RU2794030C1 - Method for prevention of toxic coagulopathy in rats - Google Patents

Abstract

FIELD: ecology; medicine; toxicology; experimental biology.

SUBSTANCE: invention can be used for the experimental development of methods for the prevention and pathogenetic correction of manifestations of toxic coagulopathy in rats with prolonged exposure to potassium bichromate. The method includes daily administration of an antioxidant and a heavy metal to rats. As an antioxidant, from the first day of the experiment, melaxen was introduced into the stomach through a tube at a dose of 5 mg/kg for two months. A solution of potassium dichromate is introduced as a heavy metal at a dose of 0.5 mg/kg in terms of metal.

EFFECT: invention provides reduction of pathological changes in indicators in the hemostasis system.

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 and pathogenetic correction of manifestations of toxic coagulopathy in rats with prolonged exposure to potassium bichromate.

A.M.,

M.A. et al. 2016.).Однако по поводу их эффективности или вреда ведутся многочисленные дискуссии (Vincent JB, Lukaski НС. Chromium. AdvNutr. 2018; 9(4):505-506.https://doi.org/10.1093/advances/nmx021).Chromium belongs to heavy metals of a high toxicity class. With excessive intake of its compounds in the body, multiple organ structural and functional disorders can develop. Their allergenic, mutagenic, carcinogenic, gonadotropic and other effects have been proven (Sun H, Brocato J, Costa M. 2015, Chen QY, Murphy A., Sun H. et al. 2019, DesMarais TL, Costa M. 2019). At the same time, the question of the essentiality of the trace element chromium (Cr3+) and its effect on carbohydrate, fat and protein metabolism has been discussed so far. Conditions of chromium deficiency in the body are described, but the scientific justification for the positive effects of chromium detected is insufficient (Vincent JB New Evidence against Chromium as an Essential Trace Element. The Journal of Nutrition. 2017 Dec; 147(12):2212-2219. https:/ /doi.org/10.3945/jn.117.255901). Reagent "Samokhotsky", "Kaliumbichromicum", picolinatechromium and other chromium-containing drugs are available in pharmacy chains in the form of dietary supplements (San Mauro-Martin I.,

am,

MA et al. 2016.). However, there are numerous discussions about their effectiveness or harm (Vincent JB, Lukaski NS. Chromium. AdvNutr. 2018; 9(4):505-506.https://doi.org/10.1093/advances/nmx021) .

The mechanisms of pathogenic action of chromium ions are associated with the generation of reactive oxygen species and high membrane toxicity (Zeng M, Xiao F, Zhong X, et al. Reactive oxygen species play a central role in hexavalent chromium-induced apoptosis in Hep 3B cells without the functional roles of p53 and caspase-3 Cell PhysiolBiochem 2013;32(2):279-290 https://doi.org/10.1159/000354436). It has been shown that the memranotropic effect is accompanied by the entry into the bloodstream of a large amount of thrombogenic substances, the sources of which can be components of endothelial membranes, blood cells, as well as cellular structures of various organs (Pretorius, L., Taute, N., Van Rooy, M., & Oberholzer, N.M., Investigating the ultrastructural and viscoelastic characteristics of whole blood after exposure to the heavy metals cadmium, lead and chromium, alone and in combination, Ultrastructuralpathology, 2022, 1-11, Advanceonlinepublication, https://doi.org /10.1080/01913123.2022.2075999). Relevant is the search for means of pathogenetic prevention of the toxic effect of chromium on the body among antioxidants (Gagloeva E.M., Brin V.B., Akhpolova V.O. Processes of hemocoagulation and lipid peroxidation and mechanisms of violation of the osmoregulatory function of the kidneys in chronic intoxication with chromium compounds // Medical Bulletin of the North Caucasus, 2020;15(3):320-324).

As a means of preventing toxic coagulopathy under the influence of chromium in rats, we used an analogue of the pineal hormone melatonin - Melaxen. In modern literature, the role of melatonin in almost all life processes, the effect on sleep, the activity of the cardiovascular, endocrine, immune systems, as well as the hemostasis system is widely studied (Arushanyan E.B. Effect of melatonin on platelet hemostasis and its circadian organization 2013.76( 5); Kumari S, Dash D. Melatonin elevates intracellular free calcium in human platelets by inositol 1,4,5-trisphosphate independent mechanism. FEBS Lett. 2011;585(14):2345-2351. https://doi.org /10.1016/j.febslet.2011.05.067). The possibilities of its use as a drug are being actively studied. The combination of traditional therapies with melatonin proved to be more effective than without it. A pronounced adaptogenic effect, desensitizing, anti-inflammatory, hepatoprotective effect, anti-stress effects, etc. are described. The pronounced membrane-protective properties of melaxen are due to its pronounced antioxidant effect (Arushanyan E.B. 2006-2019). Taking into account the diversity of the described properties of melatonin, one can expect high efficiency of melatonin in chronic intoxication, however, we did not find data on the possibility of its use in order to prevent disorders in the hemostatic system during long-term administration of chromium into the body in the available literature.

There is a way to prevent hemostasis disorders in the toxic effects of lead with the help of antioxidant vitamins (Byshevsky A.Sh., Galyan S.L., Ralchenko I.V. and others. The effect of a combination of antioxidant vitamins on hemostasis during experimental hyperoxidation // Experimental and Clinical pharmacology, 2005.68(3), 34-36.). The animals were administered Complivit at a dose of 1.8 mg/kg per day with a diet based on vitamin A, at the same time with daily administration of lead acetate at a dose of 0.5 mg/kg for 15 days with food. An excess of vitamin A against the background of the toxic effect of lead causes a weakening of shifts in the hemostasis system caused by metal, inhibits platelet activation and causes a decrease in the level of markers of intravascular blood coagulation (fibrin degradation products, soluble complexes of fibrin monomers, D-dimers, factors P3 and P4). The disadvantage of this method is that with its help, it is impossible to study the prevention of coagulopathy caused by the introduction of potassium bichromate, since this work did not consider the effect of chromium on the blood coagulation system.

Closest to the proposed invention is a method for the prevention of toxic coagulopathy in rats using one of the components of propolis with antioxidant and anti-inflammatory properties (phenyl ester of caffeic acid at a dose of 10 mg/kg) (Ashour, TariqHelal. Preventative effects of caffeic Acid phenyl ester on cadmium intoxication induced hematological and blood coagulation disturbances and hepatorenal damage in rats //ISRN Hematol.2014 Mar30;2014:764754.http://dx.doi.Org/10.1155/2014/764754) simultaneously with intraperitoneal administration of cadmium chloride at a dose of 0.05 mg/kg for 21 days.

The disadvantages of the prototype are:

• The impossibility of developing the prevention of chromium coagulopathy, as the prototype did not consider the preventive effect of caffeic acid phenyl ester on the blood coagulation system with the toxic effects of chromium on the body of experimental animals. It is known that different metals have predominant organs and "targets" of toxic action. Accordingly, the effectiveness of this method in cadmium intoxication does not prove the possibility and effectiveness of the prophylactic use of an antioxidant during prolonged exposure to chromium.

• Under conditions of technogenic environmental pollution, chromium enters the body mainly with food and water orally, respectively, intragastric administration is more consistent with the natural conditions of intoxication. Modeling of intoxication with intraperitoneal injection of a heavy metal is also more technically complicated than intragastric administration through an atraumatic probe.

• prophylactic administration of an antioxidant for a short time does not allow to obtain a method for the prevention of chronic coagulopathy. A chronic experiment carried out for two months is more informative, and allows you to study in more detail at different times in dynamics the mechanisms of the preventive action of an antioxidant on the formation of disorders in the hemostasis system under conditions of toxic exposure, which is especially important when studying the hemostasis system.

The claimed invention is aimed at solving the problem of developing a method for the prevention of chronic toxic coagulopathy in rats under the influence of chromium.

The solution to this problem provides a significant reduction in pathological changes in the hemostasis system in chronic poisoning with potassium bichromate in rats, which was accompanied by a decrease in the intensity of lipid peroxidation processes and restoration of the activity of antioxidant defense enzymes.

To achieve this technical result, the claimed method for the prevention of chronic toxic coagulopathy in rats, including the daily administration of an antioxidant and a heavy metal, differs in that as an antioxidant, from the first day of the experiment, melaxen is administered to animals through a stomach tube at a dose of 5 mg/kg for two months. and a solution of potassium bichromate at a dose of 0.5 mg/kg in terms of metal.

Daily administration of melatonin through a tube into the stomach at a dose of 5 mg/kg simultaneously with intragastric administration of an aqueous solution of potassium bichromate helps to reduce the degree of pathological changes in plasma and cellular hemostasis, prevents a decrease in the activity of the anticoagulant and fibrinolytic components of the hemostasis system, reducing the degree of thrombinemia. Positive effects are associated with a pronounced antioxidant and membrane-protective effect of melatonin, a decrease in the cytotoxicity of chromium. The claimed method is efficient, cost-effective and easily reproducible.

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

According to the authors, the essence of the claimed invention does not follow for specialists explicitly from the known level of medicine, since it does not reveal the above possibility of obtaining a method for the prevention of chronic toxic coagulopathy, characterized in that rats are given 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 an aqueous solution of potassium bichromate at a dose of 0.5 mg/kg, in terms of metal, which allows us to conclude that the criterion "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, which consists in an effective and easily reproducible method for the prevention of chromium-induced toxic coagulopathy, preventing pathological activation of the hemostasis system and the restoration of the anticoagulant and fibrinolytic potential of the blood, reducing the level of markers thrombinemia with prolonged exposure to chromium, by reducing the intensity of lipid peroxidation processes, restoring the activity of the antioxidant system, 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 chromium in distilled water, potassium bichromate is dissolved in such a way that 0.05 mg of chromium is accounted for per unit of solution, equal to 0.5 ml, in terms of metal. For every 100 g of rat weight, 0.5 ml of a toxic 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 accounted for per solution unit equal to 1 ml.

Two groups of rats are injected with a solution of potassium bichromate at a dose of 0.5 mg/kg through an atraumatic probe into the stomach daily 1 time per day for 60 days. Simultaneously with the first day of administration of chromium salt of one of the groups, a solution of melaxen is administered every day at a dose of 5 mg/kg through an intragastric tube.

To study the state of the hemostasis system, the number of platelets and their aggregation activity are determined; fibrinogen content; APTT - activated partial thromboplastin time; PT - prothrombin time; VPFM - relative polymerization time of fibrin monomer to assess the state of the final stage of blood coagulation; AT(III) antithrombin (III) activity and protein C activity - to assess the state of the anticoagulant system; according to the time of spontaneous euglobulin lysis, the state of the fibrinolytic link is investigated; RFMK concentration of soluble fibrin-monomer complexes was determined by a quantitative version of the phenanthroline test - to assess the level of thrombinemia (Petrishchev N.N., Papayan L.P. 1999; Momot A.P., 2009). The intensity of lipid peroxidation (LPO) is judged according to the concentration of malonic dialdehyde (MDA) in erythrocytes and the level of hydroperoxides in blood plasma. In order to evaluate the effectiveness of antioxidant protection (AOZ), the activity of superoxide dismutase and catalase in erythrocytes is determined.

The results of a study with the introduction of Melaxen and potassium bichromate in rats demonstrate the effectiveness of the use of an antioxidant in the prevention of chronic chromium-induced toxic coagulopathy. Melatonin helps to reduce the severity of changes in cellular and plasma hemostasis, reduces the level of thrombinemia, restoring the activity of the processes of the anticoagulant system and fibrinolysis, due to the pronounced antioxidant activity in conditions of chronic intoxication with potassium bichromate.

An example of a specific implementation.

Male rats of the line Wistarmassoy 300-350 g were injected through an atraumatic probe into the stomach with a solution of potassium dichromate at a dose of 0.5 mg/kg every day for two months. Simultaneously, from the first day of administration of potassium dichromate, half of the rats were intragastrically injected every day with melaxen at a dose of 5 mg/kg. Intact animals (10 rats) kept under standard vivarium conditions served as controls. Stabilization and obtaining of blood plasma samples was carried out taking into account international standards for clinical laboratory diagnostics for studies in the field of hemostasis (Petrishchev N.N., Papayan L.P. 1999; Momot A.P. Tsyvkina L.P., Taranenko I.A. 2011). The material for the study was whole blood, as well as blood plasma rich and poor in platelets. The indicators of the coagulation system, the state of the anticoagulant and fibrinolytic components of the hemostasis system, the activity of lipid peroxidation and antioxidant protection processes were studied according to standard methods.

To study the state of the hemostasis system, the number of platelets and their aggregation activity (ADP inducer at a final concentration of 10.0 μg/ml), fibrinogen, APTT, PT, VPFM, protein C and AT (III) activity, SEL, RFMC, using diagnostic sets of the company "Technology Standard", Barnaul, Russia. The tests were performed on a CGL-2110 Solar turbidimetric coagulometer, an AP 2110 Solar aggregometer (Belarus). The state of lipid peroxidation was assessed by the content of hydroperoxides in blood plasma (diene conjugates and diene ketones) and MDA in erythrocytes. The antioxidant status was assessed by the activity of catalase and SOD in erythrocytes. The measurements were carried out on a PV1251A spectrophotometer, Solar, Belarus.

Statistical data analysis was carried out using Microsoft Excel 2016 and STATISTICA 10.0., StatSoft programs, taking into account the distribution of signs in groups according to the Shapiro-Wilk test. Variables are presented as median (Me) and interquartile range (Q25÷Q75). Differences between animal groups were assessed using the Mann-Whitney U-test. Spearman's rank correlation coefficient (rs) was used to estimate the statistical dependence of observation series. The critical significance level was considered to be p≤0.05.

Experiments have shown that intragastric administration of a solution of potassium bichromate at a dose of 0.5 mg/kg of body weight for two months causes disturbances in the hemostasis system in rats, contributes to the development of toxic coagulopathy (Table 1.). Two weeks later, in rats, the concentration of fibrinogen in the blood plasma significantly increased, the degree of ADP-aggregation of platelets increased. The hypercoagulable orientation of blood coagulation processes after a month was confirmed by shortening of the chronometric indicators of APTT, PT, high fibrinogen levels, and shortening of VPMF. A decrease in the number of platelets was recorded, with an increase in their functional activity. The shift of the hemostatic potential towards hypercoagulability was compensated by the activation of the anticoagulant and fibrinolytic systems. A significant increase in the activity of protein C, AT(III) and a shortening of the time of spontaneous euglobulin lysis were revealed.

Hypercoagulation changes after two months of the experiment progressed, APTT and PT were shortened, the concentration of fibrinogen was higher than the control. High aggregation activity of platelets persisted and a sharp increase in the concentration of thrombinemia markers - RFMK was detected, which may indicate the development of a state of thrombotic readiness. At the same time, a decrease in antithrombin activity was detected against the background of suppression of fibrinolytic activity (Table 1.) (Momot A.P., Tsyvkina L.P., Taranenko I.A. Modern methods of recognizing thrombotic readiness. Barnaul, 2011).

The introduction of the antioxidant melatonin for two months contributed to a decrease in stress in the hemostasis system, a decrease in the likelihood of developing a state of thrombotic readiness, compared with the data in rats with isolated administration of chromium. This is evidenced by a decrease in the severity of changes in RFMC, the restoration of the activity of physiological anticoagulants and fibrinolysis (Table 1).

In modern literature, information is widely presented on the relationship between lipid peroxidation processes in blood plasma, erythrocytes, leukocytes and platelets with changes in the activity of blood coagulation processes, thrombinemia under various conditions in normal and pathological conditions (Byshevsky A.Sh. 2006).

Intragastric administration of chromium caused an increase in the concentration of MDA in erythrocytes and an increase in the level of hydroperoxides in blood plasma. At the same time, a decrease in the activity of superoxide dismutase and catalase was detected, which is probably associated with the depletion of the enzymatic activity of antioxidants (Table 2.).

Prophylactic use of the natural antioxidant melaxen prevented a decrease in the level of catalase and SOD activity in erythrocytes, with a simultaneous decrease in the concentration of MDA and restoration of the level of hydroperoxides in the blood plasma, which indicates a pronounced antioxidant effect of melaxen under conditions of long-term administration of chromium to the body of rats.

Statistical analysis of the obtained experimental data revealed correlations between the positive dynamics of the hemostasis system parameters with the introduction of melatonin with a decrease in the level of lipid peroxidation products and the restoration of the activity of antioxidant blood enzymes in chronic intoxication with potassium bichromate.

After two months of the experiment, an increase in AT(III) activity and restoration of the time of spontaneous euglobulin lysis was associated with a decrease in the concentration of MDA (rs=-0.65; p<0.05; rs=0.71; p<0.001) and an increase in SOD activity ( rs=0.68; p<0.05; rs=-0.72; p<0.05) and catalase (rs=0.65; p<0.01; rs=-0.63; p<0 .05). A correlation between a decrease in the degree of thrombinemia and a decrease in the concentration of MDA (rs=0.62; p<0.05) and an increase in catalase activity (rs=-0.74; p<0.05) was established.

Experiments have shown that the prophylactic administration of melatonin in chronic chromium intoxication causes a decrease in the severity of the shift in indicators of vascular-platelet and coagulation hemostasis, anticoagulant and fibrinolytic systems, and also helps to reduce thrombinemia. Changes in the parameters of the hemostasis system correlate with the restoration of the level of lipid peroxidation processes and the activity of antioxidant enzymes - catalase and superoxide dismutase during prophylactic administration of melaxen simultaneously with heavy metal.

The results of the experiments allow us to consider it appropriate to study the possibility of using anticoagulants and antioxidants in the development of principles for the prevention and correction of disorders in the hemostasis system and urinary function of the kidneys when exposed to heavy metal compounds.

Claims (1)

A method for preventing toxic coagulopathy in rats, including daily administration of an antioxidant and a heavy metal, characterized in that from the first day of the experiment, melatonin at a dose of 5 mg/kg and a solution of potassium bichromate at a dose of 0 are administered to rats through a tube into the stomach for two months as an antioxidant .5 mg/kg in terms of metal.