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EFFECT OF D-002 ON ACETIC ACID-INDUCED COLITIS IN RATS AT SINGLE AND REPEATED DOSES
Pharmacological Research, Vol. 41, No. 4, 2000 doi:10.1006rphrs.1999.0596, available online at http:rrwww.idealibrary.com on
EFFECT OF D-002 ON ACETIC ACID-INDUCED COLITIS IN RATS AT SINGLE AND REPEATED DOSES ´ DASIY CARBAJAL and SURIA VALDES ´ MIRIAM NOAU , ROSA MAS, Department of Pharmacology, Laboratory of Histology, Center of Natural Products, National Center for Scientific Research, 25 A¨ e and 158 Street, Playa, Post Box 6990, Ha¨ ana, Cuba Accepted 31 August 1999
D-002 is a natural mixture of higher aliphatic primary alcohols purified from beeswax, with mild anti-inflammatory and effective antiulcer effects experimentally proved. Furthermore, it reduces leukotriene ŽLTB 4 . in the exudate of carrageenan-induced pleurisy and has a protective effect on the pre-ulcerative phase of carrageenan-induced colonic ulceration in the guinea pig. This study was conducted to determine the effect of D-002 on acetic acid-induced colitis in rats at single and repeated doses. In a first series, D-002 was orally administered at 25 and 50 mg kgy1 , 24 h before the induction of colitis, meanwhile, in a second series, it was administered 24 h after the induction of colitis. Two other series ŽIII and IV. examined the protective and therapeutic effect of D-002 administered for 7 days at the same doses, before or after colitis induction. Significant reductions in wet weight, macroscopic injury, polymorphonuclear infiltration and wall thickness were observed in colonic mucosa of D-002-treated animals compared with controls in both protective and therapeutic alternatives. It is concluded that D-002 was effective to protect or prevent the damage associated to acetic acid-induced colitis. Q 2000 Academic Press KEY
WORDS:
D-002, acetic acid-induced colitis.
INTRODUCTION D-002 is a natural mixture of higher aliphatic primary alcohols isolated and purified from beeswax; it contains 1-triacontanol, 1-octacosanol, 1-dotriacontanol, 1-hexacosanol and 1-tetracosanol. In addition, 1-tetratriacontanol is present as a minor component. D-002, has anti-ulcer activity in different experimental models w1x, and reduces thromboxane B 2 ŽTxB 2 . in the gastric mucosa of rats with ethanol induced-ulceration w2x. On the other hand, D-002 shows moderate anti-inflammatory properties and reduces leukotriene ŽLTB 4 . levels in exudated carrageenan-induced pleurisy w3x. Inflammation and release of mediators, such as leukotrienes is a common occurrence in different pathological processes, including ulcerative diseases of the colon w4, 5x. In a previous work, a protective U
Corresponding author.
1043]6618r00r040391]05r$35.00r0
effect of D-002 on the pre-ulcerative phase of carragenan-induced colonic ulceration in guinea pigs, was demonstrated w6x. The intracolonic infusion of dilute solutions of acetic acid is one of the most used animal models of inflammatory bowel disease w7]9x, so, it has been used to evaluate the putative action of different drugs on this process. Taking into account these facts, this study was undertaken to investigate the effects of D-002 on the acetic acidinduced colitis in rats at single and repeated doses.
MATERIALS AND METHODS Male Sprague]Dawley rats, 200]230 g body wt. obtained from the National Center for Laboratory Animals ŽCenpalab, Cuba. were housed in environmentally controlled rooms Ž25 " 28C, 12 h light]dark cycle., with free access to standard chow ŽCenpalab.. They were randomly allocated in three different experimental groups for each experimental series Ž n s 8 per group. Q 2000 Academic Press
Pharmacological Research, Vol. 41, No. 4, 2000
392
Administration and dosage D-002 batch composition was as follows: 1-triacontanol Ž26.63%., 1-octacosanol Ž17.49%., 1dotriacontanol Ž16.95%., 1-hexacosanol Ž15.34%. and 1-tetracosanol Ž13.24%.. 1-tetratriacontanol Ž2.24%. is also present as a minor component. D-002 was suspended in a 2% Tween 20 in H 2 O Žas vehicle.. Treatments were orally administered through gastric gavage Ž1 ml kgy1 . in all experimental series. The experimental groups included in each series were: ŽI. a control group of rats treated with the vehicle Tween 20 in water, and two groups treated with D-002 at 25 mg kgy1 Žgroup II.; and at 50 mg kgy1 Žgroup III..
Experimental design In the present work four experimental series were performed to evaluate the possible protective or therapeutic effect of D-002 in experimental colitis with single or repeated doses. In all studies, eight animals were assessed in each treatment group. Series I and II investigated the protective or therapeutic effect of single doses of D-002 on the acute colonic injury induced by the intrarectal instillation of dilute acetic acid. In series I, D-002 was orally administered at 25 and 50 mg kgy1 24 h before the induction of colitis, meanwhile in series II it was administered 24 h after the instillation of acetic acid. Series III and IV examined the protective or therapeutic effect of repeated doses of D-002 on the same model. For such an aim, series III and IV examined the effects of D-002 orally administered daily for 7 days at 25 and 50 mg kgy1 before or after colitis induction, respectively.
Colitis induction Colonic inflammation was induced in rats using the technique described w5x. Rats were fasted for 36 h and anaesthetized with ether. A rubber cannula Ž8 cm long. was inserted into the colon, via the anus and a solution of 4% acetic acid was instilled into the lumen of the colon Žtotal volume, 2 ml.. The rats were then allowed to recover from the anaesthetic and returned to their cages. The different measure-
ments were evaluated 24 h after colitis induction in series I and III, 48 h after in series II, and 8 days after in series IV. At these times, rats were killed.
Assessment of macroscopic injury The colons were excised, opened down their mesenteric borders, and cleansed of luminal contents. Severity of gross macroscopic injury was graded according to the following score system w10x: 0, normal appearance; 1, focal hyperaemia, no ulcers, single site of ulceration without associated inflammation; 3, ulceration with inflammation at one site; 4, two or more sites of discrete ulceration and inflammation; 5, major site of injury or inflammation extending 1]2 cm along length of colon; 6]10, score increased by one for each additional centimetre of damage or injury beyond 2 cm.
Assessment of the wet weight The wet weight of the colon was assessed as described w5x. A 5-cm segment of the distal colon, 3 cm proximal to the anus was resected, the lumen rinsed with ice-cold saline and weighed. Results are expressed as the mean " SD from eight samples.
Microscopical studies Sections of distal colon were selected for morphological studies. Tissues were fixed in phosphatebuffered formaldehyde, embedded in paraffin, stained with haematoxylin and eosin and evaluated by light microscopy. As an index of tissue oedema, colonic wall thickness was measured in properly oriented H & Estained sections using a calibrated eye-piece reticle positioned perpendicular to the serosal and mucosal surfaces w10x. Values were expressed in micrometres. To quantify infiltration by polymorphonuclear neutrophils ŽPMNs., cells were counted in each complete cross-section of mucosal as described w11x. The mean PMN count was determined for three sections per animal in each group. The expressed mean was calculated from the mean observed for each animal and afterwards averaged for each group. All measurements were carried out by an observer unaware of the treatment condition of the animals.
Table I Effect of administration of D-002 on acetic acid-induced colitis in rats on macroscopic injury Groups
Control D-002 D-002
Doses (mg kgy 1)
] 25 50
Macroscopic injury Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
5.62" 0.74 1.12" 0.99UU 0.75" 0.70UU
5.37" 1.18 1.12" 0.99UU 1.00" 1.06UU
6.12" 0.99 1.25" 0.88UU 0.62" 0.51UU
5.75" 1.16 1.00" 1.06UU 1.00" 1.06UU
Note: Values are expressed as the mean " SD of eight samples. P- 0.0001 comparison with controls ŽMann]Whitney U-test..
UU
Pharmacological Research, Vol. 41, No. 4, 2000
393
Table II Effect of administration of D-002 on acetic acid-induced colitis in rats on wet weight of 5-cm colon segments Groups
Control D-002 D-002
Wet wt. (g)
Doses (mg kgy 1)
] 25 50
Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
1.59" 0.003 0.71" 0.004UU 0.69" 0.006UU
1.59" 0.003 0.71" 0.004UU 0.68" 0.003UU
1.59" 0.003 0.71" 0.003UU 0.68" 0.004UU
1.58" 0.003 0.71" 0.004UU 0.68" 0.006UU
Note: Results are expressed as the mean " SD of eight samples. P- 0.0001 comparisons with control ŽMann]Whitney U-test..
UU
Statistics Comparisons between groups of wet weight, macroscopic injury, colonic wall thickness and PMN counts were performed by the use of the non-parametric Mann]Whitney U-test.
RESULTS
perimental model conditions. In D-002 treated rats, the colonic damage score was reduced significantly compared with the controls in all the experimental series. ŽTable I.. Administration of D-002 on acetic acid-induced colonic ulceration in rats, significantly reduced the wet weight of 5-cm distal colon segments compared with the controls in all experimental series ŽTable II..
Macroscopic changes Intracolonic administration of 2 ml of acetic acid caused macroscopic damage of the distal colon. The mucosa appeared haemorrhagic and with some ulcers. Comparison of control groups in the different experimental series did not show significant differences, which supports the reproducibility of the ex-
Histological changes Following the intrarectal application of 4% acetic acid to the control animals of all the studies, the following reproducible findings were observed: sections of colons manifested histological changes consisting of extensive epithelial disruption with sub-
Table III Effect of administration of D-002 on acetic acid-induced colitis in rats on colonic PMN infiltration Groups
Control D-002 D-002
Doses (mg kgy 1)
] 25 50
PMNs Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
29.62" 3.62 16.00" 2.92UU 12.75" 1.28UU
28.37" 2.26 16.00" 2.39UU 12.50" 1.92UU
29.00" 2.61 15.25" 1.58UU 12.12" 1.55UU
30.25" 2.81 15.12" 2.64UU 11.50" 1.51UU
Note: Values are expressed as the mean " SD of eight rats. P- 0.0001 comparison with controls ŽMann]Whitney U-test..
UU
Table IV Effect of administration of D-002 on acetic acid-induced colitis in rats on wall thickness Groups
Control D-002 D-002
Wall thickness (mm)
Doses (mg kgy 1)
] 25 50
Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
0.79" 0.0 0.48" 0.03U 0.44" 0.03U
0.80" 0.01 0.47" 0.03U 0.45" 0.03U
0.79" 0.0 0.48" 0.05U 0.43" 0.01U
0.79" 0.01 0.46" 0.02U 0.42" 0.02U
Note: Results are expressed as the mean " SD of eight samples. P- 0.001 ŽMann]Whitney U-test..
U
Pharmacological Research, Vol. 41, No. 4, 2000
394
epithelial haemorrhage and extravasation of red blood cells, colonic glands appeared mildly dilated and the submucosa was markedly thickened with submucosal oedema. A moderate inflammatory infiltrate Žpredominantly neutrophilic. was observed within the lamina propria and, to a lesser extent, in the submucosa. In D-002-treated rats, occasionally a weak surface epithelium damage and submucosa oedema were present. Inflammatory cell infiltration was significantly reduced in all the cases ŽTable III.. In addition, wall thickness was significantly reduced in treated animals compared with that of the controls ŽTable IV..
The basis of the efficacy of D-002 demonstrated in this model could be explained through different mechanisms such as its ability to prevent the migration of PMNs to the site of injury andror the release of toxic mediators such as leukotrienes by these inflammatory cells, or the inhibition of free radical formation induced by PMNs. Increase of LTB 4 level is associated to the infiltration of inflammatory cells occurring in this model and it has been postulated that it may play a role in colitis w5x. As it has been demonstrated, D-002 reduces LTB 4 levels, so, it could ameliorate colitis damage. Nevertheless, further experiments are needed to elucidate it and to evaluate the contribution of a putative antioxidant effect of D-002 on the present results.
DISCUSSION
These results demonstrate that D-002 administered orally either in single or repeated doses shows preventive or therapeutic effects on induced colitis. The acetic acid-induced colitis model has been extensively used by other authors w7]9x as a model which causes severe diffuse distal colitis. The present work was able to find similar results to those aforementioned. The wet weight of the inflamed colonic tissue is considered a reliable and sensitive indicator of the severity and extent of the inflammatory response w12x. In such regard, D-002, significantly reduced the wet weight of distal colon segments compared with the controls in all experimental series. In addition, rats treated with D-002, also showed a significant reduction of macroscopic lesions. A significant reduction of the colonic wall thickness was also observed in animals treated with D-002 at the doses of 25 and 50 mg kgy1 in all experimental series, indicating reduced oedema and tissue injury w10x. On the other hand, a significant reduction of PMNs was observed in these D-002 treated animals. PMNs may play a crucial role in the development of colonic injury, as has been demonstrated in many models of gastric w13, 14x and small intestinal injury w15, 16x. The occurrence of increased numbers of mucosal PMN in many forms of naturally occurring and experimental colitis has frequently been observed w7, 17x. Neutrophils infiltrating into the inflamed colonic mucosa are capable of producing superoxide and a cascade of various reactive species leading to a very reactive hydroxyl radical and peroxide w18, 19x. Another possible source is the oxidation of arachidonic acid through the lipoxygenase reaction Žproducing leukotrienes . w20x. In experimental colitis the cascade of free-radical production induces a constant self-maintaining lipoperoxidation and consumes the cellular antioxidant capability w21x.
REFERENCES 1. Carbajal D, Molina V, Valdes S, Aruzazabala ML, Mas R. Anti-ulcer activity of higher primary alcohols of beeswax. J Pharm Pharmacol 1995; 47: 731]3. 2. Carbajal D, Molina V, Valdes S, Arruzazabala ML, Rodeiro I, Mas R, Magraner J. Possible mechanism cytoprotective of D-002. An antiulcerogenic product isolated from beeswax. J Pharm Pharmacol 1996; 48: 858]60. 3. Carbajal D, Molina V, Valdes S, Arruzazabala ML, Mas R, Magraner J. Anti-inflammatory activity of D-002, an active product isolated from beeswax. Prostagland Leukotrienes Essent Fatty Acids 1998; 4: 235]8. 4. Wallace J, Keenan C, Gale D, Shoupe TS. Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotriene B4 synthesis. Gastroenterology 1992; 102: 18]27. 5. Mascolo N, Izzo A, Autore G, Maiello F, Di Carlo G, Capasso F. Acetic acid-induced colitis in normal and essential fatty acid deficient rats. J Pharmacol Exp Ther 1995; 272: 469]75. 6. Noa M, Mas ´ R. Effect of D-002 on the pre-ulcerative phase of carrageenan-induced colonic ulceration in the guinea pig. J Pharm Pharmacol 1998; 50: 549]53. 7. Mac Pherson B, Pfeiffer C. Experimental production of diffuse colitis in rats. Digestion 1978; 17: 135]50. 8. Sekizuka D, Grisham MB, Li M, Deitch E, Granger, D. Inflammation-induced intestinal hyperemia in the rat: role of neutrophils. Gastroenterology 1988; 95: 1528]34. 9. Yamada T, Zimmerman BJ, Specian R, Grisham MB. Role of neutrophils in acetic acid-induced colitis in rats. Inflammation 1991; 15: 399]411. 10. Buell M, Berin C. Neutrophil-independence of the initiation of colonic injury. Comparison of results from three models of experimental colitis in the rat. Digest Dis Sci 1994; 39: 2575]88. 11. Nygard G, Anthony A, Piasecki C, Trevethick M, Hudson M, Dhillon A, Pounder R, Wakefield A. Acute indomethacin-induced jejunal injury in the rat. Early morphological and biochemical changes. Gastroenterology 1994; 106: 567]75. 12. Rachmilewitz D, Simon P, Schwartz L, Don Griswold
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14. 15. 16. 17.
E, Fondacaro J, Wasserman, M. Inflammatory mediators of experimental colitis in rats. Gastroenterology 1989; 97: 326]37. Wallace J, Keenan C, Granger D. Gastric ulceration induced by nonsteroidal anti-inflammatory drug is a neutrophil-dependent process. Am J Physiol 1990; 259: G462]7. Kvietz P, Twohig B, Danzell J, Specian R. Ethanolinduced injury to the rat gastric mucosa. Gastroenterology 1990; 98: 909]20. Grisham M, Benoit J, Granger D. Assessment of leukocyte involvement during ischemia and reperfusion of intestine. Methods Enzymol 1990; 186: 729]41. Kubes P, Arfors K, Granger D. Platelet-activation factor-induced mucosal dysfunction: role of oxidants and granulocytes. Am J Physiol 1991; 260: G965]71. Morris G, Beck P, Herridge M, Depew W, Szewczuk
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18. 19.
20. 21.
M, Wallace J. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 1989; 96: 795]803. Baker SS, Campbell CL. Rat enterocyte injury oxygen-dependent processes. Gastroenterology 1991; 101: 716]20. Salim AS. Role of oxygen-derived free radical scavengers in the management of recurrent attacks of ulcerative colitis: a new approach. J Lab Clin Med 1992; 119(6): 710]7. Simmons NJ, Rampton DS. Inflammatory bowel disease}a radical view. Gut 1993; 34: 865]8. Loguercio C, D’Argenio, Delle Cave M, Cosenza V, Della Valle N, Mazzacca G. Direct evidence of oxidative damage in acute and chronic phases of experimental colitis in rats. Dig Dis Sci 1996; 41(6): 1204]11.
EFFECT OF D-002 ON ACETIC ACID-INDUCED COLITIS IN RATS AT SINGLE AND REPEATED DOSES ´ DASIY CARBAJAL and SURIA VALDES ´ MIRIAM NOAU , ROSA MAS, Department of Pharmacology, Laboratory of Histology, Center of Natural Products, National Center for Scientific Research, 25 A¨ e and 158 Street, Playa, Post Box 6990, Ha¨ ana, Cuba Accepted 31 August 1999
D-002 is a natural mixture of higher aliphatic primary alcohols purified from beeswax, with mild anti-inflammatory and effective antiulcer effects experimentally proved. Furthermore, it reduces leukotriene ŽLTB 4 . in the exudate of carrageenan-induced pleurisy and has a protective effect on the pre-ulcerative phase of carrageenan-induced colonic ulceration in the guinea pig. This study was conducted to determine the effect of D-002 on acetic acid-induced colitis in rats at single and repeated doses. In a first series, D-002 was orally administered at 25 and 50 mg kgy1 , 24 h before the induction of colitis, meanwhile, in a second series, it was administered 24 h after the induction of colitis. Two other series ŽIII and IV. examined the protective and therapeutic effect of D-002 administered for 7 days at the same doses, before or after colitis induction. Significant reductions in wet weight, macroscopic injury, polymorphonuclear infiltration and wall thickness were observed in colonic mucosa of D-002-treated animals compared with controls in both protective and therapeutic alternatives. It is concluded that D-002 was effective to protect or prevent the damage associated to acetic acid-induced colitis. Q 2000 Academic Press KEY
WORDS:
D-002, acetic acid-induced colitis.
INTRODUCTION D-002 is a natural mixture of higher aliphatic primary alcohols isolated and purified from beeswax; it contains 1-triacontanol, 1-octacosanol, 1-dotriacontanol, 1-hexacosanol and 1-tetracosanol. In addition, 1-tetratriacontanol is present as a minor component. D-002, has anti-ulcer activity in different experimental models w1x, and reduces thromboxane B 2 ŽTxB 2 . in the gastric mucosa of rats with ethanol induced-ulceration w2x. On the other hand, D-002 shows moderate anti-inflammatory properties and reduces leukotriene ŽLTB 4 . levels in exudated carrageenan-induced pleurisy w3x. Inflammation and release of mediators, such as leukotrienes is a common occurrence in different pathological processes, including ulcerative diseases of the colon w4, 5x. In a previous work, a protective U
Corresponding author.
1043]6618r00r040391]05r$35.00r0
effect of D-002 on the pre-ulcerative phase of carragenan-induced colonic ulceration in guinea pigs, was demonstrated w6x. The intracolonic infusion of dilute solutions of acetic acid is one of the most used animal models of inflammatory bowel disease w7]9x, so, it has been used to evaluate the putative action of different drugs on this process. Taking into account these facts, this study was undertaken to investigate the effects of D-002 on the acetic acidinduced colitis in rats at single and repeated doses.
MATERIALS AND METHODS Male Sprague]Dawley rats, 200]230 g body wt. obtained from the National Center for Laboratory Animals ŽCenpalab, Cuba. were housed in environmentally controlled rooms Ž25 " 28C, 12 h light]dark cycle., with free access to standard chow ŽCenpalab.. They were randomly allocated in three different experimental groups for each experimental series Ž n s 8 per group. Q 2000 Academic Press
Pharmacological Research, Vol. 41, No. 4, 2000
392
Administration and dosage D-002 batch composition was as follows: 1-triacontanol Ž26.63%., 1-octacosanol Ž17.49%., 1dotriacontanol Ž16.95%., 1-hexacosanol Ž15.34%. and 1-tetracosanol Ž13.24%.. 1-tetratriacontanol Ž2.24%. is also present as a minor component. D-002 was suspended in a 2% Tween 20 in H 2 O Žas vehicle.. Treatments were orally administered through gastric gavage Ž1 ml kgy1 . in all experimental series. The experimental groups included in each series were: ŽI. a control group of rats treated with the vehicle Tween 20 in water, and two groups treated with D-002 at 25 mg kgy1 Žgroup II.; and at 50 mg kgy1 Žgroup III..
Experimental design In the present work four experimental series were performed to evaluate the possible protective or therapeutic effect of D-002 in experimental colitis with single or repeated doses. In all studies, eight animals were assessed in each treatment group. Series I and II investigated the protective or therapeutic effect of single doses of D-002 on the acute colonic injury induced by the intrarectal instillation of dilute acetic acid. In series I, D-002 was orally administered at 25 and 50 mg kgy1 24 h before the induction of colitis, meanwhile in series II it was administered 24 h after the instillation of acetic acid. Series III and IV examined the protective or therapeutic effect of repeated doses of D-002 on the same model. For such an aim, series III and IV examined the effects of D-002 orally administered daily for 7 days at 25 and 50 mg kgy1 before or after colitis induction, respectively.
Colitis induction Colonic inflammation was induced in rats using the technique described w5x. Rats were fasted for 36 h and anaesthetized with ether. A rubber cannula Ž8 cm long. was inserted into the colon, via the anus and a solution of 4% acetic acid was instilled into the lumen of the colon Žtotal volume, 2 ml.. The rats were then allowed to recover from the anaesthetic and returned to their cages. The different measure-
ments were evaluated 24 h after colitis induction in series I and III, 48 h after in series II, and 8 days after in series IV. At these times, rats were killed.
Assessment of macroscopic injury The colons were excised, opened down their mesenteric borders, and cleansed of luminal contents. Severity of gross macroscopic injury was graded according to the following score system w10x: 0, normal appearance; 1, focal hyperaemia, no ulcers, single site of ulceration without associated inflammation; 3, ulceration with inflammation at one site; 4, two or more sites of discrete ulceration and inflammation; 5, major site of injury or inflammation extending 1]2 cm along length of colon; 6]10, score increased by one for each additional centimetre of damage or injury beyond 2 cm.
Assessment of the wet weight The wet weight of the colon was assessed as described w5x. A 5-cm segment of the distal colon, 3 cm proximal to the anus was resected, the lumen rinsed with ice-cold saline and weighed. Results are expressed as the mean " SD from eight samples.
Microscopical studies Sections of distal colon were selected for morphological studies. Tissues were fixed in phosphatebuffered formaldehyde, embedded in paraffin, stained with haematoxylin and eosin and evaluated by light microscopy. As an index of tissue oedema, colonic wall thickness was measured in properly oriented H & Estained sections using a calibrated eye-piece reticle positioned perpendicular to the serosal and mucosal surfaces w10x. Values were expressed in micrometres. To quantify infiltration by polymorphonuclear neutrophils ŽPMNs., cells were counted in each complete cross-section of mucosal as described w11x. The mean PMN count was determined for three sections per animal in each group. The expressed mean was calculated from the mean observed for each animal and afterwards averaged for each group. All measurements were carried out by an observer unaware of the treatment condition of the animals.
Table I Effect of administration of D-002 on acetic acid-induced colitis in rats on macroscopic injury Groups
Control D-002 D-002
Doses (mg kgy 1)
] 25 50
Macroscopic injury Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
5.62" 0.74 1.12" 0.99UU 0.75" 0.70UU
5.37" 1.18 1.12" 0.99UU 1.00" 1.06UU
6.12" 0.99 1.25" 0.88UU 0.62" 0.51UU
5.75" 1.16 1.00" 1.06UU 1.00" 1.06UU
Note: Values are expressed as the mean " SD of eight samples. P- 0.0001 comparison with controls ŽMann]Whitney U-test..
UU
Pharmacological Research, Vol. 41, No. 4, 2000
393
Table II Effect of administration of D-002 on acetic acid-induced colitis in rats on wet weight of 5-cm colon segments Groups
Control D-002 D-002
Wet wt. (g)
Doses (mg kgy 1)
] 25 50
Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
1.59" 0.003 0.71" 0.004UU 0.69" 0.006UU
1.59" 0.003 0.71" 0.004UU 0.68" 0.003UU
1.59" 0.003 0.71" 0.003UU 0.68" 0.004UU
1.58" 0.003 0.71" 0.004UU 0.68" 0.006UU
Note: Results are expressed as the mean " SD of eight samples. P- 0.0001 comparisons with control ŽMann]Whitney U-test..
UU
Statistics Comparisons between groups of wet weight, macroscopic injury, colonic wall thickness and PMN counts were performed by the use of the non-parametric Mann]Whitney U-test.
RESULTS
perimental model conditions. In D-002 treated rats, the colonic damage score was reduced significantly compared with the controls in all the experimental series. ŽTable I.. Administration of D-002 on acetic acid-induced colonic ulceration in rats, significantly reduced the wet weight of 5-cm distal colon segments compared with the controls in all experimental series ŽTable II..
Macroscopic changes Intracolonic administration of 2 ml of acetic acid caused macroscopic damage of the distal colon. The mucosa appeared haemorrhagic and with some ulcers. Comparison of control groups in the different experimental series did not show significant differences, which supports the reproducibility of the ex-
Histological changes Following the intrarectal application of 4% acetic acid to the control animals of all the studies, the following reproducible findings were observed: sections of colons manifested histological changes consisting of extensive epithelial disruption with sub-
Table III Effect of administration of D-002 on acetic acid-induced colitis in rats on colonic PMN infiltration Groups
Control D-002 D-002
Doses (mg kgy 1)
] 25 50
PMNs Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
29.62" 3.62 16.00" 2.92UU 12.75" 1.28UU
28.37" 2.26 16.00" 2.39UU 12.50" 1.92UU
29.00" 2.61 15.25" 1.58UU 12.12" 1.55UU
30.25" 2.81 15.12" 2.64UU 11.50" 1.51UU
Note: Values are expressed as the mean " SD of eight rats. P- 0.0001 comparison with controls ŽMann]Whitney U-test..
UU
Table IV Effect of administration of D-002 on acetic acid-induced colitis in rats on wall thickness Groups
Control D-002 D-002
Wall thickness (mm)
Doses (mg kgy 1)
] 25 50
Experimental series I 24 h after
II 48 h after
III 24 h after
IV 8 days after
0.79" 0.0 0.48" 0.03U 0.44" 0.03U
0.80" 0.01 0.47" 0.03U 0.45" 0.03U
0.79" 0.0 0.48" 0.05U 0.43" 0.01U
0.79" 0.01 0.46" 0.02U 0.42" 0.02U
Note: Results are expressed as the mean " SD of eight samples. P- 0.001 ŽMann]Whitney U-test..
U
Pharmacological Research, Vol. 41, No. 4, 2000
394
epithelial haemorrhage and extravasation of red blood cells, colonic glands appeared mildly dilated and the submucosa was markedly thickened with submucosal oedema. A moderate inflammatory infiltrate Žpredominantly neutrophilic. was observed within the lamina propria and, to a lesser extent, in the submucosa. In D-002-treated rats, occasionally a weak surface epithelium damage and submucosa oedema were present. Inflammatory cell infiltration was significantly reduced in all the cases ŽTable III.. In addition, wall thickness was significantly reduced in treated animals compared with that of the controls ŽTable IV..
The basis of the efficacy of D-002 demonstrated in this model could be explained through different mechanisms such as its ability to prevent the migration of PMNs to the site of injury andror the release of toxic mediators such as leukotrienes by these inflammatory cells, or the inhibition of free radical formation induced by PMNs. Increase of LTB 4 level is associated to the infiltration of inflammatory cells occurring in this model and it has been postulated that it may play a role in colitis w5x. As it has been demonstrated, D-002 reduces LTB 4 levels, so, it could ameliorate colitis damage. Nevertheless, further experiments are needed to elucidate it and to evaluate the contribution of a putative antioxidant effect of D-002 on the present results.
DISCUSSION
These results demonstrate that D-002 administered orally either in single or repeated doses shows preventive or therapeutic effects on induced colitis. The acetic acid-induced colitis model has been extensively used by other authors w7]9x as a model which causes severe diffuse distal colitis. The present work was able to find similar results to those aforementioned. The wet weight of the inflamed colonic tissue is considered a reliable and sensitive indicator of the severity and extent of the inflammatory response w12x. In such regard, D-002, significantly reduced the wet weight of distal colon segments compared with the controls in all experimental series. In addition, rats treated with D-002, also showed a significant reduction of macroscopic lesions. A significant reduction of the colonic wall thickness was also observed in animals treated with D-002 at the doses of 25 and 50 mg kgy1 in all experimental series, indicating reduced oedema and tissue injury w10x. On the other hand, a significant reduction of PMNs was observed in these D-002 treated animals. PMNs may play a crucial role in the development of colonic injury, as has been demonstrated in many models of gastric w13, 14x and small intestinal injury w15, 16x. The occurrence of increased numbers of mucosal PMN in many forms of naturally occurring and experimental colitis has frequently been observed w7, 17x. Neutrophils infiltrating into the inflamed colonic mucosa are capable of producing superoxide and a cascade of various reactive species leading to a very reactive hydroxyl radical and peroxide w18, 19x. Another possible source is the oxidation of arachidonic acid through the lipoxygenase reaction Žproducing leukotrienes . w20x. In experimental colitis the cascade of free-radical production induces a constant self-maintaining lipoperoxidation and consumes the cellular antioxidant capability w21x.
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