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Effect of sucralfate on the colonic mucosal hypoxia in the iodoacetamide-induced ulcerative colitis in rats
April 1995
Immunology, Microbiology, and Inflammatory Disorders A887
• KErOTIFEN, A MAST CELL STABILIZER, PROTECTS DEXTRAN SULFATE SODIUM (DSS)-INDUCED COLITIS IN RATS. T ODA, T KODAMA, N ARIZONO, K KASHIMA D e p a r t m e n t of Medicine a n d Medical Zoology, K y o t o P r e f e c t u r a l U n i v e r s i t y o f Medicine, Kyoto, J a p a n Ketotifen p r e v e n t s T N B / e t h a n o l - o r a c e t i c a c i d - i n d u c e d colitis in rats. In mice, h o w e v e r , k e t o t i f e n w a s r e p o r t e d to w o r s e n DSSi n d u c e d colitis. In this s t u d y we e x a m i n e d the p r o t e c t i v e effect of k e t o t i f e n in t h e d e v e l o p m e n t o f D S S - i n d u c e d colitis in rat. Methods: Colitis was i n d u c e d in W i s t a r f e m a l e r a t s b y giving 5% DSSin d r i n k i n g w a t e r f o r 5 d a y s . Ketotifen (20 o r 100 ~*g/100 g b o d y weight) was a d m i n i s t e r e d (p,o.) twice d a i l y f r o m d a y 7 b e f o r e to d a y 5 a f t e r the s t a r t o f DSS a d m i n i s t r a t i o n . In a n o t h e r g r o u p of rats, ketotifen ( 1 0 0 t t g / 1 0 0 g) w a s g i v e n twice d a l l y o n d a y s 0-5 of DSS a d m i n i s t r a t i o n . A g r o u p o f r a t s w a s g i v e n DSS alone. All a n i m a l s w e r e killed o n d a y 6, a n d h i s t o l o g i c a l f i n d i n g s w e r e e v a l u a t e d b y a s c o r i n g s y s t e m . Lesion l e n g t h was m e a s u r e d u s i n g a n i m a g e a n a l y z e r . T h e d e n s i t y of m u c o s a l m a s t cells was e s t i m a t e d in tissue s e c t i o n s s t a i n e d i m m u n o h i s t o c h e m i c a l l y w i t h anti-RMCP ( r a t m a s t cell p r o t e a s e ) II a n t i b o d y . S e r u m h i s t a m i n e was measured using an HPLC-autoanalyzer. Results: In r a t s r e c e i v e d DSS alone, severe, w i d e s p r e a d e r o s i o n s w e r e i n d u c e d in t h e r e c t u m . T h e n u m b e r s o f RMCP II-positive m a s t cells w e r e s i g n i f i c a n t l y d e c r e a s e d in t h e r e c t a l m u c o s a , a n d s e r u m h i s t a m i n e levels s i g n i f i c a n t l y i n c r e a s e d . All t h e g r o u p s o f rats, w h i c h r e c e i v e d k e t o t i f e n , s h o w e d d e c r e a s e d severities of DSSi n d u c e d colitis. The m o s t s i g n i f i c a n t effect w a s o b t a i n e d b y t h e t r e a t m e n t w i t h k e t o t i f e n ( t 0 0 ixg/100 g) f r o m d a y 7 before to d a y 5 a f t e r D S S a d m i n i s t r a t i o n : h i s t o l o g i c a l s c o r e s of m u c o s a l d a m a g e were 9.8 + 3.1 vs 3.4 -+ 1.9 in mucosa, a n d 8.4 + 2.9 vs 1.0 x 0 in s u b m u c o s a , in DSS a l o n e a n d k e t o t i f e n - t r e a t e d g r o u p , r e s p e c t i v e l y . Lesion l e n g t h was also m a r k e d l y r e d u c e d : 4 6 . 6 + 13.9 vs 2.6 -+ 1.5. The n u m b e r s of RMCP II-positive m a s t cells in r e c t a l m u c o s a d i d n o t s i g n i f i c a n t l y d e c r e a s e , a n d s e r u m h i s t a m i n e levels w e r e l o w e r in k e t o t i f e n - t r e a t e d t h a n in DSS-alone g r o u p . Conclusion: T r e a t m e n t of k e t o t i f e n e f f e c t i v e l y p r o t e c t s DSSi n d u c e d colitis in r a t s . K e t o t i f e n m a y h a v e t h e r a p e u t i c i m p l i c a t i o n s in i n f l a m m a t o r y bowel diseases.
• MACROPHAGE INFLAMMATORY PROTEIN-2 (MIP-2): REGULATION IN RAT SMALL BOWEL ENTEROCYTES. Y. Ohno. R.D. Fusunyan, N. Aoki, R.P. MacDermott, I.R. Sanderson. Combined P r o g r a m in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, B o s t o n a n d Gastrointestinal Section, L a h e y C l i n i c , Burlington, M A The small intestinal epithelium is an integral part of the mucosal immune system. But little is known about the factors that regulate chemokine secretion within enterocytes. Aim: We proposed that small intestinal epithelial cells m a y secrete the chemokine, macrophage inflammatory protein-2 (MIP-2) in rats, and we sought to examine its regulation. Methods: MIP-2 expression and secretion were examined u s i n g t h e n0n-malignant rat small intestinal cell line (IEC-6). Protein secretion Was quantified by densitometry of immunoblots, rnRNA transcripts Were detected by Northern blots. Nuclear factor binding was studied by elcctrophoretic mobility shift assays. Results: Enterocytes did not secrete MIP-2 unless stimulated. Lipopolysaccharide (LPS) and IL-1 [3 (1.0 ng/ml)stimulated MIP-2 secretion, Butyrate (1.5mM and 5mM) enhanced MIP-2 secretion in stimulated enterocytes. Butyrate enhanced the production of MIP-2 mRNA in response to IL- 113(Figure) and LPS. It also extended the period over which the m R N A accumulated in the cytoplasm without altering MIP-2 stabiltiy. Butyrate induced binding of nuclear factors in IEC-6 cells to an SP-1 DNA sequence (a response element present in the MIP-2 promoter).
Butyrat¢Enhancementof MIP-2mRNAin IEC-6Cells (IL-1 stimulated) 5 5.0mMButyrate 4
EFFECT OF SUCRALFATE ON THE COLONIC MUCOSAL HYPOXIA IN THE IODOACETAMIDE-INDUCED ULCERATIVE COLITIS IN R A T S . T. Ogihara, S. Kusstatscher*, Zs. Sander ~, M. Nagata e, N. Sate and S. Szabo*. Dept. of Gastroenterology, Juntendo Univ. Med. Sch., Tokyo, Japan and *Depts. of Pathology, Brigham & Women's Hosp., Harvard Med. Sch. , Bostonj M A & VA Medical Center, Long Beach, CA,' USA. Intracolonic administration of sulfhydryl alkylator iodoacetamide (IA) induces extensive colitis in the rat (Zs. Sand0r et al.: Gastroenterology 106: A766, 1994). The gastr0protective drug sueralfate prevents endogenous suifhydryl depletion. We t h u s s t u d i e d the e f f e c t of sucralfate on the colonic hemodynamics in IA-induced colitis in rats. Me.thods: After anesthesia(pentobarbital,3.5mg/100g) and laparotomy, 0.1ml of 3% or 6% IA in 1% methyl cellulose Was administered intracolonically with Nelaton catheter at 7cm from the anus to fasted Sprague-Dawley female rats. A small incision in colonic wall at 5em from the anus was made 10min after IA to position the probe on the mucosa. Changes in mueosal blood volume (lhrb) and mueosal blood oxygenation (IS02) were m e a s u r e d using organ r e f l e c t a n c e spectrephotometry (TS-200, Sum±tome, Japan) at 15, 30, 60, 120 mln after IA administration. Sucralfa%e (20mg/100g) was given intracolonically 30 min before IA administration. Results:
Groups
3% IA
-1.5± 7.9
-7,8~18.8 - 4 . 3 ± 1 . 5 -5.6±2.4 -9.8~12.7 -18.0~iI.0 " 5 , 6 ~ 2 . 1 -18.7±3.8' 5.4~ 5.8 -10.6±15.2 -4.6±2,7 -4.8±I.I, -i0.y±14.4 -7.~ 8.7 - 3 . 5 ~ 0 . 8 -0.1±2.1 Controls(Om!n)=lO0~ , Mean±SEM, n=3-6, *=9<0.05 ISO 2 after 120min'was slightly d e c r e a s e d by 3% IA while significantly reduced by S~ IA, indicating mucosal hypoxia, IHb showed no statistically significant differences, Sucraifatecounteracted the decrease in IS0 2 induced by 8%IA, Sueralfate also prevented the development of colonic lesions, Conclusions: i) Diminished colonic mucosal blood oxygenation preceded the development of colitis induced b y 6~ IA. 2) Sucralfate prevented the disturbance of coloni~ mucosal hemodynamics caused by IA. 3) This action might be involved in theprotective effect of the drug on the colonic lesions. 6~ IA SUC + 3% IA Suc + 8% IA
• PREVALENCE OF GASTROINTESTINAL SYMPTOMS IN HIV DISEASE IN SOUTH AFRICA EA OJKeefe, E Wood. Gastrointestinal clinic and Depa[tment of Medicine, University of Cape Town, South Africa
Ninety percent of AIDS patients in Central Africa report diarrhoea but only 30 to 60% in the West. The prevalence of diarrhoea and other GI symptoms in South Africa is unknown. METHODS Outpatients attending an HIV clinic were surveyed using an adapted bowel symptom questionnaire. Control data was collected from non-medical hospital personnel. Symptom frequency was related to WHO clinical stage of disease. RESULTS There were 58 controls (16 white, 24 mixed race, 18 African) and 85 patients (28 white, 34 mixed race, 23 African). Only one response was affected by race (weight loss reporEed by 539 of African controls) and none by gender. Median CD4 count in WHO 1 & 2 = 385, WHO 3 = 208, WHO 4 = 50. Prevalence of GI symptoms
by WHO stsge
SYMPTOM
CONTROL
WHO l&2
ABDO PAIN
24%
45%'
BOWEL DYSFUNCTION
9
27*
131
BOWELS >2/DAY
2
18 ~
9
I0
NAUSEA OFTEN
7
30#
19
30
DIARRHOEA>3X
i0
21
9
30"
DYSPHAGIA
9
i0
19
40 ~
25
50 ~
z..~ 3
STOMATITIS
12
!2i
~6
WEIGHT LOSS
23
39
2 1
Control(w/oButyrate) i i i 10 20 30 IncubationTime (Hours) Conclusions: MIP-2 is secreted by small intestinal epithelial cells in response to LPS and IL-I~. Sodium butyrate enhances SP-1 nuclear binding and increases the accumulation of MIP-2 expression in stimulated cells. 0
Mucosal blood oxygenation (~ of controls) 15sin 120min
Mucosal blood volume (~ of controls) 15sin 120min
ANOREXIA 12 * p<0.05, # p<0.01
'WHO 3 41%
WHO 4 45% 50
50 #
9O
!45 # 26 stage vs control
42
CONCLUSIONS GI symptoms generally increase with BIV disease progression, however, they cause significant early morbidity. Diarrhoea in South Africa is much less prevalent than reported from Central Africa and suggests that different opportunistic infections prevail.
Immunology, Microbiology, and Inflammatory Disorders A887
• KErOTIFEN, A MAST CELL STABILIZER, PROTECTS DEXTRAN SULFATE SODIUM (DSS)-INDUCED COLITIS IN RATS. T ODA, T KODAMA, N ARIZONO, K KASHIMA D e p a r t m e n t of Medicine a n d Medical Zoology, K y o t o P r e f e c t u r a l U n i v e r s i t y o f Medicine, Kyoto, J a p a n Ketotifen p r e v e n t s T N B / e t h a n o l - o r a c e t i c a c i d - i n d u c e d colitis in rats. In mice, h o w e v e r , k e t o t i f e n w a s r e p o r t e d to w o r s e n DSSi n d u c e d colitis. In this s t u d y we e x a m i n e d the p r o t e c t i v e effect of k e t o t i f e n in t h e d e v e l o p m e n t o f D S S - i n d u c e d colitis in rat. Methods: Colitis was i n d u c e d in W i s t a r f e m a l e r a t s b y giving 5% DSSin d r i n k i n g w a t e r f o r 5 d a y s . Ketotifen (20 o r 100 ~*g/100 g b o d y weight) was a d m i n i s t e r e d (p,o.) twice d a i l y f r o m d a y 7 b e f o r e to d a y 5 a f t e r the s t a r t o f DSS a d m i n i s t r a t i o n . In a n o t h e r g r o u p of rats, ketotifen ( 1 0 0 t t g / 1 0 0 g) w a s g i v e n twice d a l l y o n d a y s 0-5 of DSS a d m i n i s t r a t i o n . A g r o u p o f r a t s w a s g i v e n DSS alone. All a n i m a l s w e r e killed o n d a y 6, a n d h i s t o l o g i c a l f i n d i n g s w e r e e v a l u a t e d b y a s c o r i n g s y s t e m . Lesion l e n g t h was m e a s u r e d u s i n g a n i m a g e a n a l y z e r . T h e d e n s i t y of m u c o s a l m a s t cells was e s t i m a t e d in tissue s e c t i o n s s t a i n e d i m m u n o h i s t o c h e m i c a l l y w i t h anti-RMCP ( r a t m a s t cell p r o t e a s e ) II a n t i b o d y . S e r u m h i s t a m i n e was measured using an HPLC-autoanalyzer. Results: In r a t s r e c e i v e d DSS alone, severe, w i d e s p r e a d e r o s i o n s w e r e i n d u c e d in t h e r e c t u m . T h e n u m b e r s o f RMCP II-positive m a s t cells w e r e s i g n i f i c a n t l y d e c r e a s e d in t h e r e c t a l m u c o s a , a n d s e r u m h i s t a m i n e levels s i g n i f i c a n t l y i n c r e a s e d . All t h e g r o u p s o f rats, w h i c h r e c e i v e d k e t o t i f e n , s h o w e d d e c r e a s e d severities of DSSi n d u c e d colitis. The m o s t s i g n i f i c a n t effect w a s o b t a i n e d b y t h e t r e a t m e n t w i t h k e t o t i f e n ( t 0 0 ixg/100 g) f r o m d a y 7 before to d a y 5 a f t e r D S S a d m i n i s t r a t i o n : h i s t o l o g i c a l s c o r e s of m u c o s a l d a m a g e were 9.8 + 3.1 vs 3.4 -+ 1.9 in mucosa, a n d 8.4 + 2.9 vs 1.0 x 0 in s u b m u c o s a , in DSS a l o n e a n d k e t o t i f e n - t r e a t e d g r o u p , r e s p e c t i v e l y . Lesion l e n g t h was also m a r k e d l y r e d u c e d : 4 6 . 6 + 13.9 vs 2.6 -+ 1.5. The n u m b e r s of RMCP II-positive m a s t cells in r e c t a l m u c o s a d i d n o t s i g n i f i c a n t l y d e c r e a s e , a n d s e r u m h i s t a m i n e levels w e r e l o w e r in k e t o t i f e n - t r e a t e d t h a n in DSS-alone g r o u p . Conclusion: T r e a t m e n t of k e t o t i f e n e f f e c t i v e l y p r o t e c t s DSSi n d u c e d colitis in r a t s . K e t o t i f e n m a y h a v e t h e r a p e u t i c i m p l i c a t i o n s in i n f l a m m a t o r y bowel diseases.
• MACROPHAGE INFLAMMATORY PROTEIN-2 (MIP-2): REGULATION IN RAT SMALL BOWEL ENTEROCYTES. Y. Ohno. R.D. Fusunyan, N. Aoki, R.P. MacDermott, I.R. Sanderson. Combined P r o g r a m in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, B o s t o n a n d Gastrointestinal Section, L a h e y C l i n i c , Burlington, M A The small intestinal epithelium is an integral part of the mucosal immune system. But little is known about the factors that regulate chemokine secretion within enterocytes. Aim: We proposed that small intestinal epithelial cells m a y secrete the chemokine, macrophage inflammatory protein-2 (MIP-2) in rats, and we sought to examine its regulation. Methods: MIP-2 expression and secretion were examined u s i n g t h e n0n-malignant rat small intestinal cell line (IEC-6). Protein secretion Was quantified by densitometry of immunoblots, rnRNA transcripts Were detected by Northern blots. Nuclear factor binding was studied by elcctrophoretic mobility shift assays. Results: Enterocytes did not secrete MIP-2 unless stimulated. Lipopolysaccharide (LPS) and IL-1 [3 (1.0 ng/ml)stimulated MIP-2 secretion, Butyrate (1.5mM and 5mM) enhanced MIP-2 secretion in stimulated enterocytes. Butyrate enhanced the production of MIP-2 mRNA in response to IL- 113(Figure) and LPS. It also extended the period over which the m R N A accumulated in the cytoplasm without altering MIP-2 stabiltiy. Butyrate induced binding of nuclear factors in IEC-6 cells to an SP-1 DNA sequence (a response element present in the MIP-2 promoter).
Butyrat¢Enhancementof MIP-2mRNAin IEC-6Cells (IL-1 stimulated) 5 5.0mMButyrate 4
EFFECT OF SUCRALFATE ON THE COLONIC MUCOSAL HYPOXIA IN THE IODOACETAMIDE-INDUCED ULCERATIVE COLITIS IN R A T S . T. Ogihara, S. Kusstatscher*, Zs. Sander ~, M. Nagata e, N. Sate and S. Szabo*. Dept. of Gastroenterology, Juntendo Univ. Med. Sch., Tokyo, Japan and *Depts. of Pathology, Brigham & Women's Hosp., Harvard Med. Sch. , Bostonj M A & VA Medical Center, Long Beach, CA,' USA. Intracolonic administration of sulfhydryl alkylator iodoacetamide (IA) induces extensive colitis in the rat (Zs. Sand0r et al.: Gastroenterology 106: A766, 1994). The gastr0protective drug sueralfate prevents endogenous suifhydryl depletion. We t h u s s t u d i e d the e f f e c t of sucralfate on the colonic hemodynamics in IA-induced colitis in rats. Me.thods: After anesthesia(pentobarbital,3.5mg/100g) and laparotomy, 0.1ml of 3% or 6% IA in 1% methyl cellulose Was administered intracolonically with Nelaton catheter at 7cm from the anus to fasted Sprague-Dawley female rats. A small incision in colonic wall at 5em from the anus was made 10min after IA to position the probe on the mucosa. Changes in mueosal blood volume (lhrb) and mueosal blood oxygenation (IS02) were m e a s u r e d using organ r e f l e c t a n c e spectrephotometry (TS-200, Sum±tome, Japan) at 15, 30, 60, 120 mln after IA administration. Sucralfa%e (20mg/100g) was given intracolonically 30 min before IA administration. Results:
Groups
3% IA
-1.5± 7.9
-7,8~18.8 - 4 . 3 ± 1 . 5 -5.6±2.4 -9.8~12.7 -18.0~iI.0 " 5 , 6 ~ 2 . 1 -18.7±3.8' 5.4~ 5.8 -10.6±15.2 -4.6±2,7 -4.8±I.I, -i0.y±14.4 -7.~ 8.7 - 3 . 5 ~ 0 . 8 -0.1±2.1 Controls(Om!n)=lO0~ , Mean±SEM, n=3-6, *=9<0.05 ISO 2 after 120min'was slightly d e c r e a s e d by 3% IA while significantly reduced by S~ IA, indicating mucosal hypoxia, IHb showed no statistically significant differences, Sucraifatecounteracted the decrease in IS0 2 induced by 8%IA, Sueralfate also prevented the development of colonic lesions, Conclusions: i) Diminished colonic mucosal blood oxygenation preceded the development of colitis induced b y 6~ IA. 2) Sucralfate prevented the disturbance of coloni~ mucosal hemodynamics caused by IA. 3) This action might be involved in theprotective effect of the drug on the colonic lesions. 6~ IA SUC + 3% IA Suc + 8% IA
• PREVALENCE OF GASTROINTESTINAL SYMPTOMS IN HIV DISEASE IN SOUTH AFRICA EA OJKeefe, E Wood. Gastrointestinal clinic and Depa[tment of Medicine, University of Cape Town, South Africa
Ninety percent of AIDS patients in Central Africa report diarrhoea but only 30 to 60% in the West. The prevalence of diarrhoea and other GI symptoms in South Africa is unknown. METHODS Outpatients attending an HIV clinic were surveyed using an adapted bowel symptom questionnaire. Control data was collected from non-medical hospital personnel. Symptom frequency was related to WHO clinical stage of disease. RESULTS There were 58 controls (16 white, 24 mixed race, 18 African) and 85 patients (28 white, 34 mixed race, 23 African). Only one response was affected by race (weight loss reporEed by 539 of African controls) and none by gender. Median CD4 count in WHO 1 & 2 = 385, WHO 3 = 208, WHO 4 = 50. Prevalence of GI symptoms
by WHO stsge
SYMPTOM
CONTROL
WHO l&2
ABDO PAIN
24%
45%'
BOWEL DYSFUNCTION
9
27*
131
BOWELS >2/DAY
2
18 ~
9
I0
NAUSEA OFTEN
7
30#
19
30
DIARRHOEA>3X
i0
21
9
30"
DYSPHAGIA
9
i0
19
40 ~
25
50 ~
z..~ 3
STOMATITIS
12
!2i
~6
WEIGHT LOSS
23
39
2 1
Control(w/oButyrate) i i i 10 20 30 IncubationTime (Hours) Conclusions: MIP-2 is secreted by small intestinal epithelial cells in response to LPS and IL-I~. Sodium butyrate enhances SP-1 nuclear binding and increases the accumulation of MIP-2 expression in stimulated cells. 0
Mucosal blood oxygenation (~ of controls) 15sin 120min
Mucosal blood volume (~ of controls) 15sin 120min
ANOREXIA 12 * p<0.05, # p<0.01
'WHO 3 41%
WHO 4 45% 50
50 #
9O
!45 # 26 stage vs control
42
CONCLUSIONS GI symptoms generally increase with BIV disease progression, however, they cause significant early morbidity. Diarrhoea in South Africa is much less prevalent than reported from Central Africa and suggests that different opportunistic infections prevail.