Discussion

Since 1975, when it was reported that a high intake of marine fish containing n-3 fatty acids is associated with a low prevalence of coronary artery disease⁽Reference Dyerberg, Bang and Hjorne¹⁵⁾, administration of fish oil-based nutritional formulas has evolved from a supportive to a therapeutic intervention in a number of critical care settings. Over the past 20 years, many studies and clinical investigations have demonstrated that n-3 PUFA are essential for normal growth and development and play an important role in the prevention and treatment of coronary artery disease, hypertension, diabetes, arthritis, other inflammatory and autoimmune disorders, and cancer⁽Reference Simopoulos, Kifer and Wykes¹⁶⁾. Several experiments have demonstrated the positive effects of n-3 PUFA in preventing the complication of chemotherapy, especially cyclosporine use⁽Reference Levy and Alexander¹⁷⁾.

The phospholipids of immune cells taken from rodents maintained on normal laboratory chow typically contain 15–20 % of fatty acids as arachidonic acid and contain very little of the very long-chain n-3 PUFA⁽Reference Calder, Yaqoob and Harvey^18, Reference Yaqoob, Newsholme and Calder¹⁹⁾. Ingestion of EPA and DHA from fish partially replaces n-6 fatty acids (especially arachidonic acid) in cell membranes, especially those of platelets, erythrocytes, neutrophils, monocytes and liver cells, leading to decreased production of PGE2 metabolites, decreased concentrations of thromboxane A2 (a potent platelet aggregator and vasoconstrictor), decreased formation of leukotriene B4 (an inducer of inflammation and a powerful inducer of leucocyte chemotaxis and adherence), increased concentrations of thromboxane A3 (a weak platelet aggregator and vasoconstrictor), increased concentrations of prostacyclin PGI3 (active vasodilators and inhibitors of platelet aggregation), and increased concentrations of leukotriene B5 (a weak inducer of inflammation and chemotactic agent)⁽Reference Weber, Fischer and von Schacky^2, Reference Lewis, Lee and Austen²⁰⁾. The recognition that n-3 PUFA possess a range of anti-inflammatory and immunomodulatory properties has prompted a series of studies investigating their efficacy in animal models of inflammatory bowel disease⁽Reference Shoda, Matsueda and Yamato²¹⁾ as well as in clinical trials⁽Reference Hillier, Jewell and Dorrell^22, Reference Haegebarth and Clevers²³⁾. The effect of n-3 PUFA during chemotherapy-induced intestinal damage has not been previously investigated.

The pathogenesis of chemotherapy-induced gastrointestinal mucositis has been described by Sonis et al. ⁽Reference Sonis, Elting and Keefe¹³⁾, and includes five phases: initiation by chemotherapy; up-regulation and generation of messenger signals; signalling by pro-inflammatory cytokines and amplification of mucosal injury; ulceration of the mucosa; and, finally, healing. The initial stages of inflammation in mucositis include increased pro-inflammatory cytokine levels, which act as a homing marker for inflammatory immune cells in the submucosa. Since anti-inflammatory properties of n-3 PUFA have been assessed in a number of disease settings⁽Reference Loeschke, Ueberschaer and Pietsch^6–Reference Schmöcker, Weylandt and Kahlke¹⁰⁾, we hypothesised in this experiment that n-3 fatty acids might reduce the severity of chemotherapy-induced mucositis.

The present study demonstrates that enteral n-3 PUFA in the control animals did not change significantly intestinal mucosal parameters. This conclusion is supported by unchanged bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth.

Consistent with our previous experiments⁽Reference Sukhotnik, Mogilner and Karry^24, Reference Sukhotnik, Mogilner and Shteinberg²⁵⁾, the present findings have shown that MTX-induced mucositis results in significant mucosal damage. This conclusion is supported by the observed increase in intestinal injury score compared with the control animals. Histologically, MTX animals exhibited degeneration and shortening of the villus length, severe villous epithelial atrophy, a significant loss of the crypt architecture, signs of crypt remodelling, and polymorphonuclear leucocyte infiltration in the lamina propria. Vacuolisation in surface epithelium cells was minimal. In addition, MTX rats showed intestinal mucosal hypoplasia. This is evident from decreased bowel and mucosal weight, decreased mucosal DNA and protein, and decreased villus height and crypt depth in this model. Decreased body weight in MTX rats may suggest a malnutrition state. Parallel decreases in mucosal DNA and protein indicate that the smaller intestinal mass of MTX animals can be attributed to cellular hypoplasia. The most significant differences were observed in the terminal ileum, while hyperplasia in the proximal jejunum was less prominent. BrdU was used in our experiment to determine an index of crypt cell proliferation. This analogue of thymidine is incorporated into the DNA of proliferating cells during the S-phase of the cell cycle. The present findings suggest that proliferation of crypt cells decreased significantly following MTX administration, which was closely correlated with decreased crypt depth. MTX and its active metabolites block tetrahydrofolate synthesis by binding to the folic acid site on the enzyme dihydrofolate reductase. This action results in the depletion of nucleotide precursors, and the inhibition of DNA, RNA, protein synthesis and cellular proliferation. Decreased cell proliferation rate was accompanied by lower β-catenin levels. Extensive experimental evidence suggests that Wnt/β-catenin signalling plays a central role in maintaining the intestinal stem-cell niche and in regulating differentiation of stem cells within the intestinal epithelium towards either enterocytes or one of three secretory cell lineages⁽Reference Haegebarth and Clevers²³⁾. Cell loss in the small intestine of MTX-induced mucositis is mainly regulated by programmed cell death. The present results show that the intrinsic pathway, with its regulation by the Bcl2 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 anti-apoptotic Bcl2 gene decreased. Changes in protein levels are in agreement with mRNA expression. Bax protein level was significantly up-regulated in MTX-treated rats compared with the control animals. These changes correlated with the enhanced enterocyte apoptosis during MTX-induced mucositis.

In the present study, administration of enteral n-3 PUFA in MTX rats decreased mucosal inflammation and reversed intestinal mucosal damage. This is evident from the decreased intestinal injury score. Although the mucosa was still severely damaged, MTX-n-3 PUFA rats showed the presence of newly formed crypts and regeneration, less significant villous epithelial atrophy and less significant polymorphonuclear leucocyte infiltration in the lamina propria. The mechanisms of this effect are unknown. Further studies are required to determine whether decreased generation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) as well as decreased activation of NF-κB signalling, decreased leucocyte chemotaxis and decreased T-cell reactivity, which were described in an animal model of inflammatory bowel disease⁽Reference Calder²⁶⁾, may be responsible for the observed protective effects of n-3 PUFA in chemotherapy-induced mucositis. In addition, reduced inflammation was coupled with enhanced intestinal repair. Enteral n-3 PUFA significantly increased overall bowel and mucosal weight, which occurred together with a synergistic increase in mucosal DNA and protein levels, which indicates that the greater intestinal mass is attributed to cellular hyperplasia. n-3 PUFA also caused a significant stimulation of cell proliferation and a concomitant decrease in cell apoptosis compared with MTX-untreated rats, which suggests an activated enterocyte turnover and may be considered as one of the main mechanisms of mucosal hyperplasia in recovering bowel. It has been suggested that incorporation of n-3 PUFA into the intestinal mucosa may have altered mucosal function and could have contributed to the regulation of growth and transduction signals such as those involving protein kinase C⁽Reference Calderaro, Parrillo and Balestrieri²⁷⁾; however, further investigation is required to ascertain the mechanisms by which n-3 PUFA enhance repair in the intestinal mucosa. The transmission of extracellular proliferation and differentiation signals into their intracellular targets is mediated by a signalling cascade culminating in mitogen-activated protein kinase (MAPK). MAPK are serine/threonine-specific protein kinases that regulate various cellular activities, such as gene expression, mitosis, cell proliferation, differentiation and apoptosis⁽Reference Pearson, Robinson and Beers Gibson²⁸⁾. One of the MAPK signalling pathways triggered by cytokines or growth factors is the ERK pathway. In the present study, the observed increase in enterocyte proliferation rate in MTX-n-3 PUFA animals was accompanied by elevated β-catenin and phosphorylated ERK protein levels, which suggests activated stem cell activity and a stimulated MAPK signalling pathway. Treatment with n-3 PUFA also resulted in decreased cell apoptosis. Bax mRNA levels were down-regulated, while Bcl2 mRNA was up-regulated in n-3 PUFA-treated rats compared with MTX-non-treated animals. Thus, the Bax:Bcl2 ratio decreased in MTX-n-3 PUFA rats compared with MTX animals, suggesting increased enterocyte survival. These results suggest that n-3 PUFA may inhibit chemotherapy-induced apoptosis by concomitant stimulation of the anti-apoptotic Bcl2 gene and inhibition of the pro-apoptotic Bax gene expression. Both elevated cell proliferation and decreased cell apoptosis may be considered as one of the main mechanisms responsible for compensatory hyperplasia and, thus, responsible for increased intestinal cell mass.

The mechanisms of the positive effects of PUFA on intestinal mucosal homeostasis are poorly understood. Several experiments have demonstrated that exposure of animals to a PUFA-enriched diet results in an increased PUFA concentration in ileal mucosa. In the present study, we have also shown the stimulating effect of PUFA on intestinal mucosa in the ileum. Since PUFA are absorbed mostly in the proximal intestine, it should be emphasised that the effects in the distal intestine are probably systemic. However, the local effect of small amounts of PUFA that did not get absorbed in the proximal jejunum cannot be excluded. Since changes in serum and tissue levels of NF-κB, TNF, IL-1β and IL-6 have been demonstrated during chemotherapy-induced mucositis⁽Reference Logan, Stringer and Bowen²⁹⁾, further studies are required to evaluate cytokine and key inflammatory protein tissue levels to define trafficking pathways of anti-inflammatory effects of PUFA in this model.

In conclusion, enteral n-3 fatty acids reverse intestinal damage and stimulate intestinal recovery following MTX-induced intestinal damage in a rat. Enhanced cell proliferation and inhibited programmed cell death (through the up-regulation of Bcl2 and the down-regulation of Bax expression) may be responsible for this effect. Enteral n-3 fatty acids may be clinically beneficial in preventing intestinal damage and in stimulating intestinal recovery in patients with chemotherapy-induced mucositis.