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UDC 615.324:616.61:616.33-002.44-085-092.4
EVALUATION OF MORPHOFUNCTIONAL ACTIVITY OF NUCLEOLUS ORGANIZERS IN CELLS OF THE GASTRIC MUCOSA OF RATS ON THE BACKGROUND OF PHARMACOLOGICAL CORRECTION OF "INDOMETACIN" ULCER WITH A PEPTIDE COMPLEX OF PORCINE KIDNEY TISSUE
Altai State Medical University, Barnaul
S.E. Lorents, A.Yu. Zharikov, I.P. Bobrov, O.N. Mazko, O.G. Makarova, M.E. Siryachenko
To assess the changes in the proliferative activity of cells of the gastric mucous membrane of rats during the preventive use of the peptide complex from the tissues of pig kidneys under the conditions of the "indometacin" ulcer, the morpho-functional activity of the nucleolar organizers in cells was studied. According to the results of the study, it was found that against the background of the seven-day preventive administration of the drug, the parameters of the morpho-functional activity of the nucleolar organizers are significantly reduced in comparison with the control, which indicates a decrease in the proliferation of the epithelium. Thus, it can be assumed that the peptide complex, having an anti-ulcerogenic effect, does not have the ability to directly stimulate the regeneration of tissues.
Key words: gastric ulcer, nucleolar organizers, peptide extract, gastroprotective effect.
Introduction
Earlier in the experiment, it was established that the use of the peptide complex from the tissues of porcine kidneys for the purpose of pharmacological correction of the "indometacin" ulcer was accompanied by a significant improvement in the course of the pathology. This was mainly evidenced by a decrease in the number of deep striplike erosions of the gastric mucosa and their depth, a decrease in the Pauls index and an increase in the thickness of the gastric mucosa [1].
In this context, we were interested in the question of studying possible mechanisms of the detected anti-ulcerogenic effect. In this connection, at the first stage, we decided to study the effect of the peptide complex on the morphofunction-al activity of the nucleolus organizers in the cells of the gastric mucosa. Nucleolus organizers are specific regions of chromosomes, containing many genes encoding rRNA. Nucleoli are formed around nucleolus organizers, and the proteins in them are responsible for activating and controlling the transcription of ribosomal genes, for regulating cell division [2,3]. By peptic ulcer disease, prolifera-tive processes significantly change in the damage zone [4]. The severity of these processes in the cell is directly related to the activity and the number of nucleolar organizers in the nucleus. This activity can be determined due to the presence of argyro-philic proteins (AgNORs), which can be detected by silvering [5,6]. Therefore, the determination of quantitative indices of AgNORs in the nuclei of cells of the gastric mucosa against the backdrop of the use of a peptide complex from the tissues of porcine kidneys can contribute to an under-
standing of the mechanisms of its anti-ulcerogenic action.
Thus, the purpose of this study is to determine the nature of the effect of the peptide complex from the porcine kidneys tissues on proliferative processes in the gastric mucosa by "indometacin" ulcer by means of morphofunctional evaluation of the activity of nucleolar organizers in cells.
Materials and methods
The study was carried out on 20 male Wistar rats (age 7-8 weeks, weight 200-250 g) divided equally into 2 groups: control (modeling of peptic ulcer disease) and experimental (modeling of gastric ulcer + prophylactic introduction of peptide complex from the tissues of porcine kidney). The animals were kept in standard conditions of a vivarium with free access to food and water. The studies were carried out in accordance with the principles of humanity set out in the directives of the European Community (86/609/EEC) and the Helsinki Declaration, in accordance with the "Rules for Working with Experimental Animals".
As an ulcer-forming factor, non-steroidal anti-inflammatory drug indometacin was used. The model of indometacin damage to the stomach of rats was reproduced by single intragastric administration of indometacin at a dose of 60 mg/kg in 1 ml of saline. The animals of the control group were injected intragastrically with 1 ml of distilled water prior to the administration of indomethacin for 7 days. The animals of the experimental group under similar conditions were injected with a pep-tide complex of porcine kidney tissues at a dose of 75 mg/kg. Six hours after the administration of the ulcer-forming factor, rats were euthanized,
the stomachs removed, washed in distilled water, the material fixed in a solution of 10% neutral formalin, and the activity of the nucleolar organizers was determined.
The nucleolus organizers (NO) were detected using the two-step method of Daskal Y. et al. [1980], in our modification [7]. When the material was stained, the round black AgNORs granules were clearly defined against the background of brown nucleoli and lighter nuclei. By zooming x 1000 under the oil immersion of the microscope, the number of nucleoli, the total area of AgNORs per nucleus and the nucleolar-nuclear ratio (Nucle-oli/Nuclei) in relative units were calculated. In each case, no less than 25-30 nuclei of the cervical gastric mucous membrane (GMM) of the rat stomach were examined.
The statistical processing of the material was carried out with the statistical package Statistica 10.0. By the normal distribution of data, the methods of parametric statistics (Student's t-test) were used to check statistical hypotheses, and if the obtained data did not meet the criteria of normal distribution (Shapiro-Wilk criterion W = 0.89, p <0.01), then the Kolmogorov-Smirnov test or the Mann-Whitney U-test were implied. The data were considered statistically significant by p <0.05.
Results and discussion
Evaluation of the morphofunctional activity of NO in the cells of the cervical parts of the GMM of the control group of rats showed that the number of nucleoli was 2.85 ± 0.3 per 1 nucleus. The AgNORs area per 1 nucleus averaged 4.5 ± 0.4 ^m2. The average nucleus area was 34.9 ± 2.5 |jm2. The ratio of Nucleoli/Nuclei was 0.18 ± 0.04 RU (Figure 1).
The assessment of morphofunctional activity of NO NO in the cells of the cervical parts of the GMM in the experimental group of animals showed that the number of nucleoli constituted 1.2 ± 0.1 per nucleus. The area of AgNORs was 1.7 ± 0.1 |jm2 per 1 nucleus. The average nucleus area constituted 17.2 ± 0.9 |jm2. The Nucleoli/Nuclei ratio constituted 0.10 ± 0.005 RU (Figure 2).
By comparing the results obtained, it was noted that all the parameters of the morphofunctional activity of the NO in the cells of the cervical parts of the GMM in the rats of the experimental group were significantly lower than the corresponding parameters in the control group: the number of nu-cleoli per nucleus is 2.4 times less, the AgNORs area is 2.6 times smaller (p <0.05), the nucleus area - 2 times smaller (p <0.05), the ratio of Nucleoli/Nu-clei - 1,8 times (p <0,05).
Thus, the results of the study showed that by indometacin damage to the rats of the control group rats in the cervical parts of the GMM, the high nucleus area and high rates of morpho-functional activity of the nucleolus organizers were
Figure 1. High activity of nucleolar organizers in cells of the cervical parts of the GMM in the rats of the control group. Silvering according to Bobrov I.P.
Figure 2. Low activity of nucleolar organizers in cells of the cervical parts of the GMM in the rats of the control group. Silvering according to Bobrov I.P.
and the rate of proliferative processes in the cervical areas. In the experimental group in the cells of the cervical parts of the GMM, the nucleus area was small, and the parameters of the morphofunc-tional activity of NO were low, which may indicate a low activity and rate of proliferative processes in the GMM.
Considering the low activity of biosynthetic processes in the GMM cells in the experimental group compared to the control, it can be assumed that the mechanism of the anti-ulcerogenic action of the peptide complex is not directly related to the stimulation of tissue regeneration. It is not excluded that other mechanisms that prevent the ulceration of the mucosa under the influence of indo-methacin are involved. The identification of these mechanisms will be the goal of further research.
Conclusion
The prophylactic intragastric administration of the peptide complex from the tissues of porcine kidneys on the model of "indometacin" gastric ulcer is accompanied by a weakening of the activi-
ty of nucleolar organizers in the nuclei of the cells of the gastric mucosa, which indicates a decrease in the activity of proliferative processes. Thus, it can be assumed that the peptide complex, possessing an anti-ulcerogenic effect, does not have the ability to directly stimulate the regeneration of tissues.
References
1. Lorents S.E., Zharikov A.Yu., Bobrov I.P., Mazko O.N., Makarova O.G., Kiselev V.I. Gas-troprotective action of the peptide complex from pig kidneys at experimental «indomethacine» ulcer in rats. Siberian Scientific Medical Journal. 2017; 6(37): 5-9.
2. Zenit-Zhuravleva E.G., Polkovnichenko E.M., Lushnikova A.A. Nucleophosmin and nucleolin: encoding genes and expression in various human and animal tissues. Molecular medicine. 2012; 4: 2533.
3. Bobrov I.P., Cherdantseva T.M., Bryukhanov V.M. Prognostic significance of argyrophilic proteins of nucleolar organizers regions (agnor) quantification in renal cell carcinoma. Russian Journal of Oncology. 2013; 1: 19-24.
4. Ageeva T.A., Maltseva Yu.G., Chernova L.N. Pathomorphological characteristic of the processes of cellular renewal in the gastric mucosa in peptic ulcer disease. Vestnik Novosibirsk State University. 2011; 3(9): 6-12.
5. Sirri V., Roussel P., Hernandez-Verdun D. The AgNOR proteins: qualitative and quantitative changes during the cell cycle. Micron. 2000; 2(31): 121-126.
6. Dolgatov A.Yu., Hervald V.Ya., Bobrov I.P. Expression of argyrophilic proteins associated with the region of the nucleolar organizer (Ag-NOR) by kidney cancer: clinical and morphological parallels. Siberian journal of oncology. 2011; 1: 45.
7. Bobrov I.P., Avdalyan A.M., Cherdantseva T.M. et al. Modified method for revealing argyro-philic proteins of the nucleolus organizer region
on paraffin sections. Morphology. 2010; 5(138): 6567.
Contacts
Corresponding author: Zharikov Aleksandr Yur-yevich, Doctor of Biological Sciences, Associate Professor, Head of the Department of Pharmacology of ASMU, Barnaul. 656056, Barnaul, ul. Papanintsev, 126. Tel.: (3852) 241859. E-mail: [email protected]
Lorents Samira Elshadovna, lecturer of the Department of Pharmacology of ASMU, 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 of ASMU, 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 of ASMU, 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 of ASMU, Barnaul. Tel.: (3852) 669927. E-mail: [email protected]
Siryachenko Mariya Evgenyevna, 5th year student of the Faculty of Pharmacy of ASMU, Barnaul. Tel.: (3852) 669927. E-mail: [email protected]
