- Email: [email protected]
Antiulcerogenic and antiinflammatory actions of fatty acids on the gastrointestinal tract
Prostaglandios Leukotrienes and Essential 0 Longman Group UK Ltd 1991
Fatty Acids
(1991) 43. 135-140
Review
Antiulcerogenic and Antiinflammatory Actions of Fatty Acids on the Gastrointestinal Tract A. Lugea, S. Videla, J. Vilaseca and F. Guarner Digestive System Research Unit, Hospital General I/all d’Hebron, Barcelona, 08035 Spain (Reprint requests to FG)
INTRODUCTION Eicosanoids are mediators of defensive and inflammatory processes of the gut mucosa, and their role in the pathogenesis of gastrointestinal ulceration has been documented in a large number of studies (1). The local eicosanoid system is modulated by intraluminal, neural and hormonal factors. Among the intraluminal factors, the diet might have a significant relevance in the regulation of mucosal eicosanoid biosynthesis, since the dietary intake of precursor fatty acids could directly influence the rate and pattern of eicosanoid generation (2-4). Marine lipids have a high proportion of longchain poly-unsaturated fatty acids called n-3 or omega-3 because of the double bond located between the third and fourth carbon atom from the methyl end of the fatty acid chain. N-3 fatty acids are scarce in the normal Western diet, which mainly includes polyunsaturated fatty acids from the n-6 series. Recent evidence that chronic intake of fishderived n-3 fatty acids inhibits neutrophil and monocyte function (5,6) suggests that n-3 fatty acids have antiinflammatory properties. A hypdthetical mechanism that has been proposed to explain these observations is the competition between eicosapentaenoic acid (EPA, 20:5 n-3) and arachidonic acid (AA, 2O:4 n-6) as precurso rs of eicosanoid synthesis. Eicosapentaenoic acid competitively inhibits AA metabolism and is transformed into eicosanoid analogues with diverse biological activities when compared to AA-derivatives (7, 8). Thus, we set up an experimental study to investigate the influence of the dietary fatty acid content on eicosanoid biosynthesis by the gastrointestinal mucosa. Secondly, we tested the effect of the diet on the development of duodenal mucosal ulcers induced by acid hypersecretion (cysteamine-induced
Autonomous
University of
duodenal ulcers). In addition, we studied if diet supplementation with fatty acids would modify the duodenal mucosal resistance to acid. Finally, the current review shows data on the effect of the diet on the colonic mucosal ulcerations induced by an inflammatory challenge (TNB model of granulomatous colitis). Mucosal eicosanoid synthesis Two experimental diets were prepared using a rodent low-fat diet supplemented at 8% by weight with either sunflower or cod liver oil, as sources of n-6 or n-3 polyunsaturated fatty acids, respectively. Oils were added to the standard mixture during the manufacturing process (Letica, Barcelona, Spain). The final fatty acid content of the chow pellets was determined by gas chromatography (Table 1). Two groups of male Sprague-Dawley rats were fed the experimental diets (9). Both diets were well tolerated by the laboratory animals. During the first Table 1 Fatty acid content of the rat chow pellets Fatty acid
Sunflower oil diet
Cod liver oil diet
l4:O 16:0 l6:l 18:O 18:l 18:l 18:2 18:3 18:4 2O:l 20:4 20:5 22:l 22~6 Total
0.33 4.76 0.22 4.22 24.34 1.04 51.51 0.78 0.24 0.03 88.77
2.88 10.68 5.49 3.51 13.69 3.52 12.21 1.22 1.10 7.57 0.26 5.95 5.04 6.91 84.90
n-7 n-9 n-7 n-6 n-3 n-3 n-9 n-6 n-3 n-11 n-3
Values are expressed as mg per g of diet. 135
136
Prostaglandins Leukotrienes
and Essential Fatty Acids
w/d1
501
l
l
Fig. 1 Plasma fatty acid levels after 4 weeks on sunflower oil or cod liver oil supplemented diet. Five rats per point (mean 2~ sem). (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
week on diet, daily food intake averaged 18.8 + 2.1 g in the sunflower oil group and 19.8 + 1.8 g in the cod liver oil group. Animals fed the sunflower oil diet had a body weight gain of 22.9 f 5.1 g/week on the first week, which was similar to that achieved by cod liver oil fed rats (21.3 + 3.6). There was no significant difference in body weight between the two experimental groups after 4 weeks on diet. Faecal fat was measured in serial samples from both groups of rats, and was found to be undetectable in every instance. Plasma fatty acid levels after 4 weeks on either diet are shown in Figure 1. Sunflower oil fed rats had significantly higher levels of linoleic acid (18:2 n-6) and AA (20:4 n-6) than cod liver oil fed animals. As expected, the cod liver oil group showed high levels of n-3 fatty acids (EPA, 20:5 n-3, and docosahexaenoic, 22:6 n-3) which were almost absent in sunflower oil fed rats. Thus, the plasma fatty acid profile became similar to that of the respective diet (see Table 1). After 1 week on either diet, generation of PGE2, 6-keto-PGF,,, TXB2 and LTB4 by gastric mucosa specimens was significantly lower in the cod liver oil group than in sunflower oil fed rats (9). The differences became even more marked after 4 weeks on the experimental diets. In contrast, generation of AA-derived eicosanoids by the colonic mucosa was not reduced after 1 week on the cod liver oil diet. Only LTB4 production was significantly decreased in cod liver oil fed rats. At 4 weeks, however, all eicosanoids measured were significantly reduced in the cod liver oil group as compared to sunflower oil fed rats. Generation of PGE3 and LTB5 was undetectable in rats fed the sunflower oil diet. Homogenates from colonic mucosa generated significant amounts of these EPA-derived eicosanoids only in the cod liver oil group. Interestingly, this group of rats still showed a higher production of PGEZ than that of
PGE3 after 4 weeks on the cod liver oil diet, suggesting that AA remained the main substrate for cyclooxygenase activity (9). By contrast, generation of LTCS was quantitatively more important than that of LTC4, indicating that the shift to the n-3 substrate was predominant in the lipoxygenase pathway. Thus, changes in the dietary intake of precursor fatty acids may modify the eicosanoid system in the gastrointestinal tract. It is interesting to remark that generation of n-6 fatty acid derived eicosanoids rapidly decreases in the stomach after fish oit feeding, while changes in the colonic mucosa take more time. A second remarkable conclusion is that the shift from n-6 to n-3 substrate appears to be more important in the lipo- than in fhe cyclooxygenase pathway. Duodenal ulcers The cysteamine model of duodenal ulcers was first described by Selye and Szabo (10). A single S.C. injection of cysteamine to laboratory rats induces acute mucosal damage in the duodenum mediated by an increase in gastric acid output and a reduction of duodenal bicarbonate release, among other factors. We used this model to test the effect of the fish oil diet on the development of acute mucosal ulcerations (11). After 4 weeks on cod liver or sunflower oil diet, matched groups of rats were subjected to the following protocols: (a) duodenal damage induced by 400 mg/Kg S.C. cysteamine in 9 rats killed 24 h after cysteamine and in 16 rats followed for up to 14 days; (b) gastric acid and duodenal bicarbonate secretion after 100 mg/Kg i.v. cysteamine in anaesthetized rats. Duodenal lesions were assessed microscopically and scored as erosions, ulcers or perforated/ penetrated ulcers. As shown in Figure 2, duodenal number
of rats
iIIr _ -AL..-
EROSION 0
St Jllf lower
ULCER
Diet
m
PEN/PER
Cod
Liver
ULCER
Diet
Fig. 2 Duodenal lesions induced by 400 mg/Kg SC. cysteamine in rats fed with either the sunflower oil or the cod liver oil supplemented diet. Lesions were assessed microscopically and scored as erosions, ulcers or penetrated/perforated - ulcers. The difference between both distributions is s@icant (p < 0.05, Chi-square test).
Fatty Acids and the Gastrointestinal Tract
1 ,
PERFORATION
EPITHELIALIZATION
3
Sunflower
Diet
m
Cod Liver
Diet
Outcome after a single 400 mg/Kg S.C. cysteamine injection to rats fed either sunflower oil or cod liver oil diet. Rats were followed for up to 14 days after cysteamine and killed for microscopical assessment of duodenal damage. Animals with perforation died during the first week of the follow-up. The difference between groups is significant (p < 0.01, Fisher’s exact test). Fig. 3
lesions assessed 24 h after cysteamine were deeper in sunflower oil fed rats when compared to the
cod liver oil group. In addition, the outcome of the rats followed for up to 14 days showed a significantly higher rate of epithetialization in rats fed with the cod liver oil diet than in rats fed with the sunflower oil diet (Fig. 3). Gastric acid output was significantly stimulated by cysteamine in both groups (p < 0.01) b u t t h e increase was more marked in the sunflower group (6.1 f 2.8 to 26.4 + 1.1 pmo!/h) than in cod liver oil fed rats (4.2 f 0.8 to 16.1 + 2.6, p < 0.05 versus sunflower oil group). There were no differences between both groups in basal and 10 mM hydrochloric acid (HCl)-stimulated duodenal bicarbonate secretion, and after cysteamine both groups of animals failed to release duodenal bicarbonate in response to 10 mM HCI. In summary, the fish oil diet reduced the increase in gastric acid output stimulated by cysteamine and decreased the severity of the duodenal lesions. Experimental data by other investigators suggested that prostaglandins derived from n-3 fatty acids have more potent antisecretory action than those derived from arachidonic acid (12, 13). Our findings would be consistent with those data. Duodenal resistance to acid The duodenal mucosa is normally challenged by intermittent exposure to acid because of periodic gastric emptying. Preliminary studies by our group demonstrated that mild acid irritation of the rat duodenum increases mucosal resistance to acid (14).
137
This mechanism, first described in the gastric mucosa by Robert et al (15) and named adaptive cytoprotection, was suppressed by indomethacin pretreatment suggesting the participation of prostaglandin synthesis. We studied the resistance of the duodenal mucosa to acid in three groups of male Sprague-Dawley rats fed a rodent diet supplemented with either oleic acid (18: 1 n-9), linoleic acid (18:2 n-6) or EPA (20:5 n-3). The diets were prepared on the basis of a standard diet with a fat content of 6% by weight (1.5% linoleic acid) supplemented with an additional 2% of pure oleic, linoleic or eicosapentaenoic acid. The diets were isocaloric and well tolerated by the animals, as assessed by a similar daily food intake and body weight gain throughout the three experimental dietary groups. Under anesthesia, matched groups of rats were subjected to two experimental protocols. Protocol A consisted of dose-response curves of duodenal damage induced by instillation of hydrochloric acid (HCl) into the duodenal lumen after perfusion with saline. This protocol evaluated the resistance of the duodenal mucosa to acid injury in the three dietary groups. Protocol B consisted of dose-response curves of duodenal damage by acid instillation after pretreatment with a mild dose of acid. This protocol evaluated the adaptive cytoprotection of the duodenal mucosa after a brief exposure to mild acid in the three experimental groups. The mucosal damage was assessed macroscopically and histologically, and scored according to the criteria shown in Table 2.
Table 2 Criteria for assessment of duodenal damage
Macroscopical score Mucosal appearance
Normal Petechiae Necrotic
Damaged area
0 cm along the duodenum < 3 cm 3-7 cm > 7 cm Maximum score Histological score
Depth of necrosis
None < 33% of the villi 33%-66% > 66%
Thrombotic phenomena
None Mild Severe Maximum score
The macroscopic score was obtained by the product of mucosal appearance times damaged area scores. Likewise, the histologic score was the product of depth of necrosis times thrombotic phenomena scores. A global score was obtained by summation of macroscopic and histologic scores (O-15).
138
Prostaglandins Leukotrienes and Essential Fatty Acids Damage
Damage
Oleic diet
lcore
200
300
400
500
Linoletc diet
smre
600
700
200
300
400
500
600
700
HCI dose (rmol)
HCI dose (rmol) -
Saline pretreatment
- - -
100 pmol HCI pretreatment
Fig. 4 Duodenal damage scores after intraluminal instillation of hydrochloric acid at different doses in rats fed either an oleic acid supplemented diet (left) or a linoleic acid, diet (right). Pretreatment with saline (solid line) or mild acid (100 pmol of HCI, dotted line) was given 30 min prior to acid instillation. Difference between saline and mild acid pretreatment is significant among oleic and linoleic rats (p < 0.01, analysis of covariance). Difference between oleic and linoleic curves after acid pretreatment is significant (p < 0.05). Mean f sem of five rats per point. Damage
Oleic diet
200
300
400
500
EPA diet
score
600
700
200
300
400
500
600
700
HCI dose (umol)
HCI dose (~tmol) -
Saline pretreatment
- - -
100 cmol HCI pretreatment
Fig. 5 Duodenal damage scores after intraluminal instillation of hydrochloric acid at different doses in rats fed either an oleic acid supplemented diet (left) or an eicosapentaenoic acid diet (right). Pretreatment with saline (solid line) or mild acid (100 pmol of HCI, dotted line) was given 30 min prior to acid instillation. Difference between saline and mild acid pretreatment is significant among oleic and EPA rats (p < 0.01). Difference between oleic and EPA curves after acid pretreatment is significant (p < 0.05). Mean + sem of five rats per point.
The duodenal damage induced by instillation of HCl at different doses is shown in Figures 4 and 5. The solid lines represent the mucosal resistance to direct acid instillation (Protocol A). There were no differences in the mucosal resistance to acid among the three experimental groups. The dotted lines show the damage after adaptive cytoprotection by pretreatment with a 100 pmol pulse of HCl (Protocol B). Interestingly, the pretreatment with HCl induced a shift of the curves to the right. A higher dose of acid was needed to achieve similar damage when the duodenum had been pretreated with mild acid. Hence, the dose of HCI that induces a half maximal damage increases when the duo-
denum has been pretreated with mild acid (Figs 4 & 5). The shift to the right is more marked in rats fed the linoleic diet than in the oleic group (Fig. 4). Likewise, the gap between the curves is wider in EPA fed rats than in the oleic acid fed, group (Fig. 5). Analysis of covariance of the adaptive cytoprotection curves demonstrated a significant difference between the oleic diet and the linoleic or EPA diet. Additional experiments showed that after pretreatment with indomethacin there was no adaptive cytoprotection, i.e. no significant protection could be achieved by a brief exposure to mild acid. Thus, indomethacin pretreatment suppressed differences
Fatty Acidsand the GastrointestinalTract
139
lSdrn’ T
between the three dietary groups. In conclusion, these studies suggest that duodenal adaptation to acid is enhanced in animals fed the linoleic or the EPA diet when compared to rats fed the oleic diet. It seems likely that the beneficial effect of the linoleic or EPA supplemented diets is mediated by prostaglandin generation.
POE-2
.
Experimental ulcerative colitis
0
In another series of experiments (17), we used the TNB model of inflammatory colitis which features chronic ulcerations of the colonic mucosa that persist for up to 8 weeks (18). Colonic inflammatory lesions were induced by a single intracolonic administration of 50 mg trinitrobenzene sulphonic acid diluted in 10% ethanol, in rats fed with the experimental diets for at least 4 weeks. On days 0, 3, 20, 30, 40 and 50 after TNB, the luminal release of eicosanoid mediators was measured by intracolonic dialysis, as described elsewhere (19). On days 20, 30 and 50, ten rats per group were killed and the distal colon was removed to assess mucosal damage macroscopically and histologically according to the criteria stated in Table 3.
Table 3 Criteria for assessment of colonic lesions
0
10
0
10
so
20
Sunflowu
Dial
l
-•
M 30 MYSAFERTNB
so
40 Cod Llvu
40
Did
*‘---*
so
Fig. 6
Changes in luminal release of PGE, (upper panel) and TXBz (lower panel) after the induction of colitis by intracolonic administration of TNB on day 0. Values represent ng per ml of fluid obtained after 1 h of in vivo dialysis of the colon. Ten rats per point (mean + sem) but on days 40 and 50. in which 7 rats from the sunflower oil group and 6 rats from cod liver oil group were subjected to dialysis. (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
Macroscopical score Adhesions
None Minimal Involving several
0 1 bowel loops
2
Strictures
None Mild Severe. proximal dilatation
0 2 3
Ulcers
None Linear ulceration < 1 cm Two linear ulcers < 1 cm More sites of ulceration or one large ulcer > 1 cm
0 I 2
Less than 1 mm l-3 mm More than 3 mm
0
Maximum score
IO
Wall thickness
3
1 2
Histological score Ulceration
No ulcer, epithelialization Small ulcers < 3 mm Large ulcers > 3 mm
0 1 2
Inflammation
None Mild Moderate Severe
0 I 2 3
Depth of the lesion
None Submucosa Muscularis propria Serosa
0 1 2 3
Fibrosis
None Mild Severe
0 1 2
Maximum score
10
As shown by Figure 6, luminal release of PGE, and TXB2 changed as time elapsed from TNB instillation. For PGE*, peak release occurred earlier (approximately day 3 after TNB) than for TXBZ (approximately day 20 after TNB). For PGEZ, there were no significant differences between both experimental groups throughout the study, although mean levels in the dialysis fluid from sunflower oil fed rats were always above those detected in cod liver oil fed rats. In contrast, luminal release of TXB2 markedly increased from day 3 to days 20 and 30 in sunflower oil fed rats but this change did not occur in cod liver oil fed animals. Hence, significant differences between the sunflower and cod liver oil groups occurred on days 20 and 30. Changes in the intracolonic release of LTB4 ware also different in both groups of rats. In the sunflower oil group, levels increased from day 0 (0.167 f 0.034 ng/ml) to day 3 (5.965 + 1.316) and persisted l$gh on day 20 (5.787 k 1.870). Cod liver oil fed rats showed a similar pattern (day 0: 0.082 + 0.021; day 3: 6.441 + 1.016), but mean levels on day 20 (3.674 + 0.573) were lower than on day 3, suggesting that the inflammatory activity had decreased. Figure 7 depicts the mean macroscopical and histological scores obtained from rats sacrificed on days 20, 30 and 50 after TNB. On day 20, macroscopical damage scores were significantly lower in
140
. -k d
Prostaglandins Leukotrienes
and Essential Fatty Acids
1
l
1 HIstological Score
SunflowerDlst 0
.
Cod LiverDiet
I
Fig. 7 Morphological lesion scores of colonic damage induced by TNB, assessed according to the criteria shown in Table 3. Means + sem are shown; (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
cod liver oil fed animals than in the sunflower oil group, but there was no difference in the histological scores. With the progression of the inflammatory colitis, differences between both experimental groups became more evident and cod liver oil fed rats showed lower macroscopical and histological lesion scores than sunflower oil fed rats. By day 50, inflammation and ulcerations were almost absent in cod liver oil fed animals, while the sunflower oil group still showed flaring mucosal lesions. Thus, the initial injury was similar in both groups of rats, but the development of chronic inflammatory lesions in the colon was mitigated by the cod liver oil diet. In summary, these experiments suggest that dietary supplementation with a fish oil rich in n-3 fatty acids palliates the progression of chronic ulcerative lesions in the TNB-model of inflammatory colitis, and shortens the course of the disease. The beneficial effects are associated with a reduction of the luminal release of thromboxane and leukotriene B4 during the chronic stage of the inflammatory lesions. CONCLUSIONS Changes in the pattern of eicosanoid synthesis by the gastrointestinal mucosa can be induced by altering the dietary intake of their precursor fatty acids. These changes may be relevant for the expression of diseases involving gastrointestinal ulceration. Acknowledgements The authors wish to thank Ms. Celid Liria and Mrs. Dolors Soldevila for their excellent technical assistance. This work was partly supported by a Grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
References 1. Hawkey C J, Rampton D S. Prostaglandins and the gastrointestinal mucosa: are they important in its function, disease, or treatment?. Gastroenterology
89: 1162-88.1985. 2. Grant H W, Palmer K R, Kelly R W, Wilson N H, Misiewicz J J. Dietary linoleic acid, and gastric acid, and prostaglandin secretion. Gastroenterology 94: 955-9,1988. 3. Prichard P, Brown G, Bhaskar N, Hawkey C. The effect of dietary fatty acids on the gastric production of prostaglandins and aspirin-induced injury. Aliment Pharmacol Therap 2: 179-84, 1988. 4. Schepp W, Steffen B, Ruoff H J, Schusdziarra V, Classen M. Modulation of rat gastric mucosal prostaglandin E, release by dietary linoleic acid: effects on gastric acid secretion and stress-induced mucosal damage. Gastroenterology 95: 18-25, 1988. 5. Lee T H, Hoover R L, Williams J D, et al. Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function. N Eng J Med 312: 1217-24, 1985. 6. Endres S, Ghorbani R, Kelley V E, et al. The effect of dietary supplementation with n-3 polyunsaturated fattv acids on the synthesis of intcrleukin-1 and tumor necrosis factor by mononuclear cells. N Ene J Med 320: 265-71. 1989. Needleman P, Raz A, Mmkes M S, Ferrendeh J A, Sprecher H. Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological urooerties. Proc Nat1 Acad Sci USA 76: 944-8, i97i). Terano T. Salmon J A, Moncada S. Biosynthesis and biological activity of leukotriene B,. Prostaglandins 27: 217-32, 1984. Rodriguez R, Martinez M, Guarner F. Malagelada J R. Modification of gut mucosal eicosanoid synthesis by a fish oirdiet. J Clin Nutr Gastroenterol 5: 11-19. 1990. 10. Selye H. Szabo S. Experimental model for production of perforating duodenal ulcers by cysteamine in the rat. Nature 244: 458-9. 1973. 11. Guarner F. Vilaseca J, Salas A, Rodriguez R, Malagelada J R. Reduction of cysteamine-induced duodenal ulcers by dietary fish oil. Eur J Gastroenterol Hepatol (in press), 1991. 12. Whittle B J R. Moncada S, Vane J R. Biological activities of some metabolites and analogues of prostacyclin. p 141 in Medicinal Chemistry Advances (De Las Heras F G. Veea S eds) Pergamon Press, Oxford, 1981. ’ 13. Boughton-Smith N K. Whittle B J R. Inhibition of gastric acid secretion in the rat by protaglandins derived from arachidonic acid or eicosapentaenoic acid. Gastroenterology 98: A24. 1990. 14. Lugea A, Salas A, Guarner F, Rodriguez R, Azpiroz F, Malagelada J R. Duodenal resistance to acid load: a role for adaptive cytoprotection. Gastroenterology 98: A82, 1990. 15. Robert A, Nezamis J E, Lancaster C, Davis J P, Field S 0, Hanchar A J. Mild irritants prevent gastric necrosis through “adaptive cytoprotection” mediated by prostaglandins. Am J Physiol 245: G113-G121,1983. 16. Lugea A, Salas A, Guarner F, Rodriguez R, Malagelada J R. Dietary polyunsaturated fatty acids enhance adaptive cytoprotection in the duodenum. Gastroenterology 98: A420, 1990. 17. Vilaseca J. Salas A, Guarner F, Rodriguez R, Martinez M, Malaaelada J R. Dietary fish oil reduces progression of chronic inflammatory lesions in a rat model of colitis. Gut 31: 539-544, 1990. 18. Morris G P, Beck P L, Herridge M S, Depew W T, Szewezuk M R, Wallace J L. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 79.5-803, 1989. 19. Vilaseca J, Salas A, Guarner F, Rodriguez R, Malagelada J R. Participation of thromboxane and other eicosanoid synthesis in the course of experimental colitis. Gastroenterology 98: 269-277, 1990.
Fatty Acids
(1991) 43. 135-140
Review
Antiulcerogenic and Antiinflammatory Actions of Fatty Acids on the Gastrointestinal Tract A. Lugea, S. Videla, J. Vilaseca and F. Guarner Digestive System Research Unit, Hospital General I/all d’Hebron, Barcelona, 08035 Spain (Reprint requests to FG)
INTRODUCTION Eicosanoids are mediators of defensive and inflammatory processes of the gut mucosa, and their role in the pathogenesis of gastrointestinal ulceration has been documented in a large number of studies (1). The local eicosanoid system is modulated by intraluminal, neural and hormonal factors. Among the intraluminal factors, the diet might have a significant relevance in the regulation of mucosal eicosanoid biosynthesis, since the dietary intake of precursor fatty acids could directly influence the rate and pattern of eicosanoid generation (2-4). Marine lipids have a high proportion of longchain poly-unsaturated fatty acids called n-3 or omega-3 because of the double bond located between the third and fourth carbon atom from the methyl end of the fatty acid chain. N-3 fatty acids are scarce in the normal Western diet, which mainly includes polyunsaturated fatty acids from the n-6 series. Recent evidence that chronic intake of fishderived n-3 fatty acids inhibits neutrophil and monocyte function (5,6) suggests that n-3 fatty acids have antiinflammatory properties. A hypdthetical mechanism that has been proposed to explain these observations is the competition between eicosapentaenoic acid (EPA, 20:5 n-3) and arachidonic acid (AA, 2O:4 n-6) as precurso rs of eicosanoid synthesis. Eicosapentaenoic acid competitively inhibits AA metabolism and is transformed into eicosanoid analogues with diverse biological activities when compared to AA-derivatives (7, 8). Thus, we set up an experimental study to investigate the influence of the dietary fatty acid content on eicosanoid biosynthesis by the gastrointestinal mucosa. Secondly, we tested the effect of the diet on the development of duodenal mucosal ulcers induced by acid hypersecretion (cysteamine-induced
Autonomous
University of
duodenal ulcers). In addition, we studied if diet supplementation with fatty acids would modify the duodenal mucosal resistance to acid. Finally, the current review shows data on the effect of the diet on the colonic mucosal ulcerations induced by an inflammatory challenge (TNB model of granulomatous colitis). Mucosal eicosanoid synthesis Two experimental diets were prepared using a rodent low-fat diet supplemented at 8% by weight with either sunflower or cod liver oil, as sources of n-6 or n-3 polyunsaturated fatty acids, respectively. Oils were added to the standard mixture during the manufacturing process (Letica, Barcelona, Spain). The final fatty acid content of the chow pellets was determined by gas chromatography (Table 1). Two groups of male Sprague-Dawley rats were fed the experimental diets (9). Both diets were well tolerated by the laboratory animals. During the first Table 1 Fatty acid content of the rat chow pellets Fatty acid
Sunflower oil diet
Cod liver oil diet
l4:O 16:0 l6:l 18:O 18:l 18:l 18:2 18:3 18:4 2O:l 20:4 20:5 22:l 22~6 Total
0.33 4.76 0.22 4.22 24.34 1.04 51.51 0.78 0.24 0.03 88.77
2.88 10.68 5.49 3.51 13.69 3.52 12.21 1.22 1.10 7.57 0.26 5.95 5.04 6.91 84.90
n-7 n-9 n-7 n-6 n-3 n-3 n-9 n-6 n-3 n-11 n-3
Values are expressed as mg per g of diet. 135
136
Prostaglandins Leukotrienes
and Essential Fatty Acids
w/d1
501
l
l
Fig. 1 Plasma fatty acid levels after 4 weeks on sunflower oil or cod liver oil supplemented diet. Five rats per point (mean 2~ sem). (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
week on diet, daily food intake averaged 18.8 + 2.1 g in the sunflower oil group and 19.8 + 1.8 g in the cod liver oil group. Animals fed the sunflower oil diet had a body weight gain of 22.9 f 5.1 g/week on the first week, which was similar to that achieved by cod liver oil fed rats (21.3 + 3.6). There was no significant difference in body weight between the two experimental groups after 4 weeks on diet. Faecal fat was measured in serial samples from both groups of rats, and was found to be undetectable in every instance. Plasma fatty acid levels after 4 weeks on either diet are shown in Figure 1. Sunflower oil fed rats had significantly higher levels of linoleic acid (18:2 n-6) and AA (20:4 n-6) than cod liver oil fed animals. As expected, the cod liver oil group showed high levels of n-3 fatty acids (EPA, 20:5 n-3, and docosahexaenoic, 22:6 n-3) which were almost absent in sunflower oil fed rats. Thus, the plasma fatty acid profile became similar to that of the respective diet (see Table 1). After 1 week on either diet, generation of PGE2, 6-keto-PGF,,, TXB2 and LTB4 by gastric mucosa specimens was significantly lower in the cod liver oil group than in sunflower oil fed rats (9). The differences became even more marked after 4 weeks on the experimental diets. In contrast, generation of AA-derived eicosanoids by the colonic mucosa was not reduced after 1 week on the cod liver oil diet. Only LTB4 production was significantly decreased in cod liver oil fed rats. At 4 weeks, however, all eicosanoids measured were significantly reduced in the cod liver oil group as compared to sunflower oil fed rats. Generation of PGE3 and LTB5 was undetectable in rats fed the sunflower oil diet. Homogenates from colonic mucosa generated significant amounts of these EPA-derived eicosanoids only in the cod liver oil group. Interestingly, this group of rats still showed a higher production of PGEZ than that of
PGE3 after 4 weeks on the cod liver oil diet, suggesting that AA remained the main substrate for cyclooxygenase activity (9). By contrast, generation of LTCS was quantitatively more important than that of LTC4, indicating that the shift to the n-3 substrate was predominant in the lipoxygenase pathway. Thus, changes in the dietary intake of precursor fatty acids may modify the eicosanoid system in the gastrointestinal tract. It is interesting to remark that generation of n-6 fatty acid derived eicosanoids rapidly decreases in the stomach after fish oit feeding, while changes in the colonic mucosa take more time. A second remarkable conclusion is that the shift from n-6 to n-3 substrate appears to be more important in the lipo- than in fhe cyclooxygenase pathway. Duodenal ulcers The cysteamine model of duodenal ulcers was first described by Selye and Szabo (10). A single S.C. injection of cysteamine to laboratory rats induces acute mucosal damage in the duodenum mediated by an increase in gastric acid output and a reduction of duodenal bicarbonate release, among other factors. We used this model to test the effect of the fish oil diet on the development of acute mucosal ulcerations (11). After 4 weeks on cod liver or sunflower oil diet, matched groups of rats were subjected to the following protocols: (a) duodenal damage induced by 400 mg/Kg S.C. cysteamine in 9 rats killed 24 h after cysteamine and in 16 rats followed for up to 14 days; (b) gastric acid and duodenal bicarbonate secretion after 100 mg/Kg i.v. cysteamine in anaesthetized rats. Duodenal lesions were assessed microscopically and scored as erosions, ulcers or perforated/ penetrated ulcers. As shown in Figure 2, duodenal number
of rats
iIIr _ -AL..-
EROSION 0
St Jllf lower
ULCER
Diet
m
PEN/PER
Cod
Liver
ULCER
Diet
Fig. 2 Duodenal lesions induced by 400 mg/Kg SC. cysteamine in rats fed with either the sunflower oil or the cod liver oil supplemented diet. Lesions were assessed microscopically and scored as erosions, ulcers or penetrated/perforated - ulcers. The difference between both distributions is s@icant (p < 0.05, Chi-square test).
Fatty Acids and the Gastrointestinal Tract
1 ,
PERFORATION
EPITHELIALIZATION
3
Sunflower
Diet
m
Cod Liver
Diet
Outcome after a single 400 mg/Kg S.C. cysteamine injection to rats fed either sunflower oil or cod liver oil diet. Rats were followed for up to 14 days after cysteamine and killed for microscopical assessment of duodenal damage. Animals with perforation died during the first week of the follow-up. The difference between groups is significant (p < 0.01, Fisher’s exact test). Fig. 3
lesions assessed 24 h after cysteamine were deeper in sunflower oil fed rats when compared to the
cod liver oil group. In addition, the outcome of the rats followed for up to 14 days showed a significantly higher rate of epithetialization in rats fed with the cod liver oil diet than in rats fed with the sunflower oil diet (Fig. 3). Gastric acid output was significantly stimulated by cysteamine in both groups (p < 0.01) b u t t h e increase was more marked in the sunflower group (6.1 f 2.8 to 26.4 + 1.1 pmo!/h) than in cod liver oil fed rats (4.2 f 0.8 to 16.1 + 2.6, p < 0.05 versus sunflower oil group). There were no differences between both groups in basal and 10 mM hydrochloric acid (HCl)-stimulated duodenal bicarbonate secretion, and after cysteamine both groups of animals failed to release duodenal bicarbonate in response to 10 mM HCI. In summary, the fish oil diet reduced the increase in gastric acid output stimulated by cysteamine and decreased the severity of the duodenal lesions. Experimental data by other investigators suggested that prostaglandins derived from n-3 fatty acids have more potent antisecretory action than those derived from arachidonic acid (12, 13). Our findings would be consistent with those data. Duodenal resistance to acid The duodenal mucosa is normally challenged by intermittent exposure to acid because of periodic gastric emptying. Preliminary studies by our group demonstrated that mild acid irritation of the rat duodenum increases mucosal resistance to acid (14).
137
This mechanism, first described in the gastric mucosa by Robert et al (15) and named adaptive cytoprotection, was suppressed by indomethacin pretreatment suggesting the participation of prostaglandin synthesis. We studied the resistance of the duodenal mucosa to acid in three groups of male Sprague-Dawley rats fed a rodent diet supplemented with either oleic acid (18: 1 n-9), linoleic acid (18:2 n-6) or EPA (20:5 n-3). The diets were prepared on the basis of a standard diet with a fat content of 6% by weight (1.5% linoleic acid) supplemented with an additional 2% of pure oleic, linoleic or eicosapentaenoic acid. The diets were isocaloric and well tolerated by the animals, as assessed by a similar daily food intake and body weight gain throughout the three experimental dietary groups. Under anesthesia, matched groups of rats were subjected to two experimental protocols. Protocol A consisted of dose-response curves of duodenal damage induced by instillation of hydrochloric acid (HCl) into the duodenal lumen after perfusion with saline. This protocol evaluated the resistance of the duodenal mucosa to acid injury in the three dietary groups. Protocol B consisted of dose-response curves of duodenal damage by acid instillation after pretreatment with a mild dose of acid. This protocol evaluated the adaptive cytoprotection of the duodenal mucosa after a brief exposure to mild acid in the three experimental groups. The mucosal damage was assessed macroscopically and histologically, and scored according to the criteria shown in Table 2.
Table 2 Criteria for assessment of duodenal damage
Macroscopical score Mucosal appearance
Normal Petechiae Necrotic
Damaged area
0 cm along the duodenum < 3 cm 3-7 cm > 7 cm Maximum score Histological score
Depth of necrosis
None < 33% of the villi 33%-66% > 66%
Thrombotic phenomena
None Mild Severe Maximum score
The macroscopic score was obtained by the product of mucosal appearance times damaged area scores. Likewise, the histologic score was the product of depth of necrosis times thrombotic phenomena scores. A global score was obtained by summation of macroscopic and histologic scores (O-15).
138
Prostaglandins Leukotrienes and Essential Fatty Acids Damage
Damage
Oleic diet
lcore
200
300
400
500
Linoletc diet
smre
600
700
200
300
400
500
600
700
HCI dose (rmol)
HCI dose (rmol) -
Saline pretreatment
- - -
100 pmol HCI pretreatment
Fig. 4 Duodenal damage scores after intraluminal instillation of hydrochloric acid at different doses in rats fed either an oleic acid supplemented diet (left) or a linoleic acid, diet (right). Pretreatment with saline (solid line) or mild acid (100 pmol of HCI, dotted line) was given 30 min prior to acid instillation. Difference between saline and mild acid pretreatment is significant among oleic and linoleic rats (p < 0.01, analysis of covariance). Difference between oleic and linoleic curves after acid pretreatment is significant (p < 0.05). Mean f sem of five rats per point. Damage
Oleic diet
200
300
400
500
EPA diet
score
600
700
200
300
400
500
600
700
HCI dose (umol)
HCI dose (~tmol) -
Saline pretreatment
- - -
100 cmol HCI pretreatment
Fig. 5 Duodenal damage scores after intraluminal instillation of hydrochloric acid at different doses in rats fed either an oleic acid supplemented diet (left) or an eicosapentaenoic acid diet (right). Pretreatment with saline (solid line) or mild acid (100 pmol of HCI, dotted line) was given 30 min prior to acid instillation. Difference between saline and mild acid pretreatment is significant among oleic and EPA rats (p < 0.01). Difference between oleic and EPA curves after acid pretreatment is significant (p < 0.05). Mean + sem of five rats per point.
The duodenal damage induced by instillation of HCl at different doses is shown in Figures 4 and 5. The solid lines represent the mucosal resistance to direct acid instillation (Protocol A). There were no differences in the mucosal resistance to acid among the three experimental groups. The dotted lines show the damage after adaptive cytoprotection by pretreatment with a 100 pmol pulse of HCl (Protocol B). Interestingly, the pretreatment with HCl induced a shift of the curves to the right. A higher dose of acid was needed to achieve similar damage when the duodenum had been pretreated with mild acid. Hence, the dose of HCI that induces a half maximal damage increases when the duo-
denum has been pretreated with mild acid (Figs 4 & 5). The shift to the right is more marked in rats fed the linoleic diet than in the oleic group (Fig. 4). Likewise, the gap between the curves is wider in EPA fed rats than in the oleic acid fed, group (Fig. 5). Analysis of covariance of the adaptive cytoprotection curves demonstrated a significant difference between the oleic diet and the linoleic or EPA diet. Additional experiments showed that after pretreatment with indomethacin there was no adaptive cytoprotection, i.e. no significant protection could be achieved by a brief exposure to mild acid. Thus, indomethacin pretreatment suppressed differences
Fatty Acidsand the GastrointestinalTract
139
lSdrn’ T
between the three dietary groups. In conclusion, these studies suggest that duodenal adaptation to acid is enhanced in animals fed the linoleic or the EPA diet when compared to rats fed the oleic diet. It seems likely that the beneficial effect of the linoleic or EPA supplemented diets is mediated by prostaglandin generation.
POE-2
.
Experimental ulcerative colitis
0
In another series of experiments (17), we used the TNB model of inflammatory colitis which features chronic ulcerations of the colonic mucosa that persist for up to 8 weeks (18). Colonic inflammatory lesions were induced by a single intracolonic administration of 50 mg trinitrobenzene sulphonic acid diluted in 10% ethanol, in rats fed with the experimental diets for at least 4 weeks. On days 0, 3, 20, 30, 40 and 50 after TNB, the luminal release of eicosanoid mediators was measured by intracolonic dialysis, as described elsewhere (19). On days 20, 30 and 50, ten rats per group were killed and the distal colon was removed to assess mucosal damage macroscopically and histologically according to the criteria stated in Table 3.
Table 3 Criteria for assessment of colonic lesions
0
10
0
10
so
20
Sunflowu
Dial
l
-•
M 30 MYSAFERTNB
so
40 Cod Llvu
40
Did
*‘---*
so
Fig. 6
Changes in luminal release of PGE, (upper panel) and TXBz (lower panel) after the induction of colitis by intracolonic administration of TNB on day 0. Values represent ng per ml of fluid obtained after 1 h of in vivo dialysis of the colon. Ten rats per point (mean + sem) but on days 40 and 50. in which 7 rats from the sunflower oil group and 6 rats from cod liver oil group were subjected to dialysis. (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
Macroscopical score Adhesions
None Minimal Involving several
0 1 bowel loops
2
Strictures
None Mild Severe. proximal dilatation
0 2 3
Ulcers
None Linear ulceration < 1 cm Two linear ulcers < 1 cm More sites of ulceration or one large ulcer > 1 cm
0 I 2
Less than 1 mm l-3 mm More than 3 mm
0
Maximum score
IO
Wall thickness
3
1 2
Histological score Ulceration
No ulcer, epithelialization Small ulcers < 3 mm Large ulcers > 3 mm
0 1 2
Inflammation
None Mild Moderate Severe
0 I 2 3
Depth of the lesion
None Submucosa Muscularis propria Serosa
0 1 2 3
Fibrosis
None Mild Severe
0 1 2
Maximum score
10
As shown by Figure 6, luminal release of PGE, and TXB2 changed as time elapsed from TNB instillation. For PGE*, peak release occurred earlier (approximately day 3 after TNB) than for TXBZ (approximately day 20 after TNB). For PGEZ, there were no significant differences between both experimental groups throughout the study, although mean levels in the dialysis fluid from sunflower oil fed rats were always above those detected in cod liver oil fed rats. In contrast, luminal release of TXB2 markedly increased from day 3 to days 20 and 30 in sunflower oil fed rats but this change did not occur in cod liver oil fed animals. Hence, significant differences between the sunflower and cod liver oil groups occurred on days 20 and 30. Changes in the intracolonic release of LTB4 ware also different in both groups of rats. In the sunflower oil group, levels increased from day 0 (0.167 f 0.034 ng/ml) to day 3 (5.965 + 1.316) and persisted l$gh on day 20 (5.787 k 1.870). Cod liver oil fed rats showed a similar pattern (day 0: 0.082 + 0.021; day 3: 6.441 + 1.016), but mean levels on day 20 (3.674 + 0.573) were lower than on day 3, suggesting that the inflammatory activity had decreased. Figure 7 depicts the mean macroscopical and histological scores obtained from rats sacrificed on days 20, 30 and 50 after TNB. On day 20, macroscopical damage scores were significantly lower in
140
. -k d
Prostaglandins Leukotrienes
and Essential Fatty Acids
1
l
1 HIstological Score
SunflowerDlst 0
.
Cod LiverDiet
I
Fig. 7 Morphological lesion scores of colonic damage induced by TNB, assessed according to the criteria shown in Table 3. Means + sem are shown; (*) denotes p < 0.05 between sunflower oil and cod liver oil fed animals.
cod liver oil fed animals than in the sunflower oil group, but there was no difference in the histological scores. With the progression of the inflammatory colitis, differences between both experimental groups became more evident and cod liver oil fed rats showed lower macroscopical and histological lesion scores than sunflower oil fed rats. By day 50, inflammation and ulcerations were almost absent in cod liver oil fed animals, while the sunflower oil group still showed flaring mucosal lesions. Thus, the initial injury was similar in both groups of rats, but the development of chronic inflammatory lesions in the colon was mitigated by the cod liver oil diet. In summary, these experiments suggest that dietary supplementation with a fish oil rich in n-3 fatty acids palliates the progression of chronic ulcerative lesions in the TNB-model of inflammatory colitis, and shortens the course of the disease. The beneficial effects are associated with a reduction of the luminal release of thromboxane and leukotriene B4 during the chronic stage of the inflammatory lesions. CONCLUSIONS Changes in the pattern of eicosanoid synthesis by the gastrointestinal mucosa can be induced by altering the dietary intake of their precursor fatty acids. These changes may be relevant for the expression of diseases involving gastrointestinal ulceration. Acknowledgements The authors wish to thank Ms. Celid Liria and Mrs. Dolors Soldevila for their excellent technical assistance. This work was partly supported by a Grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.
References 1. Hawkey C J, Rampton D S. Prostaglandins and the gastrointestinal mucosa: are they important in its function, disease, or treatment?. Gastroenterology
89: 1162-88.1985. 2. Grant H W, Palmer K R, Kelly R W, Wilson N H, Misiewicz J J. Dietary linoleic acid, and gastric acid, and prostaglandin secretion. Gastroenterology 94: 955-9,1988. 3. Prichard P, Brown G, Bhaskar N, Hawkey C. The effect of dietary fatty acids on the gastric production of prostaglandins and aspirin-induced injury. Aliment Pharmacol Therap 2: 179-84, 1988. 4. Schepp W, Steffen B, Ruoff H J, Schusdziarra V, Classen M. Modulation of rat gastric mucosal prostaglandin E, release by dietary linoleic acid: effects on gastric acid secretion and stress-induced mucosal damage. Gastroenterology 95: 18-25, 1988. 5. Lee T H, Hoover R L, Williams J D, et al. Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function. N Eng J Med 312: 1217-24, 1985. 6. Endres S, Ghorbani R, Kelley V E, et al. The effect of dietary supplementation with n-3 polyunsaturated fattv acids on the synthesis of intcrleukin-1 and tumor necrosis factor by mononuclear cells. N Ene J Med 320: 265-71. 1989. Needleman P, Raz A, Mmkes M S, Ferrendeh J A, Sprecher H. Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological urooerties. Proc Nat1 Acad Sci USA 76: 944-8, i97i). Terano T. Salmon J A, Moncada S. Biosynthesis and biological activity of leukotriene B,. Prostaglandins 27: 217-32, 1984. Rodriguez R, Martinez M, Guarner F. Malagelada J R. Modification of gut mucosal eicosanoid synthesis by a fish oirdiet. J Clin Nutr Gastroenterol 5: 11-19. 1990. 10. Selye H. Szabo S. Experimental model for production of perforating duodenal ulcers by cysteamine in the rat. Nature 244: 458-9. 1973. 11. Guarner F. Vilaseca J, Salas A, Rodriguez R, Malagelada J R. Reduction of cysteamine-induced duodenal ulcers by dietary fish oil. Eur J Gastroenterol Hepatol (in press), 1991. 12. Whittle B J R. Moncada S, Vane J R. Biological activities of some metabolites and analogues of prostacyclin. p 141 in Medicinal Chemistry Advances (De Las Heras F G. Veea S eds) Pergamon Press, Oxford, 1981. ’ 13. Boughton-Smith N K. Whittle B J R. Inhibition of gastric acid secretion in the rat by protaglandins derived from arachidonic acid or eicosapentaenoic acid. Gastroenterology 98: A24. 1990. 14. Lugea A, Salas A, Guarner F, Rodriguez R, Azpiroz F, Malagelada J R. Duodenal resistance to acid load: a role for adaptive cytoprotection. Gastroenterology 98: A82, 1990. 15. Robert A, Nezamis J E, Lancaster C, Davis J P, Field S 0, Hanchar A J. Mild irritants prevent gastric necrosis through “adaptive cytoprotection” mediated by prostaglandins. Am J Physiol 245: G113-G121,1983. 16. Lugea A, Salas A, Guarner F, Rodriguez R, Malagelada J R. Dietary polyunsaturated fatty acids enhance adaptive cytoprotection in the duodenum. Gastroenterology 98: A420, 1990. 17. Vilaseca J. Salas A, Guarner F, Rodriguez R, Martinez M, Malaaelada J R. Dietary fish oil reduces progression of chronic inflammatory lesions in a rat model of colitis. Gut 31: 539-544, 1990. 18. Morris G P, Beck P L, Herridge M S, Depew W T, Szewezuk M R, Wallace J L. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 79.5-803, 1989. 19. Vilaseca J, Salas A, Guarner F, Rodriguez R, Malagelada J R. Participation of thromboxane and other eicosanoid synthesis in the course of experimental colitis. Gastroenterology 98: 269-277, 1990.