RU2712761C1 - Agent for pulmonary endothelial regeneration stimulation at metabolic syndrome combined with chronic obstructive pulmonary disease - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine. Disclosed is the use of glucagon-like peptide 1 (GLP-1) for stimulating pulmonary endothelial regeneration in a combined pathology of metabolic syndrome and chronic obstructive pulmonary disease.EFFECT: technical result consists in the implementation of the prescription, wherein the introduction of GLP-1 to the mice with the above pathology resulted in a significant increase in endothelial progenitor cells in the lungs, comparable to their number in the intact animals.1 cl, 3 dwg, 10 tbl

Description

The invention relates to medicine, cell technology and regenerative medicine, and can be used to stimulate the regeneration of pulmonary endothelium with a combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease.

Recently, the attention of researchers and practitioners has been attracting the problem of comorbid pathologies, usually associated with several links of pathogenesis with each other. The relationship between metabolic syndrome (MS) and lung diseases, including COPD, has been confirmed by a number of clinical studies [1,2]. A common feature of metabolic syndrome and COPD is called systemic inflammation [3]. In addition, in patients with metabolic syndrome and obesity [4] and in patients with COPD [5] there are vascular complications mediated by impaired function and a decrease in the number of circulating endothelial cells.

To date, effective approaches to the treatment of MS patients with complications such as COPD do not exist. The clinical practice of MS is mainly focused on the appointment of anti-inflammatory drugs, the treatment of cardiovascular complications, maintenance therapy. The chronic and progressive nature of MS and COPD, when combined, the high incidence of complications leading to disability of the population, determine the need to develop effective approaches to treat this category of patients and to search for drugs with the ability to stimulate the regeneration of cells and tissues, and especially lung endothelium.

The problem solved by this invention is the expansion of the arsenal of tools with the ability to stimulate the regeneration of endothelial lung with a combination of metabolic syndrome and chronic obstructive pulmonary disease.

The problem is achieved by the use of glucagon-like peptide 1 to stimulate the regeneration of pulmonary endothelium with a combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease.

New in the present invention is the use of a course of administration once a day for 40 days of glucagon-like peptide 1 with a combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease.

We first revealed the ability of glucagon-like peptide 1 to stimulate endothelial regeneration, which is reduced with combined pathology: metabolic syndrome and chronic obstructive pulmonary disease.

Glucagon-like peptide-1 (GLP-1) is a hormone secreted by L-cells of the ileum of the ileum in response to food intake. This hormone causes both short-term and long-term pleiotropic effects. GLP-1 stimulates the production of insulin by β-cells, blocks the release of glucagon by pancreatic a-cells through somatostatin, slows down gastric emptying, improves peripheral glucose tolerance, suppresses appetite in the hypothalamus and tonsils, increases beta-cell mass, provides protection against glucolipotoxicity and glucose lipotoxicity [6], has anti-inflammatory properties [7]. The ability of GLP-1 to improve microvascular perfusion and counteract insulin resistance may be one of the reasons for the effectiveness of therapy based on analogues of GLP-1 in type 2 diabetes mellitus [8]. This range of biological effects is due to the widespread representation of GLP-1 receptors in the body. The GLP-1 receptor is a membrane receptor associated with the G-protein, found not only in the cells of the pancreatic islets, but also in some other tissues, such as the central nervous system, kidneys, heart, blood vessels, lungs, and is present on the endothelial, neuronal cells [9, 10]. We previously demonstrated the ability of GLP-1 to influence the regeneration of pancreatic beta-cell progenitors in type 1 diabetes mellitus [11].

The use of glucagon-like peptide 1 for a new purpose was made possible due to the new property identified by the authors to stimulate the regeneration of endothelial lungs with a combination of metabolic syndrome and chronic obstructive pulmonary disease.

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

The invention can be used to stimulate the regeneration of pulmonary endothelium with a combination of 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 60 female mice of the C57BL / 6 line. Animals came from the Department of Experimental Biological Models NIIIFiRM them. E.D. Goldberg of Tomsk State Scientific Medical Center (veterinary certificate is available). The keeping of animals and experimental design are approved by the Ethics Committee of the NIIIFiRM them. E.D. Goldberg Tomsk State Scientific Research Center 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, 2 and 3.

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, 6 - middle pulmonary field, c - lower pulmonary field; g, e, e - with induced metabolic syndrome and chronic obstructive pulmonary disease, where g is the upper pulmonary field, d - middle pulmonary field, e - lower pulmonary field, g, h, and - with induced metabolic syndrome and chronic obstructive pulmonary disease receiving glucagon-like peptide-1, where w is the upper pulmonary field, s is the middle pulmonary field, and is the lower pulmonary field. Magnification x 100.

In FIG. 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, b, c) :. a, intact; b - with induced metabolic syndrome and chronic obstructive pulmonary disease; c - with induced metabolic syndrome and chronic obstructive pulmonary disease receiving glucagon-like peptide-1. Magnification x 100.

In FIG. 3 shows 20-fold images of CD31 positive lung cells of mice after cultivation, stained with: Hoechst (blue) to identify cell nuclei (a, d, g); CFSE (green) (b, d, s); (Hoechst + CFSE) composite image using all colors (c, f, and). Determination of the percentage of cells by exterase activity is made by the ratio of cells counted in the green channel to the total number of cells counted using the blue (DAPI) channel. All scale bars are 100 microns. a, 6, c — CD31 positive lung cells of intact mice; g, d, e, - CD31 positive lung cells of mice with induced metabolic syndrome and chronic obstructive pulmonary disease; g, h, and - CD31 positive lung cells of mice with induced metabolic syndrome and chronic obstructive pulmonary disease receiving glucagon-like peptide-1.

The invention is carried out 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 / t) [12]. Monosodium glutamate, a salt of glutamic acid, is widely used on the one hand as a food supplement (E621) [13]. On the other hand, sodium glutamate is actively used to model obesity and metabolic disorders in experimental animals, and it is noted that newborn animals are more sensitive to the damaging effects of the drug [14]. The date of birth of animals is considered 1 day of the experiment. On the 124th day of the experiment, the Lie 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 extracting, the cigarette filter is removed, the length of the filter cigarette is 80 mm, and the filter is removed 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 0.22 μm. 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 a 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 [15].

Chronic obstructive pulmonary disease is caused by the course of intratracheal administration of lipopolysaccharide and cigarette smoke extract [16, 17]. LPS at a dose of 3 μg / mouse in 50 μl of phosphate buffer and 50 μl of cigarette smoke extract is administered intratracheally [18]. 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 levels, measurements of the lipid profile in the blood and standard histological studies of the lungs with the calculation of the emphysema area [18]. 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.

Glucagon-like peptide-1 (GLP-1) is a neuropeptide and incretin obtained from transcription of the proglucagon gene product (Sigma, St. Louis, Missouri, USA). GLP-1 is administered daily intraperitoneally into the pancreas at a dose of 3 mmol / kg from the 147th to the 187th day of the experiment.

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 [19]. Micropreparations from each experimental animal are examined under an Axio Lab light microscope. A1 ("Carl Zeiss", Germany) at 100 and 400x 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 [20, 21].

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 microscope. A1 (“Carl Zeiss”, Germany) with an AxioCam ERc5s video camera (“Carl Zeiss”, Germany) connected to a personal computer. The resulting images are processed using Axio Vision Rel. 4.8.2. 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 [22-24].

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 the membrane protein CD31 (ab28364, 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. An Axio Lab microscope is used to obtain micrographs. A1 (Carl Zeiss, Germany) with an AxioCam ERc5s camera (Carl Zeiss, Germany). 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.

The expression of membrane receptors of pulmonary and peripheral blood mononuclear cells is analyzed using murine monoclonal antibodies by flow cytometry. For analysis, a FACS Canto II instrument with FACS Diva software (BD Biosciences), USA) is used. Cell suspensions are stained with the following antibodies: CD45 PerCP, CD31 APC, CD34 FITC. The study uses appropriate isotypic controls.

On the 188th day of the experiment, the effect of GLP-1 on CD31 + endothelial cells of the lungs of mice in vitro is studied. For this, the lungs of mice are isolated, the lung tissue is cut into pieces, digested with a collagenase / dispase solution (StemCell Technologies) and mechanically dispersed into a suspension of individual cells. After removing the supernatant, the cell pellet was washed once with complete DMEM, and then resuspended in 10 ml with full DMEM and placed in a T-25 gelatin-coated plastic tissue culture plate for 24 hours. The next day, the cells were removed from the plate using trypsin for magnetic sorting.

Magnetic sorting in order to enrich the CD31 + lung cell suspension with endothelial cells is performed standardly using positive selection with anti-CD31 antibodies (StemCell Technologies, Canada). The CD31 + cell fraction was isolated using an EasySep® magnet (StemCell Technologies, Canada). The procedure allows to increase the number of CD31 + cells by almost 3 times compared with the level before separation.

The saturated suspension of CD31 ⁺ cells (10 ⁶ cells / 1 ml of medium) obtained after magnetic sorting was cultivated on gelatin-coated plastic tablets for T-25 cell cultures in M199 medium for 6 days under standard gas (3.5% CO2) and temperature conditions (37 ° C). The environment is changed every 1-2 days.

After a 6-day cycle of culturing CD31 ⁺ , mouse cells were removed from the plate using trypsin, the M199 medium was changed, and the cells were seeded at a concentration of 3 × 10 ⁵ cells / 1 ml of medium on gelatin-coated T-25 plastic plates. Prior to cultivation on medium with CD31 + cells, we add GLP-1 (10 ^-7 M). The cultivation cycle under standard conditions (3.5% CO ₂ , 37 ° C) is 24 hours. At the end of the cultivation, the effect of GLP-1 on CD31 ⁺ cells is evaluated using flow cytometry and image processing in each well with Cytation ™ 3.

Images of CD31 ⁺ cells were obtained using a Cytation 3 multimode cell imager (BioTek Instruments, Inc., Winooski, VT) equipped with DAPI, GFP and Texas Red cubes.

At the end of the incubation period, the endothelial cells of the lungs are treated with Hoechst 33342, CFSE, Annexin V-iFluor ™ 350 and 7-AAD fluorescent dyes. It is then visualized using Cytation 3 under 4 × or 20 × lenses, followed by cell analysis using Gen5 ™ data analysis software. All collected images are pre-processed to smooth the background before performing analytical methods. To determine the number of cells based on the number of Hoechst33342 stained nuclei on the blue channel, a cell analysis is performed so that the default parameters lead to adequate calculated data for further analysis.

Statistical processing of the results is carried out by methods of variation statistics using the statistical processing package SPSS 12.0. Arithmetic mean (M), arithmetic mean error (m), 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.

The experiments show that the administration of sodium glutamate to newborn mice induces metabolic disturbances in mice of C57BL / 6 females by 124 days of the experiment. In animals, there is a significant increase in the Lee index, body weight (Table 1)., The concentration of triglycerides and low density lipoproteins, a decrease in the level of high density lipoproteins in blood serum (Table 2). In animals with metabolic syndrome, a more pronounced increase in glucose level is recorded and an increase in the area under the curve (AUC) is observed (Table 3).

Thus, monosodium glutamate induces dyslipidemia and obesity, causes disturbances in the glucose tolerance test in mice, which can be interpreted as metabolic disorders.

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. The introduction of glucagon-like peptide-1 from the 147th to the 187th day of the experiment leads to a decrease in body weight in mice and, accordingly, the Lie index on the 188th day of the experiment to the level of intact animals (table 4). 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 intact mice by 188 days of the experiment (Table 4).

An important criterion for confirming metabolic disorders is a change in glucose metabolism. The introduction of GLP-1 reduces the severity of the rise in glucose level and decreases the area under the curve (AUC) during the glucose tolerance test on day 186 of the experiment (Table 5), and normalizes the level of glucose in the peripheral blood of animals (table 6).

In addition to the positive effect on metabolic disorders, the administration of GLP-1 improves the performance in the lung tissue in chronic obstructive pulmonary disease. A preliminary macroscopic assessment of the lung tissue of mice with COPD shows a soft consistency and loss of tissue elasticity compared to intact animals, while the organ tissue falls off 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). Pathomorphological changes in the lungs of mice receiving GLP-1 are less pronounced than in the group with metabolic disorders and COPD (Fig. 1, table 7). The introduction of GLP-1 significantly reduces the area of emphysema-expanded tissue in the lower region of the lungs of mice compared with the pathological control group.

An immunohistochemical study of lung tissue of mice with metabolic syndrome and COPD showed a significant decrease in the number of cells expressing CD31 in the lung tissue compared with the intact control (Fig. 2, table 8). The introduction of GLP-1 normalizes the number of cells expressing CD31 in the lung tissue of mice with metabolic disorders and COPD.

On the 188th day of the experiment, in mice against the background of metabolic syndrome and COPD, the number of endothelial progenitor cells with the CD45-CD31 + CD34 + phenotype decreases in the blood and increases in the lungs compared to the intact control group (Table 9). With the introduction of GLP-1, there is a significant (1.8-fold) increase in endothelial progenitor cells circulating in the blood, and their decrease in the lungs compared with a group of mice with metabolic syndrome and COPD.

For cultural studies, enrichment of the cell population of endothelial lung cells is carried out using CD31-positive magnetic separation. Flow cytometry is used to control the separation process. The procedure allows increasing the number of CD31 ⁺ cells almost 3 times, up to 71% of all stained cells compared to the initial level (before separation). Next, the obtained enriched population of endothelial cells is cultured for 5 days, then GLP-1 is added and cultured for another day. Under the influence of GLP-1, the number of apoptotic CD31 + cells in the culture is significantly reduced (more than 2 times compared with a culture without GLP-1). GLP-1 significantly increases the expression of the marker CD34 in a culture of CD31 + lung endothelial cells (table 10).

GLP-1 increases the number of CD31 + endothelial cells with active esterases (Fig. 3, table 10). These in vitro studies indicate that the expression markers CD31 and CD34 endothelial progenitor cells may be the target for GLP-1 and are involved in incretin regeneration of damaged endothelium in mice with metabolic syndrome and COPD.

Thus, glucagon-like peptide 1 stimulates the regeneration of pulmonary endothelium with a combination of metabolic syndrome and chronic obstructive pulmonary disease.

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Claims (1)

The use of glucagon-like peptide 1 to stimulate the regeneration of pulmonary endothelium with the combined pathology of the metabolic syndrome and chronic obstructive pulmonary disease.

Images