In the last two decades, arginine has attracted major interest since it has been identified as the natural substrate of nitric oxide, and is now recognized to play a major role in many regulation processes. Nitric oxide is a multifunctional intercellular messenger molecule that plays an important role in a variety of physiological processes. Nitric oxide is synthesized from L-arginine by the inducible nitric oxide synthase (iNOS). Arginine and nitric oxide are critical to the normal physiology of the gastrointestinal tract and maintain the mucosal integrity of the intestine in various intestinal disorders. Arginine stimulates intestinal cell migration and intestinal protein synthesis through a focal adhesion kinase dependent mechanism in vitro [13
]. Arginine supplementation enhances intestinal growth and development in weanling piglets [14
]. Dietary supplementation with arginine stimulates small intestinal mucosal recovery following experimental radiation enteritis [15
] and accelerates ulcer healing in experimental ulcerative ileitis [16
]. In a recent study, we have shown that oral arginine decreases intestinal injury caused by lipopolysacharide endotoxemia in a rat [9
]. In another study, we have demonstrated that oral arginine administration improves mucosal recovery following intestinal ischemia-reperfusion injury in a rat [10
]. The mechanisms of these positive effects are still unclear; however, a stimulating effect of appropriate amounts of NO on enterocyte proliferation and a suppressive effect on enterocyte death via apoptosis may be considered as one of them.
The role of arginine in prevention of chemotherapy-induced intestinal damage is unclear. Hämäläinen Met al have shown recently that chemotherapy inhibites iNOS expression, and subsequent NO production, in a dose-dependent manner at therapeutically achievable drug concentrations in a human colon epithelial cell line [17
]. In a recent experiment, Gulgun et al. have demonstrated that proanthocyanidin, arginine and glutamine supplementation had a positive effect in the protection of the small intestine from methotrexate-induced injury [18
]. The mechanisms of this effect were not investigated. In a recent clinical trial, Izaola et al. have shown that arginine and glutamine enhanced formula decreases the rate of radiotherapy-induced oral mucositis in patients with head and neck cancer [19
Methotrexate is the most commonly used anti-metabolite agent in clinical oncology practice. Since ARG maintains the mucosal integrity of the intestine in various intestinal disorders, we hypothesized in the present study that this amino acid could prevent intestinal mucosal injury or/and improve intestinal recovery following MTX-induced mucositis. Alterations in bowel and mucosal weights, mucosal DNA and protein content and histological appearance were measured. BrdU was used in our experiment to determine an index of crypt cell proliferation. Immunohistochemistry for caspase-3 was used to characterize enterocyte apoptosis. We have demonstrated that a single dose of MTX caused a severe mucosal injury in the small bowel, indicated by severe villous atrophy, epithelial flattening, intensive crypt loss and signs of crypt remodeling, which was accompanied by marked cellularity and an increased number of blood vessels in stroma. In addition, treatment with MTX resulted in significant mucosal hypoplasia. A decrease in bowel and mucosal weight, mucosal DNA and protein, and decrease in villus height support this conclusion. Parallel decreases in mucosal DNA and protein indicate that the smaller mucosal mass of MTX animals can be attributed to cellular hypoplasia. Histologically, villus height decreased in response to MTX administration, suggesting decreased absorptive surface area. Mucosal DNA content as well as the enterocyte proliferation index decreased significantly in both jejunum and ileum following MTX administration. MTX is an analog of folic acid and works by attaching to dehydrofolate reductase to inhibit DNA production. As a result crypt proliferation in the small bowel is inhibited. In addition, the proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction. The mechanism responsible for this effect is poorly understood. Verburg and coworkers have shown in MTX-treated rats that BrdU-positive cells are not restricted to the crypts but are also found in up to one-third of the length of the villi due to migration of the cells [20
]. Our data suggest that MTX animals had lower β-catenin levels which fits with decreased cell proliferation. Extensive experimental evidence suggests that Wnt/β-catenin signaling plays a central role in maintaining the intestinal stem-cell niche and in regulating differentiation of stem cells within the intestinal epithelium toward either enterocytes or one of three secretory cell lineages (Paneth, goblet, or enteroendocrine cells [21
]. Cell loss in the small intestine MTX-induced mucositis is mainly regulated by programmed cell death. Our results show that the intrinsic pathway, with its regulation by the bcl-2 family of proteins, was altered by MTX consistent with changes in cell apoptosis: the mRNA levels of the pro-apoptotic gene bax increased, while those of the antiapoptotic bcl-2 gene decreased. With regard to the protein level, qualitative and quantitative changes were in agreement with mRNA expression. Bax protein levels were significantly up-regulated while bcl-2 protein levels were down regulated in MTX-treated rats compared to control animals. These changes correlate with the enhanced enterocyte apoptosis during MTX-induced mucositis.
Results of the present study show that dietary arginine protects the intestinal mucosa from damage caused by MTX. While MTX rats showed severe villous atrophy, epithelial flattening, extensive crypt loss and signs of crypt remodeling, marked cellularity and an increased number of blood vessels in stroma, arginine-treated rats showed more preserved architecture as well as the presence of newly formed crypts and regeneration. While the proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction, the proliferative zone of MTX-ARG rats was only mildly affected, showing a slight shift upwards within the crypts. In addition, exposure to oral arginine significantly enhanced intestinal recovery following MTX-induced damage. This is evident from the significant increase in bowel and mucosal, increased DNA and protein content in ileum. Increased mucosal weight without changes in mucosal surface area may suggest that mucosal hyperplasia rather than bowel enlargement or intestinal muscle hypertrophy is responsible for the increased intestinal mass. Increase in mucosal DNA and protein along with hypertrophy of the individual cells which we have demonstrated morphometrically is characteristic of tissues undergoing increased cell proliferation or repair. Histologically, marked increases in villus height in both jejunum and ileum suggest increased absorptive surface area and closely correlate with increased cell mass. The present data suggest that arginine did not change significantly mucosal proliferation in functioning intestine, but decreased significantly cell apoptosis rate, which may represent the main mechanism that maintains mucosal structure following MTX-induced damage. Our results show that the intrinsic pathway, with its regulation by the bcl-2 family of proteins, was altered by arginine in accordance with changes in cell apoptosis: the mRNA and protein levels of the pro-apoptotic bax decreased, while those of the antiapoptotic bcl-2 protein levels increased. Correspondingly, bax/bcl-2 ratio decreased in MTX-ARG rats compared to MTX animals, suggesting increased enterocyte survival. Further investigation is needed to define the regulation of this special apoptotic state with respect to the Fas/Fasl-mediated extrinsic pathway.
The mechanism of the positive effects of arginine is unclear. In non-operated, non-stressed animal, total food intake is 20-25 g/rat/day, total protein intake is about 6 g/rat/day, and total energy intake is 100 kcal/rat/day [22
]. We do not believe that an additional 300 mg/rat/day of protein (vs 6 g of total protein) as well as additional energy of 1.2 kcal/rat (vs total 100 kcal/rat/day) may have influenced the results. The limitation of the current experiment is a lack of isonitrogenous MTX group. Therefore, this study is not able to prove that arginine supply limits intestinal damage. It was previously reported that other amino acids (like glutamine, glycine or histidine) have protective effects on intestinal mucosa during chemotherapy or inflammatory conditions. An additional MTX-isonitrogenous amino acid group will be added in future study to determine whether other amino acids have similar effects.