Main

In food-sensitive enteropathy, villous atrophy, crypt hyperplasia, and infiltration of lymphocytes are commonly observed in patients, especially in infants and young children, who tend to develop malabsorption and chronic diarrhea(1). The pathogenesis of this enteropathy has been studied, and the roles of activated mucosal T cells(2) and the production and regulation of cytokines(3) and LTs(4) have been pointed out.

LTs are potent inflammatory mediators known to induce increased vascular permeability(5), water and electrolyte secretion(6), and vasoconstriction(7). Elevation of LTB4 and LTC4 synthesis has been confirmed in hypersensitivity reactions, including atopic dermatitis(8), celiac disease(9, 10), and inflammatory bowel diseases(11). These findings suggest that LTB4 and LTC4 are deeply involved in the pathogenesis of food-sensitive enteropathy; however, the precise mechanisms of the morphologic changes observed in hypersensitivity reactions have not been well explained.

The n-3 fatty acids, such as α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, which are abundant in perilla and fish oil, are known to decrease the production of four-series LTs including LTB4 in the chronic inflammatory diseases(12). EPA supplementation therapy has been reported effective in reducing LTB4 synthesis in patients with ulcerative colitis(13) and Crohn's disease(14); however, the clinical manifestations were not unanimous(15).

In this study, the food-sensitive mouse models were fed with different diets prepared from several types of oil sources, and we investigated the fatty acids composition, morphologic changes, and LTB4 and LTC4 productions in intestine, to confirm the effect of n-6 andn-3 fatty acids in food-sensitive enteropathy.

METHODS

Mice and diets. Female BALB/c mice at 8 wk of age were used. Five groups, consisting of 10 mice each, were prepared. One of the specially designed diets, which had a varied composition of fat produced from soybean oil (used as the control), perilla oil, lard, corn oil, or 0.125 volume of corn oil (low fat diet) was given to mice in each group for 4 wk. These test diets were supplied by Morinaga Milk Co. (Tokyo, Japan). The composition of fat, casein, and sucrose supplements is described in Table 1. In brief, in comparison with the soybean oil diet, the perilla oil diet contained a large amount of α-linolenic acid and a small amount of linoleic acid, the lard diet had a high content of oleic acid and low levels of both linoleic and α-linolenic acids, and the corn oil diet had high linoleic acid and low α-linolenic acid. The low fat diet had a low quantity of both n-6 and n-3 fatty acids. Because both the corn oil diet and the low fat diet were made from corn oil, they both show the same fatty acid composition as shown in Table 1. However, the absolute value of each fatty acid in the low fat diet is 0.125 of that of the corn oil diet because the low fat diet contains 0.125 of corn oil compared with that of the corn oil diet. These diets were designed to have the same amounts of casein, sucrose, and other elements, such as minerals and vitamin A and E, and the caloric content did not differ significantly among the groups. No particular changes were observed in nonsensitized mice when they were fed these test diets.

Table 1 Composition of test diets
Full size table

Generation of mouse model. Food-hypersensitive mice were prepared as previously described(17). CY was injected intraperitoneally (1 mg; 40 mg/kg), and 2 d later 2 mg of OVA were given orally to sensitize the mice to this antigen. After the first sensitization with OVA, each group of mice was fed one of the specially designed diets described above for 4 wk. OVA, 0.1 mg dissolved in drinking water, was given in the last 10 d to produce enteropathy. Mice were killed under anesthesia with ether, and intestinal samples were removed for further examination.

Evaluation of mucosal damage. More than 10 jejunum specimens, each about 1 cm long, were removed from every mouse and washed gently with PBS. Each sample was fixed in Clarke's solution and stained with Schiff's solution(16). More than 10 pairs of villous heights and crypt depths were measured in each sample to confirm the severity of mucosal damage. To estimate the extent of mucosal damage, the ratio between villous height and crypt depth was calculated for each sample. The ratio decreases according to the severity of mucosal damage.

Evaluation of fatty acid composition in RBC membrane. Blood samples were collected in heparinized plastic tubes. Plasma was separated from cells, and RBC membranes were obtained according to the method of Burtonet al.(18). Lipid from RBC membrane was extracted with chloroform:methanol (2:1) and methylated, then the fatty acid composition was analyzed by gas chromatography as previously described(19).

LTB4 and LTC4 production in the challenged intestinal mucosa. Ten pieces of jejunal mucosa obtained from the pre- and postchallenged mice from each group were gently washed twice in cold Ca2+/Mg2+ -free PBS, and the wet weight was measured. The specimen was then incubated with 10 mM calcium ionophore A23187(Calbiochem-Behring, La Jolla, CA) for 15 min at 37°C. After centrifugation at 4°C, the supernatant was removed and stored at -80°C until the assay was performed. LTB4 and LTC4 were purified by HPLC following the method of Odlander and Claesson(20). The elute was evaporated to dryness and resuspended in the RIA buffer. Finally LTB4 and LTC4 immunoreactivities were evaluated in duplicate, using LTB4 and LTC4 3H assay kits (Amersham International, Buckinghamshire) according to the manufacturer's instruction. Production of LTB4 and LTC4 was expressed as picograms/mg of wet weight.

Statistics. Results are expressed as the mean ± SD. Differences between means were determined by the Mann-Whitney U test.

RESULTS

After 4 wk of feeding the specially designed diets, all mice looked healthy except for some mice in the corn oil-fed group, which had become anxious, stormy, and lost fur around their mouths. Slight weight loss of 4.3 ± 1.2 (mean ± SD) g was observed in the corn oil-fed group, but there was no significant difference in weight when compared with other groups.

Mucosal damage, including villous atrophy and crypt hyperplasia, was confirmed in the CY-treated OVA-sensitive mice, and it significantly differed from that of the CY-nontreated group (Fig. 1). As both were fed with soybean oil diet, it is suggested that mucosal damage was introduced by orally administrated antigen in the OVA-sensitive mice as reported previously(17). The mice fed with the lard and corn oil diets showed significant mucosal damage (p < 0.01 in both), whereas those fed with the perilla oil diet did not. In fact, the latter group had almost no mucosal damage and was similar to the CY-nontreated group. In the low fat diet group, the villous height and crypt depth ratio was higher compared with that of corn oil-fed group (p < 0.01); however, no significant difference of this ratio was found when compared with that of the soybean oil-fed group.

Figure 1
figure 1

The ratio between villous height and crypt depth in jejunal samples was determined for the antigen challenged CY-nontreated and CY-treated mice fed with soybean oil, perilla oil, lard, corn oil, and low fat diet. Data are presented as mean ± SD of 10 animals for each group(*p<0.01). Similar results were obtained in three independent experiments.

Full size image

The influence of dietary fat on the fatty acid composition of RBC membrane lipids was evaluated by gas chromatographic analysis. The fatty acids composition of RBC membranes was measured because RBC membranes are the most feasible markers of fatty acid metabolism, and they are easily influenced by diet and organ uptake, the degree to which their analysis can reflectn-3 fatty acids changes in deeper tissue sites(21). The composition of n-6 and n-3 fatty acids in the RBC membrane lipids of mice fed with various diets is shown in Table 2. Good compliance was confirmed by the RBC membrane analysis. In the perilla oil-fed group, n-3 fatty acids such as α-linolenic acid and EPA were significantly increased(p<0.01), and the total n-6 fatty acid content was significantly decreased (p<0.01) as compared with that of the soybean oil-fed group. In contrast, the corn oil-fed group contained almost the same amount of n-6 fatty acids but lower levels of EPA, decosahexaenoic acid, and other n-3 fatty acids in comparison with the soybean oil-fed group (p < 0.01). Both n-6 andn-3 fatty acid levels were significantly lower in the low fat diet group than those of the soybean oil-fed group (p<0.01). In the lard-fed group, although the amount of n-6 fatty acids remained the same, the amount of n-3 fatty acids was reduced to a level lower than that of soybean oil-fed group (p<0.01).

Table 2 The composition of n-6 and n-3 fatty acids in RBC membrane lipids
Full size table

To confirm the precise effect of n-6 and n-3 fatty acids in the production of LTs involved in the induction of mucosal damage, mice were fed with different diets, and LTB4 and LTC4 generation from the jejunal mucosa after stimulation with calcium ionophore A23187 were measured by RIA (Figs. 2 and3). LTB4 and LTC4 was measured because these LTs were considered to be one of the major inflammatory mediators in both allergic reactions and inflammatory bowel diseases(8–11). The synthesis of LTB4 and LTC4 was most strongly stimulated in the corn oil-fed group (p<0.01). In the low fat diet group, the synthesis of LTB4 and LTC4 was significantly suppressed compared with that of corn oil-fed group (p<0.01 in both). There was no significant difference between the low fat diet group and the soybean oil-fed group in relation to these LTs syntheses. In the perilla oil-fed group, LTC4 synthesis was significantly suppressed compared with that of the soybean oil-fed group (p<0.01), but LTB4 synthesis was not. LTB4 synthesis in the lard-fed group was significantly elevated(p<0.05), but LTC4 synthesis was not significantly different compared with that of the soybean oil-fed group.

Figure 2
figure 2

LTB4 production in the mucosa from OVA-sensitive mice model fed with soybean oil, perilla oil, lard, corn oil, and low fat diet are shown. LTB4 production was measured in jejunal mucosa after stimulation with calcium ionophore A23187. Data are presented as mean ± SD of 10 animals for each group (*p<0.05 and**p<0.01). Similar results were obtained in three independent experiments.

Full size image
Figure 3
figure 3

LTC4 production in the mucosa from OVA-sensitive mice model fed with soybean oil, perilla oil, lard, corn oil, and low fat diet were shown. LTC4 production was measured in jejunal mucosa after stimulation with calcium ionophore A23187. Data are presented as mean ± SD of 10 animals for each group (*p<0.01). Similar results were obtained in three independent experiments.

Full size image

DISCUSSION

The fatty acid composition of the RBC membrane in the perilla oil-fed group was high in n-3 fatty acids and low in n-6 fatty acids compared with the soybean oil-fed group. In contrast, corn oil-fed mice contained lower levels of n-3 fatty acids compared with that of the soybean oil-fed group. It is explained because the soybean oil diet contains more α-linolenic acid than that of corn oil diet, although they both have similar n-6 fatty acid composition. Both n-6 andn-3 fatty acids decreased in the low fat diet group. The significant decrease of n-6 and n-3 fatty acid composition of the RBC membrane between the corn oil diet-fed and the low fat diet-fed mice was observed because the low fat diet-fed mice took 0.125 volume of corn oil compared with that of the corn oil diet-fed mice. These results suggest that the composition of fatty acids in dietary fat is reflected well in the fatty acid composition of cell membrane lipids after a 4-wk feeding period in these mice.

Mucosal damage was observed in mice models fed with soybean, lard, and corn oil. The production of LTs was elevated in these mice models, suggesting that locally produced LTs were deeply involved in the progression of mucosal inflammation in food-sensitive enteropathy. Increased LTs synthesis and mucosal damage were most prominent in mice in the corn oil-fed group. They contained lower levels of n-3 fatty acids in comparison with that of soybean oil-fed mice, although both groups contained the same amount ofn-6 fatty acids. This observation suggests that a lower content ofn-3 fatty acids in the cell membrane may result in the increased production of LTs, leading to an enhancement of mucosal damage. In contrast, dramatic prevention of mucosal damage was observed in the low fat diet group. Mice models fed with low fat-oil diet contained a small amount ofn-6 fatty acids compared with that of corn oil-fed group. It is suggested that a lower content of n-6 fatty acids in the cell membrane may result in reduction of LTs synthesis, leading to the prevention of mucosal damage in terms of prevention of mucosal damage in food-sensitive enteropathy, it is possible to accomplish this by either increasingn-3 fatty acids or reducing n-6 fatty acids in the diet, or a combination of both. We found that perilla oil-diet attenuates mucosal damage as successfully as the low fat diet did, because they contained a smaller amount of n-6 fatty acids and a larger amount ofn-3 fatty acids, which is an ideal combination of fatty acids composition for the prevention of mucosal damage by reducing the synthesis of LTs.

Treatments with dietary fats have produced various favorable results in inflammatory diseases. In NZB/NZW F1 immune complex nephritis model mice, a low fat diet sufficiently regulates immunoglobulin synthesis(22) and antibody production(23), and thus reduces nephritis to a minimum(24). Ingestion of EPA has shown a prophylactic effect in studies of carrageenan-induced colitis in guinea pigs(25). Dietary supplementation therapy has been tried in patients with ulcerative colitis(13) and Crohn's disease(14); however, the clinical benefits are not comparable(15). Results of these trials were not well explained in terms of the relationship with fatty acid composition and LTs synthesis. In this study, a dramatic preventive effect on enteropathy was demonstrated in perilla oil-fed and low fat diet groups, resulting in favorable alterations of RBC membrane fatty acid composition and LTs synthesis. Our studies suggest that n-3-enriched diet or low fat diet, when given properly, induces the change in fatty acid composition in the cell membrane. This alteration suppresses LTB4 and LTC4 synthesis and thus results in a clinical benefit.

In summary, this study demonstrated that reducing membrane n-6 fatty acids by means of a low fat diet, or a diet supplemented withn-3 fatty acids, attenuates the mucosal damage in relation to LTs synthesis in food-sensitive enteropathy in mice.