RU2739690C1 - Method for preventing chronic molybdenum coagulopathy - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to experimental medicine, ecology and toxicology and can be used for experimental development of prevention methods and pathogenetic correction of manifestations of chronic molybdenum coagulopathy in experimental animals with prolonged action of ammonium paramolybdate. Method for preventing chronic molybdenum coagulopathy in experimental animals involves daily introduction of antioxidant and heavy metal for two months, wherein as an antioxidant from the first experimental day, animals are introduced through a probe into stomach with 0.2 ml of solution containing 0.5 mg of melatonin for each 100 g of weight, simultaneously with daily intragastric introduction of 0.3 ml of ammonium paramolybdate solution containing 5 mg of molybdenum.EFFECT: invention provides considerable reduction of blood coagulation system shifts during prolonged action of molybdenum in experimental animals.1 cl, 2 tbl, 1 ex

Description

The invention relates to ecology, medicine, toxicology, experimental biology and can be used for the experimental development of methods for the prevention of molybdenum coagulopathy with prolonged intake of metal into the body.

Molybdenum is an essential trace element necessary for the normal implementation of vital processes. At the same time, long-term intake of molybdenum into the body can lead to excessive accumulation of heavy metal in human organs and tissues and multiple organ structural and functional disorders (Avtsin A.P. 1991). It has been shown that molybdenum intoxication is accompanied by the activation of lipid peroxidation, the formation of oxidative stress and damage to membrane structures (Brin V.B. et al. 2014). Numerous experimental and clinical studies suggest that the functional blood coagulation system is one of the first to react to any pathological processes in the body, changing the parameters of its functioning (Goodwin S.A., Wheeler-Jones SR., Namiranian S. 1995). The problem of creating and developing effective methods for the prevention of chronic intoxication with molybdenum and its compounds is urgent (Reutova N.V. 2007., Pandey R., Kumar R., Singh S., Srivastava S. 2002).

We set the task to study the preventive effect of the synthetic analogue of the pineal gland hormone Melatonin Melaxen on the manifestation of coagulopathy caused by prolonged administration of ammonium paramolybdate in an experiment in rats.

Previously, it was shown that the use of melatonin is promising for the prevention of pathological changes in the indicators of the morphofunctional state of the kidneys, liver and heart when exposed to certain heavy metals in rats in the experiment (Mitziev A.K., Brin V.B., 2017). The effectiveness of the use of melatonin for the prevention of toxic coagulopathy caused by prolonged administration of a solution of nickel chloride at a dose of 5 mg / kg in white rats was revealed (RU 2683723 C1 2018). It has been shown that the use of melatonin helps to prevent changes in the hemostatic system (Gagloeva EM, Brin VB RU 2706386 C1) with intragastric administration of lead acetate at a dose of 40 mg / kg of body weight. However, the cited sources do not say anything about the possibility of prophylactic use of melatonin when molybdenum acts on the hemostatic system. Various compounds of heavy metals have different organotropic effects, have predominant organs - "targets" of damage. The peculiarity of the effect of lead on the body is its cytotoxic effect, destruction of erythrocytes, and an increase in the thromboplastic activity of corpuscles. Disorders of hemostasis begin with a toxic effect on platelet membranes, which can lead to a change in the adhesive and aggregation activity of platelets, release of aggregation inducers and secondary activation of coagulation hemostasis. In this case, an important role in the mechanisms of activation of the hemostasis system is played by erythrocyte, leukocyte factors of hemocoagulation and hemolysis (Podolyan S.K., 1998, Byshevsky A.Sh. et al., 2005). Thus, the effectiveness of this method when exposed to lead (a substance from the first class of hazardous toxicants) does not prove the possibility of using an antioxidant to prevent the development of molybdenum coagulopathy (molybdenum is a trace element necessary for living organisms).

The closest to the proposed invention is the method (Byshevsky A.Sh. et al. Hemostasis in the absence, deficiency and excess of cobalamin in rats kept on a balanced diet // Bulletin of the Samara Scientific Center of the Russian Academy of Sciences. 2009. T. 11. No. 1 (4 P.752-757), which consists in the fact that animals with a diet are injected with vitamin B12 at a dose of 1 μg / kg per day simultaneously with the daily administration of lead acetate with food at a dose of 50 mg / kg for eight weeks. The antioxidant cobalamin weakens the shifts in the hemostasis system indicators caused by lead, helping to reduce the level of markers of intravascular coagulation (PDP, RFMK, factors P3 and P4, D-dimers), modifies lipid peroxidation in platelets, inhibiting their activation. The disadvantages of this method are: - the complexity of determining the amount of lead introduced into the body with food per unit of body weight of the animal; - the difficulty of determining the amount of vitamin B12 introduced with food per unit of animal body weight; - an excessive amount of the antioxidant vitamin B12, as a preventive agent, can have side effects.

In the available literature, we did not find information on the possibility of preventing changes in the hemostatic system in molybdenum coagulopathy using melatonin.

The claimed invention is aimed at solving the problem of developing a method for the prevention of molybdenum coagulopathy in experimental animals.

The solution to this problem provides a significant decrease in the shifts in the indicators of the blood coagulation system during prolonged exposure to molybdenum in experimental animals, which is accompanied by a decrease in the intensity of lipid peroxidation.

To achieve this technical result, the claimed method for the prevention of chronic molybdenum coagulopathy in experimental animals with chronic poisoning is characterized by the daily administration of an antioxidant and a procoagulant, while the animals are injected with melaxen once a day at a dose of 5 mg / kg through a tube into the stomach for two months simultaneously with the daily intragastric administration of a solution of ammonium paramolybdate at a dose of 50 mg / kg, in terms of metal.

Daily prophylactic administration of melaxen through a tube into the stomach at a dose of 5 mg / kg simultaneously with intragastric administration of a solution of ammonium paramolybdate leads to a decrease in pathological shifts in the hemostatic system parameters, prevents the development of thrombinemia by reducing the cytotoxicity of molybdenum, membrane protective and antioxidant effects of melatonin. The claimed method is efficient, cost effective and easily reproducible.

According to the information available to the authors, the set of essential features characterizing the essence of the claimed invention is not known, which allows us to conclude that the invention meets the "novelty" criterion.

According to the authors, the essence of the claimed invention does not follow explicitly for specialists from the known level of medicine, since it does not reveal the above possibility of obtaining a method for the prevention of chronic molybdenum coagulopathy, characterized by the fact that animals are injected with melaxen once a day at a dose of 5 mg / kg through a tube into the stomach for two months simultaneously with daily intragastric administration of a solution of ammonium paramolybdate at a dose of 50 mg / kg, in terms of metal, which allows us to conclude that the criterion of "inventive step" is met.

The set of essential features that characterize the essence of the invention, in principle, can be repeatedly used in medicine to obtain the result consisting in an effective and easily reproducible method for the prevention of chronic molybdenum coagulopathy by reducing the intensity of lipid peroxidation processes, restoring antioxidant defense enzymes, restoring the activity of the anticoagulant and fibrinolytic links of the system hemostasis, a decrease in the level of thrombinemia with prolonged introduction of molybdenum into the rat organism, which allows us to conclude that the invention meets the criterion of "industrial applicability". This method is carried out as follows: to prepare a solution of a molybdenum salt in distilled water, ammonium paramolybdate is dissolved in such a way that per unit of solution equal to 0.3 ml, there is 5 mg of molybdenum, in terms of metal. For every 100 g of rat weight, 0.3 ml of solution is injected, which is not an excessive water load on the rat organism. A solution of melatonin is prepared daily before intragastric administration, for which Melaxen tablets are dissolved in distilled water in such a way that 0.5 mg of melatonin is per unit of solution equal to 0.2 ml.

Two groups of animals (10 animals each) are injected with a solution of ammonium paramolybdate through an atraumatic intragastric tube at a dose of 50 mg / kg daily for 60 days. Simultaneously from the first day of administration of ammonium paramolybdate to one of the experimental groups, a solution of melaxen is injected every day at a dose of 5 mg / kg through a tube into the stomach.

The indicators of the state of the hemostasis system and the processes of lipid peroxidation (LPO) are investigated. The number of platelets and their aggregation activity, the content of fibrinogen, the activated partial thromboplastin time (APTT), prothrombin time (PT), the relative time of fibrin monomer polymerization (VPFM) are determined to assess the state of the final stage of blood coagulation, are determined (Momot A.P., 2009 ). By the activity of antithrombin (ATIII), the state of the anticoagulant system is judged, by the time of spontaneous euglobulin lysis, the state of the fibrinolytic link is examined, by the concentration of soluble fibrin-monomer complexes (RFMK) and D-dimers, the level of thrombinemia is judged.

The intensity of lipid peroxidation (LPO) processes is judged by the concentration of malondialdehyde (MDA) in erythrocytes and the level of hydroperoxides in blood plasma. In order to assess the effectiveness of antioxidant protection (AOD), the activity of superoxide dismutase and catalase in erythrocytes is determined.

The results of a study in rats with the combined administration of the antioxidant Melaxen and ammonium paramolybdate demonstrate the effectiveness of its use in the prevention of chronic molybdenum coagulopathy. Melatonin helps to reduce the severity of changes in cellular and plasma hemostasis, restores the activity of the anticoagulant and fibrinolytic systems, reduces the level of thrombinemia due to pronounced antioxidant activity during prolonged exposure to molybdenum.

An example of a specific implementation. Two experimental groups of white male rats weighing 200-300 g were injected through an atraumatic tube into the stomach with a solution of ammonium paramolybdate at a dose of 50 mg / kg every day for two months. Experimental group No. 2 from the first day of administration of molybdenum salt, every day intragastric injection of melaxen at a dose of 5 mg / kg. Control animals were kept in standard vivarium conditions (Control No. 1 - 10 rats).

The parameters of cellular and plasma hemostasis, the state of the anticoagulant and fibrinolytic links of the hemostasis system, the activity of the processes of lipid peroxidation and antioxidant protection were studied. The material for the study was whole blood, as well as blood plasma, rich and poor in platelets. Plasma samples were stabilized and obtained in accordance with international standards for clinical laboratory diagnostics for research in the field of hemostasis (Momot A.P. et al., 2011).

To assess the state of the hemostasis system, the aggregation activity of platelets was determined by the turbidimetric method (inductor adenosine diphosphate - 10.0 μg / ml). The fibrinogen content was determined by the Clauss method; activated partial thromboplastin time; prothrombin time, relative time of fibrin monomer polymerization (Momot A.P. et al., 2011), activity of antithrombin III; time of spontaneous euglobulin lysis; the concentration of soluble fibrin monomeric complexes was determined using standard techniques using diagnostic kits from the "Technology Standard" company (Russia). The tests were carried out using a Solar-300 spectrophotometer, a CGL-2110 Solar turbidimetric coagulometer, and an AR 2110 Solar aggregometer (Belarus). The D-dimer level was determined with the INNOVANCE D-dimer reagent kit, Siemens.

To assess the intensity of lipid peroxidation (LPO) and antioxidant potential (AOP) processes, the following were determined: the content of malondialdehyde (MDA) in erythrocytes (Asakawa T., 1980), hydroperoxides (HP) (diene conjugates and diene ketones) in blood plasma (with isp. potassium ferricyanide), superoxide dismutase (SOD) activity, catalase activity in erythrocytes according to E. Beutler's method.

Statistical analysis was performed using the Microsoft Excel and Statistica 10.0 software package (StatSoft, Inc.). Proceeding from the fact that the observed characteristics did not all obey the normal distribution, the research data are presented in the form (Me [25-75%]), where Me is the median in the sample, [25-75%] - the 25th and 75th percentile. The reliability of the results obtained was assessed using the Mann-Whitney U-test. Spearman's rank correlation coefficient (r _s ) was used to assess the statistical dependence of two series of observations. Differences were considered significant if the error probability was p <0.05. Studies have shown that intragastric administration of ammonium paramolybdate at a dose of 50 mg / kg body weight promoted the development of molybdenum coagulopathy (Table 1). Molybdenum coagulopathy was characterized by a decrease in the number of platelets and an increase in their aggregation function. The fibrinogen concentration was higher than the control. A shortening of APTT and lengthening of PT were recorded, TB did not change significantly; at the same time, a shortening of the more sensitive marker of the state of the final stage of hemocoagulation (FPM) of the time of fibrin monomer polymerization was revealed. A decrease in the activity of AT (III) and a slowdown in the time of spontaneous euglobulin lysis were recorded. A significant increase in the concentration of RFMK and D-dimers (Table 1) indicates the presence of thrombinemia. Obviously, the data of the experiments performed demonstrate the development of the state of thrombotic readiness, determined by Momot et al. (2011) as a laboratory-detectable hypercoagulation with an increase in the blood content of markers of hemostasis activation, suppression of anticoagulant and fibrinolytic activity. Hypercoagulant syndrome is characterized by a state of increased readiness of the circulating blood for coagulation, while thrombus formation in the vascular bed is not detected, but a number of clinical signs of a pre-thrombotic state can be observed in the form of an increase in blood viscosity, slowing of venous blood flow, transient and initial signs of organ dysfunction. This may be evidenced by the development of dystrophic and necrobiotic changes in the tissues of the liver, kidneys and heart under the toxic effect of molybdenum, which was shown earlier (Brin V.B. et al. 2012).

The experiments showed that the isolated intragastric administration of the melatonin preparation - Melaxen for two months did not cause changes in the parameters of the blood coagulation system in animals. There was only a slight tendency towards a decrease in the number of platelets, the degree of ADP-induced aggregation relative to the intact control did not change.

Intragastric administration of melatonin against the background of the introduction of molybdenum helps to stabilize the hemostasiological picture. A displacement of the hemostatic potential towards hypercoagulation was revealed, while the activity of the anticoagulant and fibrinolytic systems was restored. Thus, melatonin helps to reduce the likelihood of developing a state of thrombotic readiness, as evidenced by a decrease in the severity of changes in RFMK and D-dimers (Table 1). The degree of ADP-aggregation increased, the number of platelets decreased, but the changes were less pronounced. The fibrinogen content did not statistically differ from the parameters in rats with chronic intoxication, but did not significantly differ from the parameters in intact rats.

Intragastric administration of the antioxidant melaxen caused an increase in the level of catalase activity in erythrocytes (p <0.05), a decrease in the concentration of malondialdehyde (p <0.001) and a restoration of the level of hydroperoxides (p <0.01 and p <0.01) in blood plasma. When analyzing the experimental data obtained, correlations between the positive dynamics of indicators of the hemostasis system and the restoration of the activity of LPO processes were established. There was a significant positive correlation between the restoration of catalase activity and an increase in antithrombin III activity (r _s = 0.64; P <0.05), as well as a high degree of inverse correlation with the time of spontaneous euglobulin lysis (r _s = 0.57; P <0 , 05). Changes in the concentration of D-dimers and shifts in MDA (r _s = 0.65; P <0.05) after two months under the influence of the antioxidant melatonin were closely positively associated.

Thus, the prophylactic use of melatonin reduces the severity of changes in the indicators of vascular-platelet and coagulation hemostasis, restores the activity of the anticoagulant and fibrinolytic blood systems and the level of thrombinemia, helping to restore the hemostatic balance. The results of the study allow us to recommend the use of melatonin for the development of methods for correcting hemostatic disorders when exposed to molybdenum compounds.

Claims (1)

A method for the prevention of chronic molybdenum coagulopathy in experimental animals, including the daily administration of an antioxidant and a heavy metal for two months, characterized in that from the first day of the experiment, 0.2 ml of a solution containing 0.5 mg of melatonin is administered through a tube into the stomach as an antioxidant. for every 100 g of weight, simultaneously with daily intragastric administration of 0.3 ml of ammonium paramolybdate solution containing 5 mg of molybdenum.