in formalin after being washed with iced isotonic saline solution. Histopathological changes in the intestinal cells were examined via qualitative blindly histological evaluation by a pathologist. On the histological examination performed after the routine tissue processing, villus length and the crypt depth were morphometrically evaluated analyzing the tissue sections stained with hematoxylin eosin (H&E), measurements were done on 40 villi from 40 crypts per animal. To examine apoptotic events, sections were examined under light microscopy. Counts of apoptotic cells and apoptotic bodies were performed using 40× objective. Cells were scored as apoptotic if they exhibited cellular shrinkage with concurrent cytoplasmic eosinophilia, nuclear pyknosis, and fragmentation with associated apoptotic bodies. Areas of marked lymphocytic infiltration or necrosis were not used for counting.
3. Statistical Analyses
Statistical analysis was performed using GraphPad Prism 5.01 (GraphPad Software, San Diego, CA) and consisted of a two-way ANOVA with Bonferroni post hoc tests when appropriate. And groups were tested for homogeneous variance then used a Student’s t test. Data are presented as mean ± standard error of the mean. For daily monitored parameters, repeated measures two-way ANOVA was performed. For all, p < 0.05 was considered significant.
4. Results
4.1. Histomorphologic Approach
Mucositis condition was achieved at mice treated with methotrexate (2.75 mg/kg/day sc). Subcutaneous route was chosen, to avoid direct intestinal insult, but induce a systemic delivery of metabolites inhibitors of cell proliferation at barrier function, reproducing an inflammatory small intestine status (Panel 1(b)) when compared with PBS treated group (Panel 1(a)—Foliaceous villi with many goblet cells). No changes were found at ileum segments at all groups. In addition, vitamin E and CS supplemented animals presented more goblet cells at villous (Panels 1(c) and (d)—black single arrows). Methotrexate induced mild goblet cell depletion, and villi atrophy in the small intestine during a period of epithelial damage and apoptotic events (Panel 1(b)—white doubleheaded arrow).
4.2. Intestinal Morphometry
Similarly, in this present study showed at Figure 1 (duodenum) and Figure 2 (jejunum), villus length was found shortened in the MTX group, at duodenum (D; 364.8 ± 19.90 vs 437 ± 19.92 μm) and at jejunum (J; 363.3 ± 20.65 μm as compared to the control group (426.0 ± 15.14 μm; p < 0.01). Crypt depth was found as hyperplasic feature at mice with intestinal mucositis. CS (D; 155 ± 6.88; J, 126.8 ± 5.65 μm) and VITE (D; 241.5 ±
16.2 J; 122 ± 5.78 μm) supplementation did not alter villous atrophy when compared as control (D; 98.0 ± 5.26 μm; J vs 114.0 ± 4.76 μm), however reestablished crypt morphometry. Methotrexate (9.3 ± 0.68) administration induced a marked increase of apoptotic bodies at duodenum compared with control mice (0.88 ± 0.17). Camellia sinensis (1.64 ± 0.78) but not vitamin E (9.2 ± 0.61) ameliorates cellular death induced by methotrexate. Shortening in the villus length, as well as the decrease in the mucosal thickness and villus length/crypt ratio, is likely to occur in the MTX-induced mucositis models in rats. Villous/crypt length ratios of control group were 3.8 at duodenum and 4.27 at jejunum; in opposite to MTX animals (1.89 at duodenum and 2.42 at jejunum), in agree with intestinal morphometry.
Panel 1. The morphology and histopathology of the jejunum treated with PBS or methotrexate (MTX; 2.5 mg/kg, s.c.) (a) Normal crypts and villi from control duodenum (100×); (b) The duodenum (100×) from an animal treated with MTX, observe the blunted villi and hyperplasic crypts compared with the control. Photomicrographs of supplemented groups; (c) Vitamin E; and (d) Camellia sinensis at mucositis condition.
Duodenum
(a)
(b)
Figure 1. Morphometric analysis of vitamin E supplementation and CS decoction in mice by methotrexate-induced intestinal mucositis (MTX) at duodenum. (a) Villous height; and (b) Crypt depth (μm) of animals treated with MTX (2.75 mg/kg/24 h sc) or PBS (control) during 3 days. Mice were supplemented with vitamins E (10 IU/animal) at the same period of MTX administration. Morphometry was performed with optical microscopy using an eyepiece (scale of 1 mm). Results are presented as mean ± SEM, #p < 0.05, MTX compared with control, and *p < 0.05, MTX compared with vitamins groups (ANOVA, Bonferronis’s test).
4.3. Apoptotic Events
At green tea 20% decoction group, no mortality was achieved, similar to those results at ulceration ear mice experimental model induced by MTX. As expected, MTXinduced mucositis mice, presented (Figure 3) more intestinal cell death (9.30 ± 0.16 apoptotic bodies/field/ sample) when it compared to control (0.78 ± 0.02). Vitamin E supplementation do not alter apoptotic events (8.60 ± 0.07), however CS supplementation (2.40 ± 0.03 apoptotic bodies/field/sample) reduces intestinal cell death mainly at jejunum at inflammatory condition.
5. Discussion
Apart from fundamental basic research investigating Jejunum
(a)
(b)
Figure 2. Morphometric analysis of vitamin E supplementation and CS decoction in mice by methotrexate-induced intestinal mucositis (MTX) at jejunum. (a) Villous height; and (b) Crypt depth (μm) of animals treated with MTX (2.75 mg/kg/24 h sc) or PBS (control) during 3 days. Mice were supplemented with vitamins E (10 IU/animal) at the same period of MTX administration. Morphometry was performed with optical microscopy using an eyepiece (scale of 1 mm). Results are presented as mean ± SEM, #p < 0.05, MTX compared with control, and *p < 0.05, MTX compared with vitamins groups (ANOVA, Bonferronis’s test).
Figure 3. Duodenal apoptosis analysis in mice with intestinal mucositis induced by methotrexate (MTX, 2.75 mg/kg/ 24 h s.c). Mice were supplemented with and E (10 IU/animal). Legends: Control (PBS); MTX (methotrexate), CS (Camellia sinensis), VITE (vitamin E). Morphometry were performed by optical microscopy using an eyepiece (scale of 1 mm), HE staining. Results are presented as mean ± SEM, #p < 0.05 (MTX compared with control, and *p < 0.05, MTX compared with others groups (ANOVA, Bonferronis’s test).
mechanisms by which the latter stages of cancer can be inhibited, there are no nutraceuticals that have advanced to the point of clinical trial testing. However, this is a promising area of basic research in cell lines and in animal models. At intestinal mucositis induced by methotrexate administration, the toxic effects are not considered to be a result of direct action on the gastrointestinal tract tissues but rather to be the consequence of an inhibition in dihydrofolate reductase synthesis. This enzyme is required to maintain the intracellular pool of tetrahydrofolate during purine and thymidine synthesis. The chemical and morphologic changes in the small intestine may be triggered by crypt cell damage [13]. To minimize the side effects in patients undergoing chemotherapy, it is important to reduce mucosal damage and stimulate tissue repair. Such intestinal damage is reported to be prevented by some kinds of nutrients and growth factors. Plant products are in use for a long time in Ayurvedic and folklore medicine for the cure of ailments with minimal side effects and comparable efficacy. Green tea term (Camellia sinensis) nominate a great variety of Thernaceae family plants, including 520 species, endemic in tropical and sub-tropical areas of Asia and South America. Green tea decoction has a medicinal use in these countries due to beneficial and potent antioxidants properties. As a medicinal beverage, it is recommended to nauseas, vomiting and at diarrhea condition. Antibacterial action had been used at infections and at wounds treatment [14]. Aside from the pluripotent effects of green tea polyphenols, exist an increasing list of molecular processes, including oxidative stress [15]. Cathechins, mainly (-)-epigallocatechin-3-gallate (EGCG), represent the largest group of flavonoids (flavan-3-ols) in the tea plant, in particular in fresh leaves and green tea. Injected and orally administered green tea cathechins, in vitro, significantly decreased bacterial LPS-induced alveolar bone desorption and IL-1 expression in the gingiva of BALB/c mice [15,16]. Initially in mucositis development, direct DNA damage caused by the chemotherapy agents might lead to cellular death in the basal epithelium and submucosal cells, but clonal cell death occurs in only small part of the damaged cells. Vitamin E and CS protection occurred probably due a retardation of inflammatory process at progenitors intestinal crypt cells [17-19]. On the other hand, the methods used at present study are insufficient to explain completely the intensive damage at mucositis; but present results demonstrated clearly an amelioration of intestinal barrier morphology. Gastrointestinal impaired function could be positively modulated by Camellia sinensis [20]. At green tea 20% decoction group, no mortality was achieved, similar to those results at ulceration ear mice experimental model induced by MTX. These results agree with our finds at intestinal mucositis model. However, at this challenge, green tea treatment did not affect the main effect of methotrexate (arresting cell division in S-phase) [21]. Data suggest that an offer of CS or vitamin E supplementation could treat villous atrophy, cells crypt hyperplasia and increase of intestinal apoptosis, induced by MTX challenge.
6. Conclusion
Marked intestinal barrier function disruption was caused by a methotrexate administration. In addition, a protecting effect of Camellia sinensis decoction at small intestine of mice with mucositis, was achieved similarly to antioxidant action of vitamin E. Probably, cathechins compounds of this green tea, initiate an antioxidant protection. However more pharmacological and biochemical assays are essential to elucidate protector mechanism, and potential active compounds.
Acknowledgements
Scientific Initiation Program (CNPq; University of Fortaleza), and a histology technician José Ivan R. De Souza by his valuable work.
NOTES