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"Gastroenterology" 4 (54) 2014

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The role of bile acids in morphological changes оf gastric mucosa in rats

Authors: Rudenko A.I., Mosiychuk L.M., Oshmyanska N.Y. - SI «Institute of Gastroenterology of AMS of Ukraine», Dnipropetrovsk

Categories: Gastroenterology

Sections: Clinical researches

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Summary

The present study was conducted on 60 white male laboratory rats in order to investigate the role of bile acids in morphofunctional changes of gastric mucosa in rats with due regard to exposure of bile and its concentration. It was found that in the early stages of duodenogastric reflux in experiment bile in low concentrations carries protective adaptive-trophic function and promotes cell renewal of the surface epithelium. At the same time long-term exposure to the bile acids regardless of its concentration can lead to the significant dystrophic changes in gastric mucosa, pilorization and atrophy of the gastric glands. In the case of the oral administration of bile followed by «immobilization and cold stress» for one hour (I group) experiment’s results can confirm that the combined effects of several aggressive factors lead to the more severe pathological changes in the gastric mucosa: first erosive and later ulcerative lesions.

Настоящее исследование было проведено с целью изучения роли желчных кислот в формировании морфофункциональных изменений слизистой оболочки желудка у крыс с учетом воздействия желчи и ее концентрации. Было установлено, что на ранних этапах моделирования дуоденогастрального рефлюкса с помощью введения желчи в невысоких концентрациях она осуществляет защитную адаптивно-трофическую функцию и способствует обновлению клеток покровного эпителия. В то же время длительная экспозиция желчных кислот, независимо от ее концентрации, может приводить к существенным дистрофическим изменениям слизистой оболочки желудка, способствовать началу пилоризации фундальных желез и атрофии желез тела желудка. При пероральном введении желчи с последующим иммобилизационно-холодовым стрессом одновременное влияние нескольких агрессивных факторов приводит к ускорению развития патологических изменений в слизистой оболочке желудка: возникновению вначале эрозивных, а в дальнейшем и язвенных поражений.

Дане дослідження було проведено з метою вивчення ролі жовчних кислот у формуванні морфофункціональних змін слизової оболонки шлунка в щурів з урахуванням впливу жовчі та її концентрації. Було встановлено, що на ранніх етапах моделювання дуоденогастрального рефлюксу за допомогою введення жовчі в невисоких концентраціях вона здійснює захисну адаптивно-трофічну функцію та сприяє оновленню клітин покривного епітелію. У той же час тривала експозиція жовчних кислот, незалежно від її концентрації, може призводити до істотних дистрофічних змін слизової оболонки шлунка, сприяти початку пілоризації фундальних залоз і атрофії залоз тіла шлунка. При пероральному введенні жовчі з наступним іммобілізаційно-холодовим стресом одночасний вплив декількох агресивних чинників призводить до прискорення розвитку патологічних змін у слизовій оболонці шлунка: виникненню спочатку ерозивних, а в подальшому й виразкових уражень.


Keywords

bile acids, duodenogastric reflux, gastric mucosa.

желчные кислоты, дуоденогастральный рефлюкс, слизистая оболочка желудка.

жовчні кислоти, дуоденогастральний рефлюкс, слизова оболонка шлунка.

Статья опубликована на с. 30-33

Context

In a number of studies has been found that the effect of taurocholate on gastric mucosa is accompanied by damage to the latter with a significant decrease in transmucosal potential difference [1–3].

On top of that inhibition of gastric acid secretion and concentration of the luminal HCO were observed as well as increase in the levels of luminal prostaglandins and NO. It should be noted that the increase in said NO concentrations depends on increasing of the luminal Ca++ [4].

In these studies increased acid secretion in the damaged stomach were observed during NO-synthase blockade using L-NAME, but only under conditions of intact prostaglandin biosynthesis. However, as of today, state of gastric secretion in the dynamics of duodenogastric reflux in vivo remains unclarified, as well as participation of NO in the regulation of gastric secretion, especially in the early stages of exposing to the adverse factors.

The aim of the study was to investigate the role of bile acids in morphofunctional changes of gastric mucosa in rats with due regard to exposure of bile and its concentration.

Methods

The study was conducted on 60 white male laboratory rats (6 months old, 220–280 g). Animals did not receive food over a period of 16–20 hours before the experiment but were kept with constant access to water. The total number of animals was divided into five study groups.

Group I (n = 10) — administration of 90.0 % bile/saline solution (per os 1 ml/100 g of body weight) for 6 and 12 days and in 15 min after that undergo «immobilization and cold stress» which lasted for 1 hour (+ 4 °C). For purposes of this study medical bile (bile of cattle and swine, ZAT «Іnfuzіya», Kiev) was used.

Animals in the II (n = 10), III (n = 10) and IV (n = 10) groups recieved 25.0, 50.0 and 90 % of bile/saline solution (per os 1 ml/100 g body weight), respectively, for 6 and 12 days.

In the animals of V group (n = 20) the characteristics and composition of gastric secretion under the conditions of single and prolonged L-NNA administration (6th and 12th day of experiment) were studied. NO–synthase was blocked out by the intraperitoneal injection of NG-nitro-L-Arginine (L-NNA, Sigma-Aldrich, USA) in dosing of 40 mg/kg. After the basal portion of gastric juice during the first hour of L-NNA administration and the collection was lasted for another hour.

Sampling of gastric juice was performed under general anesthesia using a specially modified probe [19]. Pepsin levels (mg/mL), glycoproteins (mg/mL), pH and volume were determined in the gastric juice by using common techniques [7].

The study has been performed following the standards of the European Convention of Bioethics (1997), European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, general ethical principles of animal experiments, approved in the law of Ukraine (№ 1759 — VI of 15.12. 2009) «On protection of animals from cruel treatment».

Statistical processing was performed using Microsoft Excel software package and methods of variation statistics [5]. Obtained results were considered significant if p < 0.05.

Results/interpretation

It has been established that the behavior of animals in experiment was marked by a decrease in motor, orientational and exploratory activity and increased number of bowel movements and urination that indicates activity of CNS structures and mismatch of the forementioned activity with peripheral autonomic regulatory mechanisms in the digestive system.

Deterioration of the rats’ general condition was accompanied by morphological changes in the stomach. Specifically, on day 6 of the experiment erosive damage to the proximal stomach segment has been observed in the majority of rats. After 12 days of bile administration and stress a small ulcers (1–2 mm in diameter) have been observed in the gastric cardia along with erosive-ulcerative lesions in the gastric body and cardia. In addition to morphological changes in gastric mucosa, pronounced changes of the functional state of the stomach secretory apparatus has been found. Values of basal gastric secretion in control group and under the experimental conditions is shown in table 1.

After 3 days of experiment the noticeable increase in pepsin concentration (18.1 %), glycoproteins (41.2 %) and pH (32.6 %, p < 0.05) has been observed compared to the control group, along with small statistically nonsignificant increase in the gastric juice volume.

After 6 days of experiment further increase of the gastric juice pH (62.2 % compared to control group, p < 0.05) has been observed while the volume of gastric juice remains on the previous level (see 3rd day above) and concentration of pepsin and glycoproteins return to the control values.

After 12 days of experiment the volume of gastric juice was significantly higher (85.4 %) and its acidity increased, whereas the concentration of pepsin and glycoproteins was lower (36.7 % and fivefold respectively) compared to the control group (p < 0.05).

Administration of a 25 % bile/saline solution per os for 3 days (group II) does not lead to any significant changes in gastric mucosa. However, administration of a 50 and 90 % solution (groups III and IV) resulted in dilation stomach vessels and swelling which was accompanied by leukocyte/eosinophil infiltration in the gastric glands, muscle plate and submucosa. Dense eosinophilic and leukocyte infiltration has been also observed in duodenum. At the same time vessels’ thrombi and noticeable erosions was observed in duodenum, some of which reached the muscle plate (fig. 1 a, b, c).

Thus, the oral administration of bile in experiment led to the inflammatory reaction in gastric mucosa of rats with minor degenerative changes in the surface epithelium and increased cell renewal. At the same time, oral administration of bile followed by immobilization and cold stress for an hour (+ 4 °C) was the cause of noticeable erosions.

In contrast to the daily administration of bile per os on an empty stomach after 6 days of NO synthase deficiency (group V) deterioration of the rats’ general condition has not been observed. After 12 days in this group moderate inhibition of behavioral activity in a normal environment (in the cell) has been observed, which was expressed by relative passivity of movements and low appetite, alongside with slight decrease in body weight by 7.0–11.1 gram per each animal.

Alteration in the general condition was accompanied by morphological changes in gastric mucosa. Hyperaemia of the mucosa, which was most pronounced in the stomach fundus, specifically in the area of gastrooesophageal passage and large curvature, including the formation of multiple erosions and acute ulcers with round, oval or poligonal (uncommon) shapes ranging from 1 to 2.5 mm in size, has been observed in the majority of rats on the 12th day of experiment (fig. 2 a, b).

Results obtained in the V group indicated that NO could suppress intact gastric acid secretion and non-selective blockade of the NO-synthase results in the increase of gastric juice’s volume and acidity. It could also be accompanied by a decrease in the level of pepsin and a significant increase in the concentration of glycoproteins in the gastric juice. Partially such effects can be explained by the known stimulating effect of NO on cyclooxygenase, which increase the synthesis of prostaglandins. The latter have an impact on protectability of gastric mucosa (primarily prostaglandins POE, POE2 and RMB) and have a capacity to inhibit the secretion of hydrochloric acid.

As was established previously, the administration of bile during the 12 days resulted in the thick massive chronic infiltration of gastric and duodenum mucosa, beginning of pseudopylorization, gastric mucosa atrophy and violation of NO regulatory mechanisms. In reliance on this as well as on findings from the longtime blocking of NO it can be concluded that bile injures the gastric mucosa notably under conditions of disturbance in NO–regulation system (and in this case the deficit of NO).

After 3 days blocking of local NO mechanisms under conditions of experimental duodenogastric reflux in rats it was evident that all the investigated parameters of gastric secretion is increased, especially the volume of gastric juice (85.7 %, p < 0.05), pH (82.7 %, p < 0.05) and the concentration of glycoproteins (about twofold). Whereas after 6 days there was statistically significant decrease in the concentration of glycoproteins and statistically significant increase in pH (about twofold) compared to the initial value (p < 0.05). After 12 days of blocking of NO local regulatory mechanisms the significant decrease of gastric juice volume (41.5 %) compared to the initial value (p < 0.05) has been abserved, whereas levels of pepsin, glycoproteins and acidity remain the same.

On the other hand, the mechanism of gastric secretion stimulation may be mediated by nerve reflex involving vagal afferent fibers via inhibitory action of NO-synthase antagonist, as observed in the case of L-NAME [6], which similar to L-NNA, is nonselective blocker of NO regulatory –mechanisms.

The tendency to reduction in pepsin concentration and increase in glycoproteins under the action of L-NNA indicates possible stimulating effect of NO in relation to the activity of the main cells and decelerate action to the surface epithelial cells. This action in our opinion may be indirectly performed, since we did not find any information about specific cell formations that define a direct effect of nitric oxide on forementioned cell types in the available literature.

Conclusions

In reliance on the obtained results it can be stated that in the early stages of duodenogastric reflux in experiment bile in low concentrations carries protective adaptive-trophic function and promotes cell renewal of the surface epithelium. At the same time long-term exposure to the bile acids regardless of its concentration can lead to the significant dystrophic changes in gastric mucosa, pilorization and atrophy of the gastric glands.

In the case of the oral administration of bile followed by «immobilization and cold stress» for one hour (I group) experiment’s results can confirm that the combined effects of several aggressive factors lead to the more severe pathological changes in the gastric mucosa: first erosive and later ulcerative lesions.


Bibliography

1. Nitric oxide, histamine, and sensory nerves in the acid secretory response in rat stomach after damage / K. Takeuchi, S. Kato, Y. Abe et al. // J. Clin. Gastroenterol. — 1997. — Vol. 25, –Suppl 1. — P. 39–47.

2. Mechanism of acid secretory changes in rat stomach after damage by taurocholate: role of nitric oxide, histamine, and sensory neurons / K. Takeuchi, S. Kato, T. Yasuhiro, K. Yagi // Dig. Dis. Sci. — 1997. — Vol. 42, № 3. — P. 645–653.

3. Interactive roles of endogenous prostaglandin and nitric oxide in regulation of acid secretion by damaged rat stomachs / K. Takeuchi, S. Sugamoto, H. Yamamoto et al. // Aliment. Pharmacol. Ther. — 2000. — Vol. 14, Suppl 1. — P. 125–134.

4. Regulatory mechanism of acid secretion in the damaged stomach: role of endogenous nitric oxide / K. Takeuchi, H. Araki, S. Kawauchi et al. // J. Gastroenterol. Hepatol. — 2000. — Vol. 15, Suppl. — P. 37–45.

5. Петри А. Наглядная статистика в медицине / А. Петри, К. Сэбин. — М.: ГЭОТАР-Мед., 2003. — С. 143.

6. The mechanism underlying stimulation of gastric HCO3-secretion by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester in rats / K. Takeuchi, T. Ohuchi, M. Tachibana, S. Okabe // J. Gastroenterol. Hepatol. — 1994. — Vol. 9, Suppl 1. — P. 50–54.

7. Клініко-лабораторна оцінка функціонального стану секреторних залоз шлунка (методичні рекомендації) / А.І. Руденко, Т.В. Майкова, Л.М. Мосійчук та ін. — К., 2004. — 23 с.

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