RU2714679C1 - Method for simulating a combined pathology of metabolic syndrome and chronic obstructive pulmonary disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine, namely to a method for simulating a combined pathology of metabolic syndrome and chronic obstructive pulmonary disease. Method for simulating a combined pathology of metabolic syndrome and chronic obstructive pulmonary disease, where C57BL/6 female mice one day after birth, monosodium glutamate is administered subcutaneously for 10 days in dose of 2.2 mg/kg, and then on 126th day after birth, on the background of metabolic syndrome developed in animals, determined by Lie index, hyperglycemia and dyslipidemia, chronic obstructive pulmonary disease is induced by intratracheal introduction of liposaccharide in dose of 3 mcg per mouse on 126 and 129 days after birth and course intratracheal introduction of cigarette smoke extract in dose of 50 mcl per mouse on 127, 130, 133, 136, 139, 142, 149, 156, 163, 170 days after birth, followed by histological examination to determine inflammation, pulmonary emphysema, destruction of microcirculatory bed and alveolar epithelium, where in the lumen of separate alveoli there are accumulations of macrophages, there is peribronchial edema, moderate expansion of bronchioles, alveolar passages and alveoli.

EFFECT: disclosed method is effective for searching for drugs affecting metabolic syndrome and chronic obstructive pulmonary disease.

1 cl, 2 dwg, 8 tbl, 1 ex

Description

The invention relates to the field of experimental biology and medicine, and relates to the modeling of metabolic disorders complicated by chronic obstructive pulmonary disease, for the search and development of drugs for the prevention and treatment of pulmonary complications developing in the metabolic syndrome.

It is increasingly recognized that metabolic syndrome and chronic obstructive pulmonary disease (COPD) are the leading causes of morbidity and mortality worldwide [1]. It is believed that the development of COPD and metabolic syndrome includes common risk factors that can be cross-linked in an individual patient [2]. There is a negative effect of metabolic disorders and diabetes on lung function [3]. However, the relationship between the mechanisms of development of the metabolic syndrome and COPD is still unknown, which complicates drug therapy [4]. In this regard, the creation of a new experimental model covering the symptoms of metabolic syndrome and COPD, in our opinion, is necessary.

To develop effective technologies for treating patients with metabolic syndrome complicated by chronic obstructive pulmonary disease, aimed at regenerating injured tissues, it is necessary to use an adequate experimental model that combines the symptoms of metabolic syndrome (dyslipidemia, hyperglycemia) and chronic obstructive pulmonary disease (emphysema, destruction of the alveolar epithelium and endothelium). An adequate prototype was not found in the analyzed patent and medical literature.

The problem solved by this invention is the creation of a model of the metabolic syndrome complicated by chronic obstructive pulmonary disease, which is based on metabolic disorders and pathomorphological changes in the pancreas and lung tissue.

The problem is solved by simulating the combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease, characterized by the fact that female C57B L / 6 mice are injected subcutaneously with monosodium glutamate for 10 days at a dose of 2.2 mg / kg, and then on 126 days after birth, against the background of a metabolic syndrome developed in animals, determined by the Lee index, hyperglycemia and dyslipidemia, chronic obstructive pulmonary disease is induced by intratracheal administration of lipolysaccharide in 3 μg per mouse at 126 and 129 days after birth and course intratracheal administration of a cigarette smoke extract in a dose of 50 μl per mouse at 127, 130, 133, 136, 139, 142, 149, 156, 163, 170 days after birth, determined inflammation, emphysema, destruction of the microvasculature and alveolar epithelium.

New in the proposed method is the study of the combined effects of metabolic disorders induced by sodium glutamate and chronic obstructive pulmonary disease induced by lipopolysaccharide and cigarette smoke extract extract.

The proposed method for modeling the metabolic syndrome and chronic obstructive pulmonary disease most fully reveals the clinical picture of disorders characteristic of the metabolic syndrome complicated by chronic obstructive pulmonary disease.

Monosodium glutamate (lat. Natrii glutamas, English monosodium glutamate, sodium glutamate [5]) - monosodium salt of glutamic acid, a popular food supplement E621 (also called a “flavor enhancer”) is a white crystalline powder, readily soluble in water . Recent animal studies have shown that monosodium glutamate administered to a newborn animal can be used to induce an experimental model of obesity. Such animals develop severe obesity and diabetes mellitus, the clinical course of which is similar to the development of the metabolic syndrome in humans. [6, 7]. Obesity is a key component of the metabolic syndrome, a condition determined by a combination of clinical characteristics, including abdominal obesity, hyperglycemia, hypertriglyceridemia, hypertension and low levels of high density lipoproteins [6].

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with chronic inflammation of the alveolar tissue and the formation of emphysema. Cigarette smoking was recognized as the main risk factor for developing COPD [8, 9]. Various methods are used in the experimental studies for the induction of emphysema and chronic obstructive pulmonary disease: exposure to smoking, intranasal installation of elastase, intranasal installation of lipopolysaccharide, exposure to sulfur dioxide, inhalation of dry ovalbumin t powder .d. [9]. Cigarette smoke is a mixture of more than 4000 different chemical compounds, such as free radicals, toxins, etc. Cigarette smoke extract contains almost all compounds inhaled by smokers [9]. An important criterion for evaluating the model of emphysema and COPD is the histological evaluation of lung tissue. According to the American Thoracic Society, emphysema is defined as “abnormal, constant expansion of air spaces distal to the final bronchiole, accompanied by the destruction of their walls” [10]. Therefore, a histological evaluation of lung tissue is a mandatory criterion for confirming the development of COPD.

To date, an understanding of metabolic syndrome and COPD has evolved from the concept of a disease limited by impaired fat metabolism and respiratory tract, respectively, to more “complex diseases” often associated with other chronic diseases. As the most likely mechanisms for the development of complications, systemic inflammation is proposed that promotes the development of both metabolic syndrome and COPD [11]. Therefore, in the clinical management of such patients, they are trying to treat all these potential concomitant diseases and their inactive mechanisms. However, there are no drugs successfully used in the treatment of chronic diseases, such as metabolic disorders complicated by chronic obstructive pulmonary disease. In many respects, the current situation is associated with the lack of adequate experimental models of induced metabolic disorders complicated by chronic obstructive pulmonary disease, which would allow the selection of drugs effective in both pathologies.

Distinctive features showed in the claimed combination of new properties that are not explicitly derived from the prior art in this field and are not obvious to a specialist.

An identical set of features was not found in the analyzed patent and medical literature.

The method can be used to search for effective drugs that can affect metabolic syndrome and chronic obstructive pulmonary disease. Based on the foregoing, the claimed invention meets the patentability criteria of the invention of "Novelty" and "Inventive step" and "Industrial applicability".

The proposed method was studied in experiments on 40 female mice of the C57B L / 6 line. Animals came from the Department of Experimental Biological Models NIIIFiRM them. E.D. Goldberg (veterinary certificate is available). The keeping of animals and experimental design are approved by the Ethics Committee of the NIIIFiRM them. E.D. Goldberg and comply with international rules adopted by the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes.

In order to exclude seasonal variations in the studied parameters, all experiments were carried out in the autumn-winter period. Material sampling is carried out in the morning. Mice are killed by an overdose of CO ₂ . The number of animals in each group is at least 20 individuals.

The method will be clear from the following description and the attached figures 1 and 2.

In FIG. 1 shows the lung of a mouse on day 188 of the experiment, when staining lung tissue with hematoxylin and eosin: a, b, c - intact, where a - upper pulmonary field, b - middle pulmonary field, c - lower pulmonary field; g, e, e - receiving sodium glutamate and with induced chronic obstructive pulmonary disease, where g is the upper pulmonary field, e is the middle pulmonary field, e is the lower pulmonary field. Magnification × 100.

In FIG. Figure 2 shows the lung of a mouse on day 188 of the experiment, with immunohistochemical staining of lung tissue for the following markers: CD31 (a, e), α1-antitrypsin (A1AT) (b, f), insulin (c, g), pan-cytokeratin (AE1 / AE3) (g, h): a, b, c, d - intact; d, f, g, h - treated with monosodium glutamate and with induced chronic obstructive pulmonary disease. Magnification × 100.

The method is as follows:

The experimental metabolic syndrome is caused by the introduction of sodium glutamate (from 1 to 10 days of life, daily, subcutaneously at a dose of 2.2 mg / g) [12]. The date of birth of animals is considered 1 day of the experiment. On the 124th day of the experiment, the Lie index (body mass index) is calculated [12]. This index is calculated as the cubic root of body weight (g) * 10 / nasal length (mm). In female animals with a Lie index of more than 0.300 and impaired glucose tolerance test, lung emphysema is induced on day 126 of the experiment.

Pre-obtained cigarette smoke extract from L&M REDLABEL cigarettes 2 cigarettes per ml (composition of 1 cigarette: resin 10 mg / sig, nicotine 0.8 mg / sig, CO10 mg / sig). Before receiving the extract, remove the cigarette filter, the length of the cigarette with the filter is 80 mm, while removing the filter 55 mm. The extraction is carried out by pulling the smoke of a lit cigarette through a phosphate buffer at a constant speed, using a vacuum pump, the cigarette is burned to a length of 5 mm. The burning time of one cigarette is 180 seconds. To remove particles, the resulting extract is filtered through a bacterial filter with a pore size of 45 nm. To standardize the extract obtained, before and after filtering the solution, pH (pH ~ 7) and optical density are measured at wavelengths of 405 and 540 nm (D ₄₀₅ ~ 237, D ₅₄₀ ~ 123).

Lipopolysaccharide (LPS) is used to initiate the acute phase of inflammation. Lipopolysaccharide is a component of the cell wall of gram-negative bacteria E. coli O111: B4 (Lipopolysaccharides from Escherichia coli O111: B4, Sigma, USA). LPS stimulates the cells of the innate immune system with the Toll-like receptor 4, a member of the Toll-like receptor protein family, which recognizes common pathogen-related molecular structures (PAMPs), thereby causing an increase in the inflammatory response [13].

Chronic obstructive pulmonary disease is caused by the course of intratracheal administration of lipopolysaccharide and cigarette smoke extract [9, 14, 15]. LPS at a dose of 3 μg / mouse in 50 μl of phosphate buffer and 50 μl of cigarette smoke extract is administered intratracheally [16]. With the introduction of LPS and cigarette smoke extract, general anesthesia (pentobarbital, zoletil and xylazine) is used. The introduction of LPS is performed on 126 and 129 days of the experiment. Cigarette smoke extract is administered on 127, 130, 133, 136, 139, 142, 149, 156, 163, 170 days of the experiment.

The model is controlled by indicators of glucose level, measurement of the lipid profile in the blood and standard histological studies of the lungs with the calculation of the emphysema area [14]. The blood glucose level is determined using a glucometer (Accu-Chek Performa Nanu (Roche Diagnostes GmbH, Germany). The initial blood glucose level in animals is measured after 16 hours of feed deprivation, all subsequent glucose measurements are also taken after 12 and hourly food deprivation. Assessment of lipid profile (LDL, HDL, triglycerides) is carried out by standard biochemical methods.

On the 124th and 186th day of the experiment, a glucose tolerance test is performed. At the first stage, a blood sample is taken to evaluate the initial glucose level in experimental animals. 1 hour after this, an intragastric injection of glucose (D-glucose, Sigma-Aldrich, USA) is carried out at a dose of 2 g / kg. According to the literature, the response of animals to low doses of glucose during its intragastric administration is weak, therefore, a dose of glucose of 2 g / kg is used [17]. The first blood sampling for the study of glucose levels is carried out 30 minutes after glucose injection, then the glucose content is studied at intervals of 30 minutes, the duration of the study was 90 minutes.

For morphological studies, the right lobe of the lung is fixed in a 10% solution of neutral formalin, passed through ascending alcohols to xylene and poured into paraffin according to standard methods. Deparaffinized sections with a thickness of 5 μm were stained with hematoxylin-eosin [17]. Micropreparations from each experimental animal are examined under an Axio Lab.A1 light microscope (Carl Zeiss, Germany) at 100- and 400-fold magnifications. Violations of the histoarchitectonics of lung tissue, the presence of edema and inflammatory infiltration, venous stasis, thickening of the walls of blood vessels and bronchi are evaluated [18, 19].

For each experimental animal, at least 5 micrographs are taken without overlapping over the entire surface area of the lung tissue at a 100-fold increase. The system used consists of an Axio Lab.A1 microscope (Carl Zeiss, Germany) with an AxioCam ERc5s video camera (Carl Zeiss, Germany) connected to a personal computer. The resulting images are processed using AxioVision Rel.4.8.2 software. The area of emphysematous dilated alveoli in the lungs is determined using a special function to calculate the area of the object in the image. Sections of the bronchi and blood vessels are removed from the analyzed areas.

The calculation of the relative area of emphysema is made according to the formula 1:

where ∑a is the sum of pixels occupied by emphysema-dilated alveoli in all images of one preparation, S is the number of pixels corresponding to the total area of the image (using this camera and program - 4423680), b is the sum of pixels occupied by the empty part of the glass slide, all pictures of one drug [20-22].

Immunohistochemical examination of the lungs is carried out on the 188th day of the experiment. Sections of lung tissue were placed on slides with an adhesive polylysine coating (Leica biosystems, Germany). Before staining, dewaxing of tissue sections is carried out, followed by unmasking the antigen in citrate buffer (pH = 6) for 20 minutes. Incubation with primary antibodies is carried out in a humid chamber at 37 ° C. The following primary antibodies are used to detect specific cell markers: polyclonal antibodies to insulin (ab63820, Abcam, USA), polyclonal antibodies to membrane protein CD31 (ab28364, Abcam, USA), polyclonal antibodies to α1-antitrypsin (ab9373, Abcam, USA), monoclonal antibodies to pan-cytokeratin (AE1 / AE3) (ab80826, Abcam, USA). For detection of antibodies, a visualization system is used in accordance with the manufacturer's instructions (Spring bioscience, USA). Sections are contrasted using hematoxylin. After staining, the sections are dehydrogenated in xylene and enclosed in a mounting medium. To obtain micrographs, an Axio Lab.A1 microscope (Carl Zeiss, Germany) with an AxioCam ERc5s camera (Carl Zeiss, Germany) is used. Image analysis and counting of cells expressing detectable antigens is performed using the ImageJ program. For each experimental animal, a minimum of 10 micrographs are taken without overlapping over the entire surface of the lung tissue slice at a 100-fold increase. The percentage of stained cells is estimated by counting the number of stained cells in relation to the total number of lung tissue cells.

Statistical processing of the results is carried out by methods of variation statistics using the statistical processing package SPSS 12.0. The arithmetic mean (M), the arithmetic mean error (m), the probability value (P) are calculated. The difference between the two compared values is considered reliable if the probability of their identity was less than 5% (P <0.05). Using selective asymmetry and excess coefficients, the degree of approximation of the distribution law of the investigated trait to normal is estimated. In cases of a normal distribution of attributes, the Student's parametric t-test is used for statistical evaluation. With large deviations of the distribution of the trait from the normal form, for independent samples, the non-paramatric Wilcoxon criterion U is used. To determine the reliability of differences in qualitative indicators, the Fisher angular transformation criterion is used.

Example 1

The experiments performed show that the modeling of metabolic disorders leads to a significant increase in the Lie index in mice of the C57BL / 6 female line by 124 days of the experiment (Table 1).

Note. * - the differences are statistically significant compared with the intact control (p <0.05);

A criterion for the severity of impaired glucose metabolism is the glucose tolerance test. In animals of intact control and in mice with metabolic disorders, a glucose tolerance test is performed. In response to glucose injection in intact animals, a significant increase in blood glucose levels was recorded over 30 minutes of observation (Table 2). In animals with metabolic disorders, a more pronounced increase in glucose level is recorded and an increase in the area under the curve (AUC) is observed (Table 2).

Modeling of metabolic disorders causes an increase in the concentration of LDL in the blood serum on the 124th day of the experiment (table 3). Changes in HDL, triglycerides are directed towards lowering indicators.

Note. * - the differences are statistically significant compared with the intact control (p <0.05); # - differences are statistically significant compared with the initial level (p <0.05).

Note. * - the differences are statistically significant compared with the intact control (p <0.05)

Thus, the administration of sodium glutamate from 1 to 10 days of life, daily, subcutaneously at a dose of 2.2 mg / g, causes the metabolic syndrome of C57BL / 6 female mice by 124 days of the experiment, as evidenced by an increase in the Li index in animals, and violations during glucose tolerance test and disorders of lipid metabolism.

In mice of C57BL / 6 females with a Lie index of more than 0.300 and impaired glucose tolerance, COPD was induced on day 126 of the experiment.

Against the background of the development of the combined pathology of metabolic disorders and COPD in animals, indicators are recorded to confirm the development of metabolic disorders and COPD.

The Lee index increases in all groups throughout life, and, as expected, the group with metabolic syndrome and COPD shows a significantly higher value than control intact mice by 188 days of the experiment (Table 4).

Note. * - the differences are statistically significant compared with the intact control (p <0.05)

An important criterion for confirming metabolic disorders is a change in glucose metabolism. A glucose tolerance test performed on day 186 of the experiment demonstrates a decrease in glucose homeostasis in mice with metabolic syndrome and COPD (Table 5). In addition, by 188 days of the experiment, significantly higher blood glucose levels were observed in this group of mice (Table 6).

Note. * - the differences are statistically significant compared with the intact control (p <0.05);

Note. * - the differences are statistically significant compared with the intact control (p <0.05)

Further, animals are evaluated indicators indicating the development of chronic obstructive pulmonary disease. A preliminary macroscopic evaluation of the lungs reveals an increase in the volume of the organ in mice of the control group, there is a softness of its consistency and loss of tissue elasticity compared to intact animals, while the organ tissue falls and is easily damaged. In addition, there is hyperemia of the lung tissue, hemorrhagic exudate is secreted during the incision, an increase in the right ventricle of the heart and the expansion of large vessels are detected. Histological examination shows that under the conditions of the introduction of LPS and cigarette smoke extract against the background of the development of the metabolic syndrome, macrophage accumulations are detected in the lumen of individual alveoli, peribronchial edema occurs, moderate stretching of bronchioles, alveolar passages and alveoli is noted. In addition, ruptures of the alveolar septa are observed, single atelectases are noted, the alveolar capillaries are thinning and desolate (Fig. 1, table 7). As a result of the action of LPS and cigarette smoke extract on elastic fibers, diffuse emphysema develops in the lung mouse parenchyma. The most pronounced emphysema is recorded in the lower pulmonary field, then the average pulmonary field follows the intensity of the lesion and closes the row of the upper pulmonary field (table 7).

Note: * - the differences are significant compared with the intact control (p <0.05; U - Mann-Whitney test).

An immunohistochemical study of lung tissue in mice. The introduction of cigarette smoke extract significantly reduces the number of cells expressing CD31, α1-antitrypsin and pan-cytokeratin (AE1 / AE3) in the lung tissue of mice with metabolic disorders and COPD compared with the intact control on day 188 of the experiment ( Fig. 2, table 8). A study of the expression of insulin in the lung tissue of mice does not reveal differences between the groups.

Note: * - the differences are significant compared with the intact control (p <0.05; U - Mann-Whitney test).

Thus, the administration of sodium glutamate, subcutaneously at a dose of 2.2 mg / g for 10 days starting one day after birth, to female C57BL / 6 mice and the subsequent two-fold intratracheal administration of lipolisaccharide at 126 and 129 days of the experiment at a dose of 3 μg per mouse and course intratracheal administration of cigarette smoke extract on 127, 130, 133, 136, 139, 142, 149, 156, 163, 170 days of the experiment at a dose of 50 μl per mouse, causing metabolic disturbances (hyperglycemia, glucose tolerance disorders) by 188 days of the experiment test, hypercholisterinemia) and pa omorfologicheskih changes in the lungs (destructive changes in lung tissue).

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Claims (1)

A method for simulating the combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease, characterized in that C57BL / 6 female mice are injected subcutaneously with monosodium glutamate for 10 days at a dose of 2.2 mg / kg and then for 126 days after birth, against the background of a metabolic syndrome developed in animals, determined by the Lee index, hyperglycemia and dyslipidemia, chronic obstructive pulmonary disease is induced by intratracheal administration of a lipolysaccharide at a dose of 3 μg per mouse at 126 and 129 sous after birth and course intratracheal administration of cigarette smoke extract in a dose of 50 μl per mouse on 127, 130, 133, 136, 139, 142, 149, 156, 163, 170 days after birth, then a histological examination is performed to determine pneumonia and emphysema , destruction of the microvasculature and alveolar epithelium, where accumulations of macrophages are detected in the lumen of individual alveoli, peribronchial edema occurs, moderate stretching of bronchioles, alveolar passages and alveoli is noted.

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