Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis
Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor) and conventional non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO) system and intensity of oxidative stress in the mucous membrane of the large intestine (MMLI) in rats with experimental ulcer...
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Інститут молекулярної біології і генетики НАН України
2011
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| Цитувати: | Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis / SA.Ya. Sklyarov, R.B. Lesyk, N.B. Panasyuk, I.S. Fomenko, D.Ya. Havrylyuk // Вiopolymers and Cell. — 2011. — Т. 27, № 2. — С. 147-153. — Бібліогр.: 24 назв. — англ. |
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irk-123456789-1564002019-06-19T01:25:34Z Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis Sklyarov, A.Ya. Lesyk, R.B. Panasyuk, N.B. Fomenko, I.S. Havrylyuk, D.Ya. Biomedicine Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor) and conventional non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO) system and intensity of oxidative stress in the mucous membrane of the large intestine (MMLI) in rats with experimental ulcerative colitis. Methods. Ulcerative colitis was induced by administration of acetic acid. The activity of NOsynthases, content of NO, and parameters of lipoperoxidation processes were measured in MMLI. Results. COX-2/5-LOX inhibition by 2A5DHT compound did not cause considerable destructive changes of the MMLI of rats. The activity of inducible nitric oxide synthase (iNOS) declined more than 2 fold as compared to their activity in colitis. The intensity of lipoperoxidation processes was found to be much lower than under the separate effect of celecoxib or indomethacine. Conclusions. Dual COX-2/5-LOX inhibition by 2A5DHT has a significant cytoprotective effect in MMLI that is accompanied by reduction of oxidative stress and activity of NO-synthases. The substance 2А5DHT significantly overexceeds the cytoprotective effects of both selective and non-selective COX/LOX inhibitors and can be used in the treatment of inflammatory bowel disease. Keywords: сyclooxygenase-2, 5-lipooxygenase, nitric oxide, lipoperoxidation, ulcerative colitis. Мета. Порівняти вплив сполуки 2A5DHT (інгібітора ЦОГ2/5-ЛОГ) та дію традиційних нестероїдних протизапальних засобів на показники NO-синтазної системи та оксидативного стресу у слизовій оболонці товстої кишки (СОТК) щурів за умов експериментального коліту. Методи. Розвиток коліту індуковано введенням оцтової кислоти per rectum. У СОТК визначено активність NO-синтаз, вміст NO та показники ліпопероксидації. Результати. Одночасне інгібування ЦОГ-2/5- ЛОГ сполукою 2A5DHT суттєвих деструктивних змін у СОТК не спричинило. Активність iNOS знижувалася більш ніж удвічі порівняно з їхньою активністю при коліті. Інтенсивність процесів ліпопероксидації виявилася на значно нижчому рівні, ніж при самостійній дії целекоксибу чи індометацину. Висновки. Одночасне інгібування ЦОГ-2/5-ЛОГ 2A5DHT справляє виражений цитопротективний ефект у СОТК, що проявляється у зниженні оксидативного стресу та активності NO-синтаз. Вплив сполуки 2A5DHT перевищує цитопротективні ефекти селективних і неселективних інгібіторів ЦОГ/ЛОГ, внаслідок чого її можна використовувати у лікуванні запальних захворювань кишки. Ключові слова: циклооксигеназа-2, 5-ліпоксигеназа, оксиди азоту, ліпопероксидація, виразковий коліт. Цель. Сравнить влияние соединения 2A5DHT (ингибитора ЦОГ-2/5-ЛОГ) и действие традиционных нестероидных противовоспалительных препаратов на показатели NO-синтазной системы и интенсивность оксидативного стресса слизистой оболочки толстой кишки (СОТК) крыс при экспериментальном колите. Методы. Розвитие колита моделировали введением уксусной кислоты per rectum. В СОТК определяли активность NO-синтаз, содержание NO и показатели липопероксидации. Результаты. Одновременное ингибирование ЦОГ2/5-ЛОГ соединением 2A5DHT выраженных деструктивних изменений в СОТК не вызвало. Активность iNOS снижалась болем чем вдвое по сравнению с их действием при колите. Интенсивность процессов липопероксидации была значительно ниже, чем при самостоятельном влиянии целекоксиба или индометацина. Выводы. Одновременное ингибирование ЦОГ-2/ 5-ЛОГ 2A5DHT имеет выраженный цитопротекторный эффект в СОТК, проявляющийся в снижении оксидативного стресса и активности NO-синтаз. Влияние 2A5DHT превышает цитопротекторное действие селективных и неселективных ингибиторов ЦОГ/ЛОГ, вследствие чего его можно использовать в лечении воспалительных заболеваний кишки. Ключевые слова: циклооксигеназа-2, 5-липоксигеназа, оксиды азота, липопероксидация, язвенный колит. 2011 Article Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis / SA.Ya. Sklyarov, R.B. Lesyk, N.B. Panasyuk, I.S. Fomenko, D.Ya. Havrylyuk // Вiopolymers and Cell. — 2011. — Т. 27, № 2. — С. 147-153. — Бібліогр.: 24 назв. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/156400 615.015.4 + 616.33-002 DOI: http://dx.doi.org/10.7124/bc.00008E en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| language |
English |
| topic |
Biomedicine Biomedicine |
| spellingShingle |
Biomedicine Biomedicine Sklyarov, A.Ya. Lesyk, R.B. Panasyuk, N.B. Fomenko, I.S. Havrylyuk, D.Ya. Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis Вiopolymers and Cell |
| description |
Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor) and conventional
non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO) system and intensity of
oxidative stress in the mucous membrane of the large intestine (MMLI) in rats with experimental ulcerative
colitis. Methods. Ulcerative colitis was induced by administration of acetic acid. The activity of NOsynthases, content of NO, and parameters of lipoperoxidation processes were measured in MMLI. Results.
COX-2/5-LOX inhibition by 2A5DHT compound did not cause considerable destructive changes of the
MMLI of rats. The activity of inducible nitric oxide synthase (iNOS) declined more than 2 fold as compared
to their activity in colitis. The intensity of lipoperoxidation processes was found to be much lower than
under the separate effect of celecoxib or indomethacine. Conclusions. Dual COX-2/5-LOX inhibition by
2A5DHT has a significant cytoprotective effect in MMLI that is accompanied by reduction of oxidative
stress and activity of NO-synthases. The substance 2А5DHT significantly overexceeds the cytoprotective
effects of both selective and non-selective COX/LOX inhibitors and can be used in the treatment of inflammatory bowel disease.
Keywords: сyclooxygenase-2, 5-lipooxygenase, nitric oxide, lipoperoxidation, ulcerative colitis. |
| format |
Article |
| author |
Sklyarov, A.Ya. Lesyk, R.B. Panasyuk, N.B. Fomenko, I.S. Havrylyuk, D.Ya. |
| author_facet |
Sklyarov, A.Ya. Lesyk, R.B. Panasyuk, N.B. Fomenko, I.S. Havrylyuk, D.Ya. |
| author_sort |
Sklyarov, A.Ya. |
| title |
Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| title_short |
Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| title_full |
Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| title_fullStr |
Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| title_full_unstemmed |
Comparison of dual acting drugs and conventional NSAIDs towards parameters of NO-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| title_sort |
comparison of dual acting drugs and conventional nsaids towards parameters of no-synthase system and oxidative stress in mucosal membrane of large intestine of rats with experimental ulcerative colitis |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2011 |
| topic_facet |
Biomedicine |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/156400 |
| citation_txt |
Comparison of dual acting drugs and conventional
NSAIDs towards parameters of NO-synthase system
and oxidative stress in mucosal membrane of large
intestine of rats with experimental ulcerative colitis / SA.Ya. Sklyarov, R.B. Lesyk, N.B. Panasyuk, I.S. Fomenko, D.Ya. Havrylyuk // Вiopolymers and Cell. — 2011. — Т. 27, № 2. — С. 147-153. — Бібліогр.: 24 назв. — англ. |
| series |
Вiopolymers and Cell |
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2025-07-14T06:26:49Z |
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2025-07-14T06:26:49Z |
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1837602606724153344 |
| fulltext |
Comparison of dual acting drugs and conventional
NSAIDs towards parameters of NO-synthase system
and oxidative stress in mucosal membrane of large
intestine of rats with experimental ulcerative colitis
A. Ya. Sklyarov1, R. B. Lesyk2, N. B. Panasyuk1, I. S. Fomenko1, D. Ya. Havrylyuk2
1Department of Biochemistry Danylo Halytsky Lviv National Medical University
69, Pekarska St., Lviv, Ukraine, 79010
2Department of Pharmaсeutical, Organic and Bioorganic Chemistry Danylo Halytsky Lviv National Medical University
69, Pekarska St., Lviv, Ukraine, 79010
sklyarov@meduniv.lviv.ua
Aim was to compare the action of 2A5DHT compound (dual COX-2/5-LOX inhibitor) and conventional
non-steroidal anti-inflammatory drugs towards parameters of nitric oxide (NO) system and intensity of
oxidative stress in the mucous membrane of the large intestine (MMLI) in rats with experimental ulcerative
colitis. Methods. Ulcerative colitis was induced by administration of acetic acid. The activity of NO-
synthases, content of NO, and parameters of lipoperoxidation processes were measured in MMLI. Results.
COX-2/5-LOX inhibition by 2A5DHT compound did not cause considerable destructive changes of the
MMLI of rats. The activity of inducible nitric oxide synthase (iNOS) declined more than 2 fold as compared
to their activity in colitis. The intensity of lipoperoxidation processes was found to be much lower than
under the separate effect of celecoxib or indomethacine. Conclusions. Dual COX-2/5-LOX inhibition by
2A5DHT has a significant cytoprotective effect in MMLI that is accompanied by reduction of oxidative
stress and activity of NO-synthases. The substance 2А5DHT significantly overexceeds the cytoprotective
effects of both selective and non-selective COX/LOX inhibitors and can be used in the treatment of inflam-
matory bowel disease.
Keywords: сyclooxygenase-2, 5-lipooxygenase, nitric oxide, lipoperoxidation, ulcerative colitis.
Introduction. Although the etiology of inflammatory
bowel disease (IBD) remains unknown, mounting evi-
dence implicates increased eicosanoids production in
the inflammatory process of this disease [1]. Prosta-
glandines (PGs), thromboxanes, and leukotriens (LTs)
are collectively referred to as eicosanoids. PGs are pro-
duced via the cyclooxygenase (COX) pathway and ex-
hibit both pro- and anti-inflammatory effects. COX is
now known to exist in three isoforms: COX-1, COX-2,
and COX-3. COX-1 is constitutively expressed in the
large intestine under physiological conditions. At the
same time, COX-2 is also expressed under the normal
conditions in the interstitial tissue, epithelial cells, and
neurons of submucous and intermuscular plexuses.
PGs synthesized by COX-1, provide for the processes
of water and electrolyte transport, vasodilatation, proli-
feration, and intercellular integration. Considerable
amounts of PGs synthesized by COX-2 are involved in
the inflammatory process [2]. Sustained inhibition of
both COX isoforms by nonsteroidal anti-inflammatory
drugs (NSAIDs) can cause intestinal ulceration in hu-
mans and laboratory animals. However, clinical and
experimental studies indicate that selective COX-2 in-
hibitors are less ulcerogenic than nonspecific COX in-
147
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 2. P. 147–153
Institute of Molecular Biology and Genetics NAS of Ukraine, 2011
hibitors (e. g. indomethacin) [3]. For now it is generally
accepted that people with IBD should avoid NSAIDs.
Lipooxygenase (LOX) pathway also plays an im-
portant role in inflammatory processes in IBD. Leuko-
trienes (LTs) and lipoxins produced via LOX activity
are associated with leukocytes activation and adhesion
to vascular endothelium. In ulcerative colitis and
Crohn’s disease LTB4 is considered to be linked to in-
flammatory lesions created by neutrophils observed in
the colonic and small bowel mucosa. Several experi-
mental data support the postulate that COX inhibition
with NSAIDs, besides causing a reduction in the syn-
thesis of PGs, diverts arachidonate to the 5-LOX path-
way, thus increasing the formation of LTs. This causes
vasoconstriction and increases the formation of reacti-
ve oxygen radicals from the peroxidative cleavage of
hydroxyeicosatetraenoic acids, which cause further
mucosal injury [4].
Besides pro-inflammatory systems COX/PGs and
LOX/LTs, one more very important system involved in
the development of IBD is nitric oxide (NO) system.
The destructive changes of the intestinal mucosa are as-
sociated with increased numbers of active forms of
oxygen, enhanced synthesis of NO, expression of in-
ducible nitric oxide synthase (iNOS) by epithelial cells,
macrophages, and neutrophil infiltration into the dama-
ged mucous membrane. Recent investigations have es-
tablished a close interrelationship existing between
iNOS and COX-2 [5].
In the past few decades several compounds have
been developed to block both COX and 5-LOX. Dual
COX/LOX inhibitors constitute a valuable alternative
to classical NSAIDs and selective COX-2 inhibitors for
treatment of inflammatory diseases. Moreover, they
appear to be almost exempt from gastrointestinal toxi-
city [6, 7]. One of the most potent dual inhibitors is dar-
bufelone (with the active substance 2A5DHT – 2-ami-
no-5-(3,5-ditertbutyl-4-hydroxybenzylidene)-thiazol-
4-one). In a randomized, double blind, rising single-
and multiple-dose study in healthy volunteers, it was
well tolerated up to the oral daily dose of 100 mg [4].
The role of COX/PGs and LOX/LTs systems in the
pathogenesis of IBD so far remains disputable and lea-
ves much to be elucidated. That is why the aim of this
research was to compare the action of nonspecific COX
inhibitor indomethacine, COX-2 selective inhibitor ce-
lecoxib, 5-LOX blocker AA-861 and 2A5DHT, which
is the active substance of dual COX- 2/5-LOX inhibitor
2A5DHT on indexes of NO-synthase system and in-
tensity of oxidative stress in the mucous membrane of
the large intestine (MMLI) under condition of experi-
mental ulcerative colitis.
Materials and methods. Animals. The experimen-
tal protocols were approved by the Ethical Committee
of Lviv National Medical University (Ukraine). Male
albino rats weighing 200–250 g were used. The ani-
mals were kept under artificial 12-hour light/dark cycle
at a constant temperature of 21–22 °C and fed on a stan-
dard rat chow and water ad libitum. All rats were fasted
24 h prior to the experimental procedure.
The study comprised of the following series of ex-
periments: 1 – intact animals were used as controls (n =
= 10); 2 – ulcerogenic lesions in rats were induced by
4 % acetic acid as previously described (n = 10) [8]; 3 –
non-selective COX inhibitor, indomethacin («Sigma»,
USA) was introduced in a dose of 10 mg/kg (n = 8); 4 –
selective COX-2 inhibitor, celecoxib («Arterium», Uk-
raine) was introduced in a dose of 10 mg/kg (n = 10);
5 – 5-LOX blocker, AA-861 («Wako», Japan) was in-
troduced in a dose of 50 mg/kg (n = 8); 6 – dual COX-
2/5-LOX inhibitor synthesized in the laboratory of
Department of Pharmaсeutical, Organic and Bioorga-
nic Chemistry Danylo Halytsky Lviv National Medical
University – 2A5DHT was introduced in a dose of
10 mg/kg (n = 8). All preparations (celecoxib, indome-
thacin, AA-861 and 2A5DHT) were administered in
1 ml volumes per os 1 h before and 24 h after the intra-
rectal application of acetic acid.
Under general anesthesia, rats were sacrificed by
decapitation and 10 cm of the distal colon were exci-
sed, freed from adherent adipose tissue and opened lon-
gitudinally. After washing the mucosa with saline, mu-
cosal injury (macroscopically) was assessed using the
grading scale of Morris et al. [9]. No damage (score 0);
localized hyperemia but no ulcers (score 1); linear ulcer
with no significant inflammation (score 2); linear ulcer
with inflammation at one site (score 3); two or more
sites of ulceration and inflammation (score 4); two or
more sites of ulceration and inflammation or one major
site of inflammation and ulceration extending > 1 cm
along the length of the colon (score 5). Colonic tissue
samples were homogenized in phosphate buffer pH 6.0
148
SKLYAROV A. Ya. ET AL.
1:4 and centrifuged at 5000 rpm, supernatant was used
to determine values of biochemical parameters.
Determination of NO-system in colonic tissues. The
content of NO in homogenate was determined as nitri-
tes by the method of Green et al. [10]. The absorbance
was read in a Stat fax at 550 nm. NO concentration was
expressed as µmol/g. NO-synthases (general NOS,
iNOS, and endothelial NOS – eNOS) activity was mea-
sured by the method described in detail [11]. NOS ac-
tivity was expressed in nmol NADPH⋅ min–1⋅ mg–1 of
protein.
Measurement of L-arginine in plasma samples. The
level of L-arginine in plasma samples was measured
according to the procedure of Alejnikova [12]. Plasma
L-arginine level was expressed as µg/ml.
Lipid peroxidation determination. Lipid peroxida-
tion level was expressed as MDA (malonic dialdehyde)
concentration in homogenates of MMLI. It was mea-
sured according to the procedure of Timirbulatow et al.
[13]. MDA levels were expressed as µmol/g. Catalase
(CAT) activity was determined by measuring of the
decrease in hydrogen peroxide concentration at 410 nm
by the Korolyuk method [14].
Colon mucosal catalase activity was expressed in
µmol H2O2⋅ min–1⋅ mg–1 of protein. Activity of super-
oxide dismutase (SOD) was determined by the reaction
of reduction of nitrotetrazoliume blue to nitroformazan
[15]. SOD activity was expressed in µmol ⋅ min–1⋅ mg–1
of protein.
Statistics. Experimental results were analyzed by
ANOVA and t-tests for multiple comparisons between
groups. The data was finally expressed as mean ± stan-
dard deviation. P value less than 0.05 was considered
statistically significant.
Results and discussion. In our study, injection of
4 % solution of acetic acid induced lesions, manifested
by ulcerative colitis, erosions and hemorrhages, with a
total area of 77.2 ± 25.1 mm2. Character of the structu-
re-hemorrhagic damage of the MMLI had in score 4.6 ±
± 0.69 (Figure).
Ulcerative colitis in the MMLI was associated
with enhancement of the activity of NO-synthases:
activity of general NOS enhanced more than threefold
(from 0.88 ± 0.09 to 2.68 ± 0.58 µmol ⋅ min–1⋅ g–1), acti-
vity of eNOS – by 58 %, and activity of iNOS – 6.9-
fold (P < 0.01). In colonic mucosa content of NO in-
creased by 64 % and, concomitantly, content of L-ar-
ginine in blood decreased by 51 % (from 37.8 ± 12.8 to
18.53 ± 4.98 mg/ml) (Tabl. 1).
MMLI, affected with ulcerative colitis, was sub-
jected to the following changes: enhanced activity of li-
poperoxidation processes manifested by a steep rise of
MDA content – by 116 % (from 255.5 ± 24.3 to 556.3 ±
± 15.7 µmol/g) (P < 0.01), at that, SOD activity enhan-
ced by 71 % (P < 0.01), and catalase activity – by 54 %
(P < 0.01) (Tabl. 2).
The destructive changes the intestinal mucosa are
subjected due to IBD and ulcerative colitis is associated
149
COMPARISON OF DUAL ACTING AND CONVENTIONAL NSAIDS TOWARDS PARAMETERS
0
20
40
60
80
100
120
11 2 3 4 5 6
0
1
2
3
4
5
6
11 2 3 4 5 6
* *
a
b
The character (in score) – a and the area – b of the structure-hemorrhagic damage of the MMLI: 1 – control; 2 – in rats with ulcerative colitis; 3 –
resulting from introduction of non-selective COX inhibitor indomethacin, at the colitis; 5 – resulting from introduction of 5-LOX blocker,
AA-861 at the background of ulcerative colitis; 6 – resulting from introduction of dual COX-2/5-LOX inhibitor, 2A5DHT compound at the
background of ulcerative colitis. *P < 0.05 versus the indexes in colitis
with increased numbers of active forms of oxygen, en-
hanced synthesis of NO, expression of iNOS by epithe-
lial cells, macrophages, and neutrophil infiltration into
the damaged mucous membrane [16].
In the present study, development of ulcerative co-
litis induced by per rectum introduction of acetic acid
was accompanied by the characteristic destructive
changes in the MMLI, enhanced processes of lipopero-
xidation, increased content of NO, and considerable ac-
tivation of iNOS. Due to activation of NO-synthases,
concentration of L-arginine, the substrate for NOS, in
the plasma of blood decreased and content of NO in the
MMLI increased.
IBD is characterized by a chronic intestinal inflam-
matory process with various components contributing
to the pathogenesis of the disease including different
factors such as stress and use of NSAIDS. NSAIDS are
among the most commonly used medications for the
treatment of various inflammatory conditions. The ma-
in factor limiting NSAIDs use is the concern for the de-
velopment of gastrointestinal toxicity including muco-
sal injury.
Several retrospective and cohort studies have imp-
licated NSAIDs in the onset or exacerbation of IBD. It
was shown, that injection of nonselective COX inhibi-
tors, the NSAIDs – indomethacin, naprofen, and diclo-
fenac caused destructive changes in the small intestine
of laboratory animals. For this reason, the content of
PGE2 considerably decreased which is evidence that
deficiency of PGs is the key factor whose impact re-
sults in damage to the small intestine induced by the
effect of NSAIDs [2]. The decrease of PGs synthesis in
the site of inflammation considerably correlated with
the reduction of COX-1 activity, but not COX-2 [17]. It
was acertained that СОХ-1, but not COX-2, plays a
role in maintaining the mucosal integrity [18].
150
SKLYAROV A. Ya. ET AL.
Groups of animals NO, µ mol/g iNOS, nmol⋅ min–1⋅ g–1
of protein
eNOS, nmol⋅ min–1⋅ g–1
of protein
NOS, nmol⋅ min–1⋅ g–1
of protein
L-arginine, µ g/ml
Control group 1.37 ± 0.14 0.239 ± 0.07 0.65 ± 0.09 0.88 ± 0.09 37.8 ± 12.8
4 % acetic acid 2.37 ± 0.43 1.65 ± 0.50 1.03 ± 0.32 2.68 ± 0.58 18.6 ± 4.7
4 % acetic acid + indomethacin 1.96 ± 0.11 1.64 ± 0.37 0.49 ± 0.27 2.13 ± 0.52 20.1 ± 6.7
4 % acetic acid + celecoxib 1.89 ± 0.27 0.898 ± 0.03* 0.623 ± 0,10 1.52 ± 0,12* 22.4 ± 3.8*
4% acetic acid + АА-861 2.12 ± 0.28 0.91 ± 0.13 0.73 ± 0.15 1.64 ± 0.39 23.1 ± 8.4
4 % acetic acid + 2A5DHT 2.10 ± 0.18 0.48 ± 0.15* 0.59 ± 0.17 1.08 ± 0.30* 26.3 ± 3.0
N o t e. *P < 0.05.
Table 1
The activity of NO-synthases, content of NO in MMLI and L-arginine in blood under conditions of separate and combined inhibition
of COX-1, COX-2 and 5-LOX at the background of ulcerative colitis
Groups of animals MDA, µ mol/g SOD, µ mol⋅ min–1⋅ mg–1 of protein CAT, µ mol H2O2⋅ min–1⋅ mg–1 of protein
Control group 255.5 ± 24.3 18.8 ± 5.5 2.42 ± 0.3
4 % acetic acid 556.3 ± 15.7 32.0 ± 4.5 3.72 ± 0.54
4 % acetic acid + indomethacin 543.4 ± 61.2 26.7 ± 3.1 3.54 ± 0.49
4 % acetic acid + celecoxib 444.0 ± 58.4* 17.2 ± 1.8* 3.18 ± 0.98*
4 % acetic acid + АА-861 543.6 ± 25.7 23.4 ± 2.4 3.34 ± 0.31
4 % acetic acid + 2A5DHT 334 ± 12.4* 21.2 ± 1.9* 3.36 ± 0.8
N o t e. *P < 0.05.
Table 2
The parameters of lipoperoxidation and the activity of SOD and CAT under conditions of separate and combined inhibition of COX-1,
COX-2 and 5-LOX at the background of ulcerative colitis
In our investigation, non-selective COX inhibition
by indomethacin resulted in significantly worse colitis
as compared with acetic acid colitis alone. Never-
theless parameters of NO-synthase system and oxi-
dative stress were practically the same like in ulcerative
colitis.
Many reports showed that СОХ-2 is the main form
responsible for PGE2 production at the site of inflam-
mation [17]. Nevertheless the role of COX-2 in the de-
velopment of ulcerative lesions so far remains dispu-
table. On the one hand, it was previously reported that
inhibition of COX-2 aggravated experimentally-in-
duced colonic lesion, that caused considerable inhibi-
tion of biosynthesis of PGs and increased area of lesi-
ons in colon [19, 20]. On the other hand, it was shown
that under inhibition of COX-2 with celecoxib, damage
to the MMLI was reduced as well as neutrophilic
infiltration and the level of IL 1β. Сelecoxib caused a
substantial reduction of the degree of colonic injury,
rose the myeloperoxidase activity in mucosa and in-
creased the tissue level of MDA [21].
In our investigations it was established that COX-2
inhibitor celecoxib decreased the area of lesions of co-
lonic mucosa, separate erosions and hemorrhages were
localized in the rugae. Under this condition activity of
iNOS, eNOS and NO content decreased, L-arginine
concentration in the blood plasma was increased. The
content of MDA and SOD activity decreased. There-
fore, as the СОХ-2 inhibition led to the increase of cy-
toprotective processes, it suggests that PGs, produced
by COX-2, are associated with inflammation and have
many proinflammatory activities that could contribute
to symptoms of ulcerative colitis.
Both the conventional NSAIDs and the selective
COX-2 inhibitors primarily exert their activity by redu-
cing the production of PGs induced in inflammatory
process. In recent years, it has been clarified that PGs
synthesis is only one part of arachidonic acid pathway,
this precursor being a substrate that gives rise to many
other lipid mediators, such as the LTs and the lipoxins.
LTs themselves have a major role in the development
and persistence of inflammatory process in MMLI [6].
Enhanced activation of LOX and increased content of
LTs are characteristic of ulcerative colitis [22]. A steep
rise in the content of proinflammatory LTs – C4, D4,
and B4, released predominantly by macrophages, mo-
nocytes, eosinophils, and mast cells, is observed in ex-
perimental ulcerative colitis in the MMLI. Although in
a number of research works a higher production of LTs
in colitis has been reported [23], but it was not always
accompanied by an increased production of PGE2.
In our investigations it was shown that admini-
stration of 5-LOX inhibitor AA-861 at the background
of colitis had not effect on the severity of lesions. Acti-
vity of NO-synthases had a tendency to decrease, whe-
reas iNOS activity was declined by 44 %, compared to
their activity in colitis. Contents of NO and MDA also
showed a tendency to decrease. Hence, under condition
of LOX-5 inhibition the intensity of lipoperoxidation
processes remained high as well as NOS activity. This
suggests that the product of 5-LOX reaction – LTB4
didn’t play a central role in pathogenesis of experimen-
tal ulcerative colitis.
In recent years it was shown, that both metabolites
of arachidonic acid PGs and LTs have complementary
effects in inflammation. We suggested that blockage
both LTs and PGs production might have synergic ef-
fects and achieve optimal anti-inflammatory activity.
Dual inhibition of COX-2 and 5-LOX may limit the
vascular changes seen during inflammation and leuko-
cytes induced damage of MMLI [6].
Dual COX-2/5-LOX inhibition with compound
2A5DHT displayed significant cytoprotective effect.
Microscopy detected separate hemorrhages with the
absence of considerable destructive changes of the
MMLI. Activity of iNOS declined more than 2 fold,
and total activity of NO-synthases decreased by 56 %
as compared to their activity in colitis. Content of NO
manifested a tendency to decrease. Intensity of lipo-
peroxidation processes were determined much lower
than under the effect of celecoxib and indomethacine,
MDA concentration declined by 40 %.
For this reason, dual COX-2/5-LOX inhibition by
2A5DHT has a significant cytoprotective effect in rats
with experimental ulcerative colitis and was accom-
panied by reduction of oxidative stress and activity of
NO-synthases in MMLI.
Conclusions. In an experimental model of inflam-
matory bowel disease, nonspecific COX inhibition
with indomethacin resulted in a significantly worse cli-
nical condition, as compared with acetic acid colitis
alone. Under COX-2 inhibition with celecoxib cyto-
151
COMPARISON OF DUAL ACTING AND CONVENTIONAL NSAIDS TOWARDS PARAMETERS
protective processes in MMLI were enhanced and oxi-
dative stress levels were reduced.
5-LOX inhibition with AA-861 revealed no signifi-
cant effect on structure-hemorrhagic lesions in MMLI
caused by the administration of 4 % acetic acid. Con-
comitantly, iNOS activity was reduced, MDA content
displayed a tendency to the decrease.
Dual COX-2/5-LOX inhibition with 2A5DHT
compound displayed significant cytoprotective effect,
manifested by the decreased area of the MMLI lesions,
and the decline of NO-synthases activities and the in-
tensity of lipoperoxidation processes. The substance
2А5DHT significantly overexceeds the cytoprotective
effects of both selective and non-selective COX/LOX
inhibitors and can be used in the treatment of inflam-
matory bowel disease.
Addendum. Synthesis of 2A5DHT. The starting 2-
aminothiazol-4-one was obtained according to method
described previously [24].
The elemental analyses (C, H, N) were performed
using the Perkin-Elmer 2400 CHN analyzer. Analyses
indicated by the symbols of the elements or functions
were within ±0.4% of the theoretical values. The 1H
NMR spectra were recorded on Varian Gemini
400 MHz in DMSO-d6 + CCl4 mixture using tetrame-
thylsilane (TMS) as an internal standard. Chemical
shifts are reported in ppm units with use of δ scale.
Mixtures of 2-aminothiazol-4-one (5 mmol), 3,5-
ditertbutyl-4-hydroxybenzaldehyde (5.5 mmol) and
anhydrous sodium acetate (5 mmol) were refluxed for
3 h in glacial acetic acid (10 ml). Obtained powder was
filtered off, washed with water and recrystallized with
glacial acetic acid.
Yield 68 %, 1H NMR (300 MHz, DMSO-d6 +
+ CCl4): δ [ppm] 9.81, 9.30, 9.03 (s, br. s, s, 2Н, NH2);
7.67 (s, 1H, OH); 7.53 (s, 1H, CH); 7.35 (s, 2H, arom);
1.40 (s, 18H, 2⋅ t-Bu).
Acknowledgements. The authors would like to
thank Professor Hiroshi Satoh (the Department of Ve-
terinary Medicine, Faculty of Agriculture, Tottori Uni-
versity, Japan) for providing the blocker of 5-lipooxi-
genase – AA-861.
А. Я. Скля ров, Р. Б. Ле сик, Н. Б. Па на сюк, І. С. Фо ме не ко,
Д. Я. Гав ри люк
Порівнян ня впливу пре па ратів подвійної дії та тра диційних
НПЗЗ на по каз ни ки сис те ми NO-син та зи і окси да тив но го
стре су у сли зовій об олонці тов стої киш ки щурів з
ек спе ри мен таль ним ви раз ко вим колітом
Ре зю ме
Мета. Порівня ти вплив спо лу ки 2A5DHT (інгібітора ЦОГ-
2/5-ЛОГ) та дію тра диційних не сте рої дних про ти за паль них
за собів на по каз ни ки NO-син таз ної сис те ми та окси да тив но го
стре су у сли зовій об олонці тов стої киш ки (СОТК) щурів за
умов ек спе ри мен таль но го коліту. Ме то ди. Роз ви ток коліту
інду ко ва но вве ден ням оцто вої кис ло ти per rectum. У СОТК ви-
зна че но ак тивність NO-син таз, вміст NO та по каз ни ки ліпо -
пе рок си дації. Ре зуль та ти. Одно час не інгібу ван ня ЦОГ-2/5-
ЛОГ спо лу кою 2A5DHT суттєвих дес трук тив них змін у СОТК
не спри чи ни ло. Активність iNOS зни жу ва ла ся більш ніж удвічі
порівня но з їхньою ак тивністю при коліті. Інтен сивність про -
цесів ліпо пе рок си дації ви я ви ла ся на знач но ни жчо му рівні, ніж
при са мостійній дії це ле кок си бу чи індо ме та ци ну. Вис нов ки.
Одно час не інгібу ван ня ЦОГ-2/5-ЛОГ 2A5DHT справ ляє ви ра -
жений ци топ ро тек тив ний ефект у СОТК, що про яв ляється у
зни женні окси да тив но го стре су та ак тив ності NO-син таз.
Вплив спо лу ки 2A5DHT пе ре ви щує ци топ ро тек тивні ефек ти
се лек тив них і не се лек тив них інгібіторів ЦОГ/ЛОГ, внаслідок
чого її мож на ви ко рис то ву ва ти у ліку ванні за паль них за хво рю -
вань киш ки.
Клю чові сло ва: цик ло ок си ге на за-2, 5-ліпо кси ге на за, окси ди
азо ту, ліпо пе рок си дація, ви раз ко вий коліт.
А. Я. Скля ров, Р. Б. Ле сик, Н. Б. Па на сюк, И. С. Фо ме не ко,
Д. Я. Гав ри люк
Срав не ние влияния пре па ра тов двой но го де йствия и
тра ди ци он ных НПВВ на по ка за те ли сис те мы NO-син та зы и
окси да тив но го стрес са в сли зис той об олочке тол стой киш ки
крыс с экс пе ри мен таль ным яз вен ным ко ли том
Ре зю ме
Цель. Срав нить вли я ние со е ди не ния 2A5DHT (ин ги би то ра
ЦОГ-2/5-ЛОГ) и де йствие тра ди ци он ных не сте ро ид ных про -
ти во вос па ли тель ных пре па ра тов на по ка за те ли NO-син таз -
ной сис те мы и ин тен сив ность окси да тив но го стрес са сли зи-
стой об олоч ки тол стой киш ки (СОТК) крыс при экс пе ри мен -
таль ном ко ли те. Ме то ды. Роз ви тие ко ли та мо де ли ро ва ли
вве де ни ем уксус ной кис ло ты per rectum. В СОТК опре де ля ли ак -
тив ность NO-син таз, со дер жа ние NO и по ка за те ли ли по пе ро-
кси да ции. Ре зуль та ты. Однов ре мен ное ин ги би ро ва ние ЦОГ-
152
SKLYAROV A. Ya. ET AL.
S
N
NH2
O
O
H
OH
Me Me
Me
Me
Me
Me
OH Me
Me
Me
Me
Me
Me
S
N
NH2
O
+
a
Synthesis of 2-amino-5-(3,5-ditertbutyl-4-hydroxybenzylidene)-thia
zol-4-one (2A5DHT). Reagents, conditions and yields: a – AcONa,
AcOH, reflux 3 h, 68 %
2/5-ЛОГ со е ди не ни ем 2A5DHT вы ра жен ных дес трук тив них из -
ме не ний в СОТК не вы зва ло. Активность iNOS сни жа лась бо -
лем чем вдвое по срав не нию с их де йстви ем при ко ли те. Интен-
сив ность про цес сов ли по пе рок си да ции была зна чи тель но ни-
же, чем при са мос то я тель ном вли я нии це ле кок си ба или ин до -
ме та ци на. Вы во ды. Однов ре мен ное ин ги би ро ва ние ЦОГ-2/
5-ЛОГ 2A5DHT име ет вы ра жен ный ци топ ро тек тор ный эф -
фект в СОТК, про яв ля ю щий ся в сни же нии окси да тив но го
стресса и ак тив нос ти NO-син таз. Вли я ние 2A5DHT пре вы ша -
ет ци топ ро тек тор ное де йствие се лек тив ных и не се лек тив -
ных ин ги би то ров ЦОГ/ЛОГ, всле дствие чего его мож но ис-
по льзо вать в ле че нии вос па ли тель ных за бо ле ва ний киш ки.
Клю че вые сло ва: цик ло ок си ге на за-2, 5-ли пок си ге на за, окси -
ды азо та, ли по пе рок си да ция, яз вен ный ко лит.
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UDC 615.015.4 + 616.33-002
Received 10.01.11
153
COMPARISON OF DUAL ACTING AND CONVENTIONAL NSAIDS TOWARDS PARAMETERS
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