RU2611936C1 - Method for simulation of hypogonadism caused by metabolic disorders - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: dose of 200 mg/kg of streptozotocin is administered subcutaneously to male mice of C57Bl/6 line once, on the next day after birth. Then, on the 28th day after birth, the animals are transferred to a diet rich in saturated fats. This diet is maintained until hyperglycemia, hyperinsulinemia, glucose tolerance disorders, spermatogenesis depression and fertility disorders occur.

EFFECT: method provides simulation of hypogonadism caused by metabolic disorders, designed to find effective medicines correction of male reproductive system disorders in case of metabolic syndrome.

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Description

The invention relates to the field of experimental biology and medicine, and concerns the modeling of hypogonadism for the study of drugs for the correction of disorders in the male reproductive system, developing with diabetes-like metabolic disorders.

The term male hypogonadism refers to the functional inferiority of the male sex glands. In primary hypogonadism, the testicular tissue is directly affected; in secondary hypogonadism, hypofunction of the gonads occurs due to damage to the hypothalamic-pituitary system (the gonadotropic function of the pituitary gland decreases) [Rozhivanov R.V., 2015]. Clinical manifestations of hypogonadism are poorly expressed, and therefore male hypogonadism is considered a poorly diagnosed disease [Dedov II, 2007].

There is a relationship between metabolic disorders and hypogonadism [Paoletti R., 2006]. Violations of carbohydrate and fat metabolism lead to the development of hypogonadism, the prevalence of which reaches 75% [Rozhivanov R.V., 2015]. In turn, hypogonadism exacerbates metabolic disorders [Gusakova DA, 2008]. There is evidence of a relationship between testosterone deficiency and the development of visceral obesity, insulin resistance, diabetes mellitus and metabolic syndrome [I. Tyuzikov, 2013].

Since the central element of the disease is a decrease in testosterone concentration, treatment is based on all possible methods of replenishing the deficiency of this hormone [Rozhivanov RV, 2015]. Due to the substitution nature of treatment, it is carried out continuously. Having successfully eliminated some symptoms of androgen deficiency, exogenous testosterone suppresses spermatogenesis and only exacerbates such a significant manifestation of hypogonadism as male infertility [Kliesch S., 2010].

To develop methods for treating male hypogonadism aimed at regenerating the testes and restoring their own androgen-producing apparatus, it is necessary to use an adequate experimental model of hypogonadism. A known method of modeling hypogonadism by pharmacological castration by the administration of folliculin at a dose of 50 U / kg / day for 30 days [Khotimchenko Yu.S., 1996]. The disadvantage of this modeling method is the mismatch of the known model with the clinical manifestations of hypogonadism caused by metabolic disorders.

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 hypogonadism, which is based on metabolic disorders and pathomorphological changes in the pancreas, leading to impaired spermatogenesis and fertility.

The problem is solved by simulating hypogonadism caused by metabolic disorders, characterized by the fact that male mice of the C57B1 / 6 line are injected streptozotocin once, subcutaneously at a dose of 200 mg / kg one day after birth and animals are transferred to a diet enriched with heavy saturated fats, from the 28th to the 70th day after birth, until hyperglycemia, hyperinsulinemia, impaired glucose tolerance, as well as inhibition of spermatogenesis and impaired fertility are achieved.

New in the proposed method is the study of the content of free testosterone in blood serum, the total number of germ cells and the percentage of mobile forms of germ cells per epididymis, testicular morphology and fertility in male C57B1 / 6 mice under the conditions of a single injection of streptozotocin and a long diet with a high fat content leading to the formation of diabetes-like metabolic disorders in blood serum (hyperglycemia, glucose tolerance, hyperinsulinemia) and pathomorphological changes neny pancreas (islet tissue destructive changes). The proposed method for modeling hypogonadism most fully reveals the clinical picture of sexual disorders of men suffering from diabetes-like metabolic disorders.

Streptozotocin is an antitumor agent with an alkylating effect from the group of nitrosourea derivatives. The mechanism of antitumor action has not been fully studied, but is probably due to the formation of methylcarbonium ions, which cause alkylation or bind to various intracellular structures, including nucleic acids. Forms cross-linking between DNA strands, which leads to inhibition of its synthesis [Vidal Handbook of Medicines, 2014]. It also has a hyperglycemic effect, causing irreversible damage to the pancreatic beta cells [Jinzi Wu, 2015; Huang S.W., 1981].

Distinctive features showed in the inventive 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 for the treatment of hypogonadism caused by metabolic disorders.

Based on the foregoing, the claimed invention meets the criteria for patentability of the invention of "Novelty", "Inventive step" and "Industrial applicability".

The proposed method was studied in experiments on 100 male mice of the C57BL / 6 line. Animals of the first category, conventional linear mice, were obtained from the nursery of the experimental biomedical modeling department of the NIIIFiRM im. E.D. Goldberg (certificate available).

The invention will be clear from the following description and the accompanying drawings.

In FIG. 1 shows the pancreas of male mice of the C57B1 / 6 line during staining with hematoxylin and eosin: a - intact control (enlarged × 100); b - metabolic disturbances on the 49th day of the experiment (sw. × 100); c - metabolic disturbances on the 70th day of the experiment (sw. × 100); g - intact control (sw. × 400); d - metabolic disturbances on the 49th day of the experiment (sw. × 400); e - metabolic disturbances on the 70th day of the experiment (sw. × 400).

In FIG. 2 shows the testis of mice of the C57B1 / 6 line when staining the tissue with hematoxylin and eosin: a - intact control (UV × 100); b - metabolic disturbances on the 70th day of the experiment (sw. × 100); in - intact control (UV. × 400); g - metabolic disturbances on the 70th day of the experiment (sw. × 400).

The method is as follows.

In a laboratory animal (male C57B1 / 6 mice), hypogonadism caused by metabolic disorders is modeled by the administration of streptozotocin one day after birth at a dose of 200 mg / kg, once, subcutaneously into the withers, the volume of the injected solution is 30 μl. From 28 days after birth, animals are transferred to a diet high in fat. For this, a feed enriched with heavy saturated fats (30% fat) is used (Siff EF R / M with 30% Fat Cat. No. El5116-34, Germany). By the content of heavy saturated fats, this food complies with standard European food. The duration of the high-fat feed is 42 days (from the 28th to the 70th day of the experiment).

On days 28, 35, 42, 49, 56, 63 and 70, blood glucose levels are assessed and the change in weight of the animals is recorded. The blood glucose level is determined using a glucometer (Accu-Chek Performs Nanu (Roche Diagnostes GmbH, Germany). The initial blood glucose level in animals is measured after 16-hour feed deprivation, all subsequent glucose measurements are also taken after 16- hourly food deprivation.

On the 28th, 49th and 70th 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. One hour later, an intravenous injection of glucose (D-glucose, Sigma-Aldrich, USA) into the tail vein at a dose of 2 g / kg is performed. The first blood sampling for the study of glucose level is carried out 30 minutes after glucose injection, then the glucose content is studied at an interval of 30 minutes, the study duration was 2.5 hours. The dose of glucose 2 g / kg was chosen due to the fact that, according to According to the literature, the response of animals to low doses of glucose when administered intravenously is weak, insulin concentration increases only in the first minute and rarely remains elevated during the first 5 minutes of observation [Pacini G. et al., 2009].

On day 70, the mass of animals is determined. Serum insulin and free testosterone levels are evaluated by enzyme immunoassay in accordance with the manufacturer's protocols (Cusabio Biotech CO., LTD, China).

For a morphological study of the pancreas, its part adjacent to the spleen was fixed in 10% formalin and poured into paraffin according to standard methods. Deparaffinized sections with a thickness of 5 μm are stained with hematoxylin and eosin. On the slices, the area of 10 consecutive islets of Langerhans is determined by the method of graphic computer analysis and the total number of cells and the number of pycnotized cells are counted in them. Then calculate the number of cells per unit area of the islet and the percentage of pyknotized cells.

The spermatogenesis productivity of male mice is assessed by the total number and number of motile forms of sperm. When calculating the total number of sperm per appendage, a homogenized cell suspension of epididymis in a dosed amount of physiological saline using a leukocyte melanger and Goryaev’s chamber is used.

For morphological examination of the testis, tissue pieces are fixed in 10% formalin. Fixed tissues are dehydrated, impregnated with paraffin and poured into paraffin blocks; using a microtome, thin serial (no more than 5 microns) sections are made and stained with hematoxylin and eosin. Histological examination of sections is carried out using light microscopy. On the preparations of the testes, the condition of the tubular apparatus, the morphological features of Leydig cells and the state of the stroma are evaluated. The inflammatory reaction of the testis tissue is assessed by the presence of inflammatory infiltrate and the state of the vascular bed. For each experimental animal, a minimum of 10 micrographs are taken without overlapping over the entire cut surface of the testicular tissue at 400x magnification. The system consists of an Axio Lab microscope. Al (“Carl Zeiss”, Germany) with an AxioCam ERc5s camcorder (“Carl Zeiss”, Germany) connected to a personal computer. The resulting images are processed using AxioVision Rel.4.8.2 software.

To assess fertility, intact females (under natural conditions) are planted in males with metabolic disorders for 10 days. The male / female ratio was 1/2 (20 females per 10 males). After 10 days, females and males are transplanted into different cells. Females are killed in the CO ₂ chamber on the 18th day of pregnancy. Examine the fruits in the uterus. To assess fertility, the fertility index (PI) is calculated:

PI = number of fertilized females / number of females hatched with males × 100%.

The results are processed by methods of variation statistics using IBM software. Arithmetic mean (X), 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). In cases of a normal distribution of attributes, the student’s parametric t-test is used for statistical evaluation. For large deviations of the distribution of the trait from the normal form for independent samples, the nonparametric Mann-Whitney U-test 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 persistent increase in blood glucose in mice of C57BL / 6 males starting from 42 days of the experiment. High glucose levels remain for up to 70 days (Table 1).

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 an intravenous injection of glucose (dose 2 g / kg) in intact animals, a significant increase in blood glucose levels was recorded over 60 minutes (Table 2).

Then - 90, 120, 150 min after glucose administration, a gradual decrease in glucose concentration follows. In mice under the conditions of modeling metabolic disorders, a glucose tolerance test was conducted on the 28th, 49th and 70th days of the experiment. In animals with metabolic disorders, hyperglycemia is noted. The maximum increase in glucose is recorded on the 70th day of the experiment. The glucose tolerance test shows the long-term maintenance of a high glucose concentration in animals with metabolic disorders on the 28th, 49th and 70th days of the experiment (Table 2). In animals on the 28th day of the experiment, a decrease in the level of hyperglycemia is observed at 2 hours of a blood glucose test. Meanwhile, the glucose tolerance test in pathological control mice on the 49th and 70th day of the experiment reveals persistent (without falling) preservation of a high level of hyperglycemia from 30 minutes to 150 minutes.

Example 2

Modeling of metabolic disorders causes an increase in the concentration of insulin in the blood serum on the 49th and 70th days of the experiment (Table 3). Changes in testosterone are not so straightforward. So, on the 49th day there is an increase in the level of free testosterone in the blood serum to 146% of the intact control, later on the 70th day of the experiment, the hormone concentration decreases and amounts to 64.3% of the intact control.

Example 3

A morphological study of the pancreas reveals that, unlike the mice of the C57B1 / 6 males of the intact control, the modeling of metabolic disorders causes edema and hyperemia in the pancreatic tissue in mice of the C57B1 / 6 males on the 70th day of the experiment (Fig. 1). In addition, in mice under model conditions, small- and medium-droplet fatty degeneration of acinar cells, thickening and proliferation of interlobular septa, and infiltration of islet tissue by inflammatory cells are recorded.

On the 70th day of the experiment in the pancreas in mice, under the conditions of modeling metabolic disorders, a decrease in the total number of islets of Langerhans is noted. When assessing the islet part of the pancreas, a decrease in the area of the islet of Langerhans and the number of islet cells is revealed in comparison with animals of the intact control (Table 4). Meanwhile, the percentage of pyknotized cells in the islets is increasing.

Example 4

A histological examination of the testes shows that in animals under the conditions of modeling metabolic disorders moderate destructive changes in the tubular apparatus develop (Fig. 2). There is a slight decrease in the number of layers of spermatogenic epithelium in the seminiferous tubules. There is a slight swelling of the interstitial tissue, vascular hyperemia, in single Leydig cells there are degenerative changes - vacuolization of the cytoplasm, hyperchromia of the nucleus. In a number of convoluted tubules, rarefaction of layers of spermatogenic epithelium or the disappearance of mature forms of germ cells are observed. There are isolated tubules, in the lumen of which cell detritus is observed, consisting of dead sperm and spermatids. In addition, among the desquamated epithelium, a small number of "seed balls" are found - large structures with multiple, often pyknotic nuclei or their fragments with intensely stained cytoplasm.

On the 49th and 70th day of the experiment, the body weight of the animals in the group of mice under conditions of metabolic disorders significantly decreases compared to the values of the intact control. Modeling of hypogonadism caused by metabolic disorders leads to a significant decrease in the number of motile forms of sperm and the total number of sperm on the 70th day of the experiment (Table 5).

Example 5

In the study of fertility, it was found that the fertility index in C57BL / 6 male mice with metabolic disorders on the 70th day of the experiment and in C57BL / 6 intact female mice was reduced by 2.7 times compared with that in intact C57BL male mice / 6 and intact female C57BL / 6 mice (Table 6).

Thus, a single administration of streptozotocin at a dose of 200 mg / kg a day after birth to male C57BL / 6 male mice and the transfer of animals from 28 days after birth to a diet enriched with heavy saturated fats (30% fat), leads to the formation of 70 days of the experiment metabolic disorders (hyperglycemia, glucose tolerance test disorders, hyperinsulinemia), pathomorphological changes in the pancreas (destructive changes in islet tissue) and a violation of the male reproductive system: inhibition of spermatogenesis (decrease increase in the total number and percentage of mobile forms of sperm, a drop in the level of free testosterone in the blood serum, interstitial tissue edema, hyperemia of vessels, degenerative changes in Leydig cells), impaired fertility.

Literature

1. Gusakova D.A., Kalinchenko S.Yu., Kurilo L.F. Primary hypogonadism as a cause of metabolic syndrome. Reproduction Issues, 2008; Number 3.

2. Dedov I.I., Melnichenko G.A., Fadeev V.V. Endocrinology. - M .: GEOTAR-Media; 2007.432 p.: Ill. - 500.

3. Rozhivanov R.V., Yashina Yu.N. Aspects of the use of androgen replacement therapy in the treatment of hypogonadism in men with diabetes mellitus and metabolic syndrome. Obesity and metabolism. 2015; 12 (1): 11-14.

4. Reference book of medicines Vidal, 2014. 1600 p.

5. Tyuzikov I.A. Metabolic syndrome and male infertility (literature review). Andrology and Genital Surgery, 2013. No. 2. S. 5-10.

6. Khotimchenko Yu.S., Kropotov A.V., Lisakovskaya A.V. The effect of the mountaineer on discriminatory indicators of rat ejaculate subjected to pharmacological castration // Vestnik FEB RAS. - 1996. - No. 5. - S. 61-65.

7. Jinzi Wu, Liang-Jun Yan. Streptozotocin-induced type 1 diabetes in rodents as a model for studying mitochondrial mechanisms of diabetic β cell glucotoxicity // Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 2015. V. 8. P. 181-188.

8. Huang S.W., Taylor G.E. Immune insulitis and antibodies to nucleic acids induced with streptozotocin in mice // Clin. exp. Immunol. (1981) 43, 425-429.

9. Kliesch S. Testosterone and infertility. Urologe A 2010; 49 (l): 32-6.

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Claims (1)

A method for modeling hypogonadism caused by metabolic disorders, characterized in that male mice of the C57Bl / 6 line are injected streptozotocin once, subcutaneously at a dose of 200 mg / kg one day after birth, and then animals are transferred to a diet rich in saturated fats, from 28 days before hyperglycemia, hyperinsulinemia, impaired glucose tolerance, as well as inhibition of spermatogenesis and impaired fertility.

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