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Fundamental medicine
UDC 615.324:615.331:611.61:616.33-002.44-085-092.4
COMPARATIVE MORPHOFUNCTIONAL ASSESSMENT OF THE GASTROPROTECTIVE ACTIVITY OF THE PEPTIDE COMPLEX OF PIG KIDNEY TISSUES, OMEPRAZOL, RANITIDINE AND MISOPROSTOL BY "INDOMETACIN"
GASTRIC ULCER IN RATS
Altai State Medical University, Barnaul
S.E. Lorents, A.Yu. Zharikov, I.P. Bobrov, O.N. Mazko, O.G. Makarova, G.V. Zharikova
Objective: a comparative assessment of the effect of the peptide complex of the tissues of pig kidney, omeprazole, ranitidine and misoprostol on the morphofunctional state of the gastric mucosa in rats against the background of experimental "indomethacin" ulcer.
Materials and Methods: The ulcerative lesion was modeled by intragastric administration of indomethacin, the studied drugs were administered preventively for seven days. The condition of the mucosa was assessed macro and microscopically. Results and conclusions: The peptide complex has a pronounced gastroprotective effect, which is manifested in a decrease in the number and depth of coolant erosion, a decrease in the Pauls index for band-like erosion and an increase in mucosal thickness. In terms of efficiency, it is inferior to omeprazole, superior to miso-prostol. The reached affect is close to the effect of ranitidine.
Key words: peptide complex, indomethacin, omeprazole, ranitidine, misoprostol, gastric ulcer.
Previously, it has been found that the introduction of a peptide complex of pig kidney tissues under the conditions of an experimental "indomethacin" ulcer in rats contributes to a significant relief of the pathology [1]. This observation has created the prerequisites for a more in-depth study of the prospects for the development of a new gastropro-tective agent on the basis of the indicated peptide complex.
It is known, that the existing arsenal of anti-ulcer drugs includes several groups, including proton pump inhibitors, H2-histamine receptor blockers and stimulants of the production of protective mucus (analogs of prostaglandins E). Their pharmacodynamics is well studied. Therefore, to study the nature of the gastroprotective activity of the peptide complex of pig kidney tissues, it was of interest to compare its pharmacological action with the effects of the main representatives of anti-ulcer drugs.
Thus, the purpose of this study was a comparative assessment of the effect of the peptide complex of pig kidney tissues, omeprazole, ranitidine and misoprostol on the morphofunctional state of the gastric mucosa (GM) in rats against the background of experimental "indomethacin" ulcer.
Materials and methods
An experimental study was performed on 40 male Wistar rats weighing 200-250 g. The animals were kept in standard vivarium conditions. The studies were carried out in compliance with the principles of humanity set forth in the directives of the European Community (86/609 / EEC) and the Helsinki Declaration. Experimental animals were divided into the following experimental groups:
1. Disease control, Gcontr (modeling of indomethacin GM damage) - 8 rats;
2. Group of treatment with peptide complex of pig kidney tissues, Gp c (modeling of indomethacin GM damage + preventive administration pep-tide complex of pig kidney tissues) - 8 rats;
3. Omeprazole treatment group, G (mod-
-T Or' omepr. ^
eling of indomethacin GM damage + preventive administration of omeprazole) - 8 rats;
4. Ranitidine treatment group, Gran (modeling of indomethacin GM damage + preventive administration of ranitidine) - 8 rats;
5. Misoprostol treatment group, Gmis (modeling of indomethacin GM damage + preventive administration of misoprostol) - 8 rats.
Modeling of indomethacin ulcer was reproduced by a single intragastric administration of indomethacin at a dose of 60 mg/kg in 1ml of saline. The day before the modeling of gastropathy, animals were deprived of food with free access to water. Prophylactic drug administration lasted for seven days before the modeling of gastropathy. Peptide complex of pig kidneys obtained by the method of acetic acid extraction was administered at a dose of 75 mg/kg [1]. Omeprazole, misoprostol and ranitidine were administered in doses of 37 mg/kg; 0.09 mg/kg, 25 mg/kg, respectively, conditionally constituting 2% of the LD50 [3].
In 4 hours after the administration of the ulcer-forming factor, the animals were euthanized by single-stage decapitation under ether anesthesia, the stomach was removed. The stomach was cleaned, washed with cold saline.
For the morphological study, the material was fixed in a 10% solution of neutral formalin. The material was dehydrated in isopropyl alcohol using
a carousel-type dehydration machine TISSUE-TEK VIPTM6 (Sakkura, Japan). The material was poured into paraffin using the paraffin filling station TISSUE-TEK TEC 5 (Sakkura, Japan). Histological sections 5-7 pm thick were obtained using a semi-automatic rotary microtome Accu-Cut SRM (Sakkura, Japan) and stained with hematoxylin and eosin in a TISSUE-TEK Prisma machine for micro-section automatic staining (Sakkura, Japan). Histochemical staining on neutral glycosaminoglycans was also carried out by Schiffreagent according to McManus and on acidic glycosaminoglycans with 1% alcian blue solution on 3% acetic acid (pH 2.5) according to Steedman. Preparations were placed under film in a device for the automatic enclosure of micro-sections TISSUE-TEK Film (Sakkura, Japan).
In each case, the total number of erosions, the number of deep strip-like erosions and the number of surface point erosions were counted on a macroscopic level in the gastric mucosa. Pauls index (PI) or ulceration index for each type of damage was calculated according to the formula: (N x K)/100, where N is the average number of destructions per animal, K is the percentage of lesions of animals in the group. There was calculated the thickness of the coolant and the depth of erosion. The density of the inflammatory infiltrate of 1 mm2 was calculated using an eyepiece grid of Avtandilov G.G.
Morphometric studies were performed using a specially created computer image analysis system consisting of a Leica DME microscope (Germany), Leica EC3 digital camera (Leica Microsystems AG, Germany) and Video Test Morphology software 5.2. Statistical processing of the results was performed using Statistica 6.0.
Results and discussion
Quantitative indicators of the morphofunctional state of GM in experimental animals of all groups and their statistical comparison are shown in the table.
The results of the experiments showed that in a macroscopic study, GM surface in Gcontr looked destructive, changes in the form of deep strip-like and surface point erosion were clearly visible (Figure 1). By microscopic examination, GM in rats of the control group looked atrophic. Necrotic changes in the ulcers reached the muscle layer. Many erosions were characterized by massive hydrochloric acid hematin deposits. The inflammatory infiltrate was weak and consisted of lymphocytes, plasma cells and macrophages. Muscle layer showed phenomena of edema and moderately pronounced inflammation. These phenomena are typical for the introduction of indomethacin, as evidenced by the literature [2-4].
Figure 1 - Deep strip-like erosion of GM in rats of the control group Gcontr Staining with hematoxylin and eosin. Zoom x 100.
Against this background, the preventive administration of the pig kidney peptide complex led to significant changes in the condition of GM. Thus, in a macroscopic study of these animals, the relief of GM was even. Although destructive changes in GM were detected in all experimental animals, they were represented mainly by shallow
strip-like and surface point erosion (Figure 2). The average number of erosions per 1 animal compared with the disease control in G was 1.4 times less
px.
(Table 1). The number of the most dangerous striplike erosions has decreased by more than 2 times, and the Pauls index for this type of erosion has decreased by 3.6 times. The depth of erosion was
85.7% less. The thickness of the mucous increased by 70 microns. In the submucosal layer, moderate
inflammation was determined. The vessels were in a state of moderate plethora.
Figure 2 - Surface point erosion with a large amount of hydrochloric acid hematin of GM in rats of Gp c group. Staining
with hematoxylin and eosin. Zoom x 100.
Macroscopically, GM of animals in G
-T J' omepr
looked even, the folds were mild, and no atrophic changes were found. Weakly pronounced destructive changes in the form of small point erosion containing hydrochloric acid hematin were determined only in some areas. Similar destructive changes were detected in 71.4% of animals. All damage was represented by point erosion (Figure
3). Submucosal layer is characterized by mild inflammation. The vessels are moderately full-blood. The number of GM destructions was almost 7 times reduced compared with the control of the disease, the thickness of the mucous increased almost by 1.8 times. The depth of point erosion is insignificant - 3.3 times less than in the control of the disease.
Figure 3 - Point surface erosion with the deposition of hydrochloric acid hematin in GM of rats of G (indicated
O 1 J omepr v
by the arrow). Staining with hematoxylin and eosin. Zoom x 100.
The preventive administration of ranitidine also led to the relief of the pathology. It turned out that during macroscopic examination, in rats of Gran., GM is mostly even, single nodular areas are defined. Destructive changes in GM were detected in all rats of this group. Damage was represented by point and strip-like erosion with massive deposition of hydrochloric acid hematin (Figure 4). The vessels were moderately full-blood. The
average number of erosions of the mucous did not decrease, but a preponderance towards point erosions was observed. In addition, the depth of erosion was reduced by an average of 100 microns, and the thickness of the mucous increased by 160 microns. The Pauls index for strip-like erosions was 4.2 times lower than for the disease control, and 2.7 times for point erosions.
Figure 4 - Strip-like erosion in GM of rats of Gran (indicated by the arrow). Staining with hematoxylin and eosin.
Zoom x 100.
In the group of experimental animals, where misoprostol was administered, during a macroscopic study, the relief of GM was nodular, uneven, and the folds were thickened. Destructive changes in GM were detected in all rats. The total number of damage to GM was even 52% more
than in the control of the disease (Figure 5). The lesions were represented by long shallow and deep strip-like erosions, reaching the muscular plate of the mucous membrane and even with penetration into the depth of the submucosal layer and point erosions. Vessels enlarged, full-blood.
Figure 5 - Deep strip-like erosion reaching the muscular GM plate in rats of the 4th study group (indicated by the arrow).
Staining with hematoxylin and eosin. Zoom x 100.
Discussing the results, it should be noted that the most pronounced gastroprotective effect among the studied agents was produced by omeprazole. This was largely expected, considering its well-known ability to weaken the secretion of hydrochloric acid by inhibiting the proton pump. Accordingly, in conditions of indomethacin ulcer, a decrease in the production of hydrochloric acid by omeprazole weakened the destructive effect of this factor of aggression on GM [5, 6].
It is noteworthy that the peptide complex pig kidney tissues and ranitidine had a similar gastroprotective effect, even with some
predominance towards the peptide complex. As follows from the table, after the application of the peptide complex, the number of strip-like erosion compared to ranitidine was 3.0 times less (p = 0.03), the depth of erosion was 1.4 times less (p = 0.001). The Pauls index for strip-like erosions practically did not differ between the groups, and the value of this indicator for point erosion was 2.7 times less in G The explanation of the obtained
ran. r
observations could be made on the basis of data on the mechanisms of the gastroprotective action of the peptide complex, but they have yet to be studied.
Finally, assessing the effect of misoprostol by the totality of the obtained data it should be concluded that this drug does not have a positive gastroprotective effect, and in a number of indicators, even shows a tendency to aggravate pathology. On the one hand, this looked rather strange, considering that misoprostol, being an analogue of prostaglandin E, stimulates the production of protective mucus in the stomach, which prevents the aggressive influence of hydrochloric acid. On the other hand, it is possible that with prophylactic use of the drug in conditions
Conclusions:
1. Peptide complex has a pronounced gastroprotective effect, which is reflected in a decrease in the number and depth of GMt erosion, a decrease in the Pauls index for strip-like erosion and an increase in mucosal thickness.
2. According to the effectiveness of the gastroprotective action, the peptide complex is
of normal physiological production of protective mucus before the administration of indomethacin, misoprostol did not have a significant stimulating effect on this process. Possibly, a more pronounced effect of this drug may be reached by therapeutic use.
Summarizing the above, we note that the results of the study indicate a high antiulcer activity of the peptide complex of pig kidney tissues, comparable in strength to the widely used inhibitor of the H2-histamine receptor ranitidine.
inferior to omeprazole, superior to misoprostol. The effect close to the effect of ranitidine is achieved.
References
1. Lorents S.E., Zharikov A.Yu., Bobrov I.P., Mazko O.N., Makarova O.G., Kiselev V.I. Gastroprotective action of the peptide complex of pig kidneys at experimental «indomethacine»
Table 1
Effect of the peptide complex of pig kidney tissues, omeprazole, ranitidine and misoprostol on the indices of the morphofunctional state of the gastric mucosa by experimental indomethacin ulcer
Disease control Gastroprotector
Index Peptide complex Omeprazole Ranitidine Misoprostol
Number of GM damages 9,6±1,7 6,8±1,2 1,4±0,4* ^c^0,0003) ^.=0,0009) 11,4±2,8 14,6±2,8* ^P^0,03)
Number of strip-like erosions 4,8±0,9 2,2±1,0 - 6,5±2,2* ^P^0,03) 8,0±2,1* ^p.^0,05)
Number of strip-like erosions (%) 51,1 32,4 - 45,6 42,5
Number of point erosions 4,6±1,4 4,6±1,2 1,4±0,4 6,2±1,6 9,2±1,7* ^p^0,05)
Number of point erosions (%) 48,9 67,6 100 54,4 57,5
Pauls index for striplike erosions 24 6,6 0 5,2 6,4
Pauls index for point erosions 16,8 16,8 1,0 6,2 9,2
GM thickness (^m) 335,6±12,4 406,7±14,4 (R0n=0,001) 609,7±13,7* ^<0.000001) ^<0.000001) 496,1±25,2* (RoT0,00001) ^p^0,01) 391,5±10,7 ^c^0,003)
Depth of erosions (^m) 385,75±23,7 207,8±13,6 (Ron<0.000001) 115,8±5,2* ^ =0,000002) ^=0,0009) 283,9±17* (Ron=0,002) ^,=0,001) 321,9±20,6* fr«^0,05) ^.=0,00005)
Density of inflammatory infiltrate in GM in 1 mm2 1333,3±70,5 1720,0±115,5 (Ron=0,05) 1040±211,7* ^,=0,001) ^,=0,04) 1520±122,2* tep.c=0,05) 2106,7±70,5 fcc^0,001)
Note: the changes that are significant relative to the control group are underlined, * - the changes are significant relative to the group that received the peptide complex.
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Contacts
Corresponding author: Lorents Samira Elshadovna, lecturer of the Department of Pharmacology, Altai State Medical University Barnaul. 656056, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 241859. E-mail: [email protected]
Author information
Zharikov Aleksandr Yuryevich, Doctor of
Biological Sciences, Associate Professor, Head
of the Department of Pharmacology, Altai State
Medical University Barnaul.
656056, Barnaul, ul. Papanintsev, 126.
Tel.: (3852) 241859.
E-mail: [email protected]
Bobrov Igor Petrovich, senior staff scientist of the Morphological Laboratory of the Center of medico-biological research, Altai State Medical University Barnaul.
Tel.: (3852) 669927. E-mail: [email protected]
Mazko Olesya Nikolayevna, Candidate of Biological Sciences, Senior Scientific Researcher of the Biomedical Laboratory of the Center of medico-biological research, Altai State Medical University Barnaul.
Tel.: (3852) 669927.
E-mail: [email protected]
Makarova Olesya Gennadyevna, Candidate of Pharmaceutical Sciences, Senior Scientific Researcher of the Biomedical Laboratory of the Center of medico-biological research, Altai State Medical University Barnaul. Tel.: (3852) 669927. E-mail: [email protected]
Zharikova Ganna Viktorovna, lecturer of the
Department of General and Biological Chemistry,
Clinical laboratory diagnosis, Altai State Medical
University Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 566938
E-mail: [email protected]
