RU2521279C1 - Method for correction of oxidative stress and no producing endothelial dysfunction accompanying vascular complications of diabetes mellitus in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves preliminary intraperitoneal single administration of 5% aqueous alloxan in a dose of 15 mg/kg of body weight into a rat's body on an empty stomach. That is followed by administering afobazol under conditions of oxidative stress after observing the rat's blood glucose gain at least twice. Afobazol is administered subcutaneously in a dose of 10 mg/kg of body weight once a day for 30 days with underlying administration of L-arginine in a dose of 10 mg/kg of body weight or with underlying NG-nitroarginine methyl ester (L-NAME)-inhibitor of NOS-3 enzyme in a dose of 25 mg/kg of animal's weight.

EFFECT: method enables correcting the oxidative stress and NO-producing endothelial dysfunction accompanying vascular complications of diabetes mellitus.

1 dwg, 6 tbl, 1 ex

Description

The invention relates to the pharmaceutical industry and medicine, in particular to endocrinology, and for the treatment of angiopathy and pathology of internal organs in experimental alloxan diabetes.

The study of biochemical parameters of hemodynamic vascular complications of diabetes remains one of the most urgent problems of modern biomedical science. Among several hypotheses explaining the pathogenesis of vascular diabetic lesions, a special place is played by the development of oxidative stress, due to increased generation of active oxygen metabolites (AMA), and violation of antioxidant defense (AOD) of cells. A risk factor under conditions of oxidative stress is impaired endothelial function, the main mechanisms of which may be changes in the activity and / or expression of endothelial NO synthase (NOS-3), decreased synthesis of NO from L-arginine, and sensitivity of smooth muscle cells (MMC) to NO or enhanced its degradation due to interaction with reactive oxygen species (ROS), including superoxide anion, as well as other lipid peroxidation products.

Given the important role in the development of diabetes mellitus (DM), oxidative stress, and impaired NO-producing function of the endothelium, we can assume that a new methodological approach based on the use of a drug with antioxidant properties and the ability to normalize NO metabolism is a necessary component of the pathogenetic treatment of diabetic angiopathy. Zenina T.A., Silkina I.V., Seredinin S.B., Mirzoyan R.S. Experimental and Clinical Pharmacology. - 2006. - No. 4. - P.45-47; Balasanyan MG, Kanayan AS, Jhopchayan AV Acta Physiol. Hung. 2002. vol. 89, No. 1-3, p .98).

According to the literature, the selective anxiolytic afobazole synthesized at the GUNII Pharmacology named after V.V.Zakusova RAMS, has the ability to positively modulate NO production by inhibiting the inducible isoform of NO synthase and stimulating constitutive endothelial NO synthase (NOS-3) and at the same time possesses antioxidant properties (Seredenin SB, Melkumyan D.S., Valdman EA The influence of afobazole on the BDNF content in the brain structures of inbred mice with different phenotypes of emotional-stress reactions. Experimental and clinical pharmacology. 2006. - T. 69. - No. 3. - C.3-6). However, afobazole was not previously used to correct endothelial dysfunction in vascular complications of neuroangiopathies caused by experimental diabetes mellitus.

It is very relevant to study in an experiment in rats with type 1 diabetes mellitus indicators of LPO activity, antioxidant system (AOC), the concentration of total nitric oxide metabolites (NOx), the role of the level of expression of endothelial NO synthase (NOS-3), blood lipid spectrum and on the basis of these fundamental data, the development of a possible way to correct disorders using afobazole in experimental diabetes mellitus (ESD) in rats. However, it should be noted that in the available literature there are no data on biochemical markers of endothelial dysfunction and a study of the effect of afobazole on metabolic parameters of endothelial dysfunction in experimental diabetes mellitus.

A known method for the correction of endothelial dysfunction in vascular complications of alloxan diabetes in the experiment, including the use of ubiquinone coenzyme Q ₁₀ as a drug, which is administered in an amount of 0.11 μl / 100 g live weight (see RF patent No. 2455702, IPC ⁹ G09D 23 / 28, G01N 33/48, A61K 31/122, A61P 5/48, A61P 9/08, published on July 10, 2012).

The disadvantage of this method is that NOS-3 expression data were not presented and the participation of L-arginine, a substrate for NO synthesis and NOS-3 expression stimulator, and the effect of the enzyme inhibitor L-NAME in rats with experimental diabetes mellitus were not investigated. Against the background of L-arginine and L-NAME and their combination with coenzyme Q10, the parameters of the POL-AOS system and the nature of hemodynamic changes were not studied.

Closest to the claimed technical solution is a method of treating nephroangiopathy with alloxan diabetes in experimental animals, which includes a preliminary single administration of a fasting rat intraperitoneally to a fasting rat 5% aqueous solution of alloxan at a dose of 15 mg / kg of animal weight, followed by the administration of a drug (see RF patent No. 2372898, IPC ⁹ A61K 31/197, A61P 13/12, G09B 23/28, published on November 20, 2009).

The disadvantages of the prototype are the lack of studies to study the effects of the NO donor - L-arginine and the L-NAME enzyme inhibitor on these parameters, while the study of these substances would clarify the new links in the pathogenesis of vascular complications, the participation of the substrate of the enzyme L-arginine and its inhibitor L-NAME . Moreover, information about the violation of cholesterol metabolism and its effect on the bioavailability of nitric oxide is not provided. In all these experimental conditions, there is no data on the nature of hemodynamic disturbances.

The technical result consists in determining the dose of afobazole, increasing the accuracy and reliability of the correction of experimental diabetes mellitus in terms of oxidative stress, NO-producing endothelial function and NO bioavailability.

The technical result is achieved by the fact that in the method of correcting oxidative stress and impaired NO-producing function of the endothelium in case of vascular complications of diabetes mellitus in an experiment that involves a preliminary single dose administration of a 5% aqueous solution of alloxan to a rat on an empty stomach at a dose of 15 mg / kg of animal weight, s subsequent administration of the drug according to the invention, afobazole is used as the drug, which is administered under conditions of oxidative stress after increasing blood glucose rat at least twice subcutaneously at 10 mg / kg of body weight once a day for 30 days at the background of daily administration of L-arginine, 10 mg / kg body weight or amid N ^G- nitroarginine methyl ester (L-NAME) -inhibitor of the NOS-3 enzyme at a dose of 25 mg / kg of animal weight.

This method will open up new links in the pathogenesis of angiopathy in chronic alloxan diabetes, offer a treatment that improves efficiency, reproducibility, convenience, affordability, safety and low cost for conducting an experiment on animals.

The essence of the proposed method is confirmed graphically and in tables: where Fig. 1 (a-d) shows the changes in the concentration of total cholesterol and its content in lipoproteins of different densities against the background of corrective therapy with afobazole for ESD.

Table number 1. Change in the level of NOS-3 expression under the influence of afobazole and its combination with L-arginine and L-NAME.

Table number 2. Changes in the POL-AOS system against the background of an inhibitor of the NOS-3 enzyme, NG-nitroarginine methyl ester (L-NAME).

Table number 3. Changes in the POL-AOS system against the background of L-arginine substrate.

Tables No. 4, 5, 6. The dynamics of changes in hemodynamic parameters are normal in experimental diabetes mellitus and during corrective therapy with afobazole against the background of L-arginine substrate and NOS-3 enzyme inhibitor - L-NAME.

A method for the correction of oxidative stress and impaired NO-producing endothelial function in case of vascular complications of diabetes mellitus in an experiment was carried out as follows.

To damage the insulinogenic β-cells of the islets of Langerhans, experimental diabetes mellitus (alloxan) was induced by intraperitoneal administration to rats of a 5% aqueous solution of alloxan (synthesized in the laboratory of the pathobiochemistry department of the Federal State Budget Scientific Institution IBMI VSC RAS and PCO-A) at a dose of 15 mg / kg of fasting animal weight background 24-48 hours of fasting with free access to water. After 48-72 hours on an empty stomach, blood was taken from the tail of the rat (microquantity) and the glucose level was determined by the glucose oxidase method (test kits). The model was considered valid with a 2-fold increase in blood glucose. For correction, afobazole was administered subcutaneously in an amount of 10 mg / kg of animal weight once a day for 30 days. To clarify the effect of afobazole on the availability of L-arginine substrate for endothelial NO synthase (NOS-3), afobazole was administered against the background of daily administration of L-arginine at a dose of 10 mg / kg of animal weight. In another version of the experiments, afobazole was introduced against the background of N ^G- nitroarginine methyl ester (L-NAME), an inhibitor of the NOS-3 enzyme at a dose of 25 mg / kg of the animal’s weight, the parameters of the antioxidant system, blood lipid spectrum, the state of micro- and macrohemodynamics were determined, and also the concentration of total NO metabolites and the expression of endothelial NO synthase (eNOS) under conditions of oxidative stress, and the change in these parameters was used to evaluate the presence of endothelial dysfunction in animals with vascular complications of diabetes.

At the end of the experiment, perfusion was studied at various points of tissue location (fluid exchange) by sounding with a 10 MHz sensor operating on the principle of “blind” Doppler in anesthetized animals. Then the rats were killed under thiopental anesthesia; blood was taken from the heart using a 2.8% EDTA solution as an anticoagulant to determine the concentration of malondialdehyde (MDA) and serum to determine the activity of SOD, catalase, ceruloplasmin and NO.

The level of NOS-3 expression in the aortic endothelium was determined by the method of Metelskaya V.A., Gumanova N.G., Litinskaya O.A. (Nitric oxide: a role in the regulation of biological functions, methods for determination in human blood // Laboratory Medicine. - 2005. - No. 7. - S.19-24). The aorta was removed, washed with physiological saline and placed in plastic tubes, which were stored in liquid nitrogen, after which the aorta was treated according to the procedure. The band corresponding to NOS-3 was detected in accordance with its molecular weight, established in comparison with the protein taps. The film was dried in air, the bands were scanned, and the area under the curve was calculated using the Total Lab program. The results were presented in arbitrary units as the ratio of the intensity of the band X to the intensity of the band taken as control on each film. The aorta of experimental rats was microscopically examined histologically. Histological changes in the structure were quantified by the Avtandilov method using a Nikon digital camera combined with a microscope.

Example.

The study was conducted in 9 groups of male Wistar rats:

1st group - control in the amount of 20 goals;

Group 2 — rats with experimental diabetes in the amount of 30 animals;

3rd experimental group - intact rats + L-arginine at a dose of 10 mg / kg for 30 days in an amount of 15 animals;

The 4th experimental group - intact rats + L-NAME at a dose of 25 mg / kg for 30 days in an amount of 15 animals;

The 5th experimental group — rats with ESD + L-arginine at a dose of 10 mg / kg for 30 days in an amount of 20 animals;

The 6th experimental group — rats with ESD + L-NAME at a dose of 25 mg / kg for 30 days in an amount of 20 animals;

The 7th experimental group - rats with ESD + Afobazole at a dose of 10 mg / kg of animal weight in an amount of 30 animals;

The 8th experimental group - rats with ESD + afobazole at a dose of 10 mg / kg of animal weight + L-arginine 10 mg / kg for 30 days in an amount of 20 animals;

The 9th experimental group - rats with ESD + Afobazole at a dose of 10 mg / kg of animal weight + L-NAME 25 mg / kg for 30 days in an amount of 20 animals.

Experimental diabetes mellitus, characterized by insulin deficiency, was caused in rats of the experimental group by intraperitoneal administration of a 5% aqueous solution of alloecane at a dose of 15 mg / kg body weight on an empty stomach on the background of 24-48-hour fasting with free access to water. After 48-72 hours on an empty stomach, blood was taken from the tail (microquantity) and the glucose level was determined by the glucose oxidase method (test kits) and at least 2-fold increase in blood glucose.

To correct violations of lipid peroxidation, AOS, the blood lipid spectrum, micro- and macrohemodynamics, as well as the concentration of total NO metabolites and the expression of endothelial NO synthase (NOS-3) under conditions of oxidative stress, rats of the experimental group with experimental diabetes were injected subcutaneously with Afobazole for 1 month. dose of 10 mg / kg body weight.

After the administration period, lipid peroxidation parameters, antioxidant system, blood lipid spectrum, the state of micro and macro hemodynamics, as well as the concentration of total NO metabolites and the expression of endothelial NO synthase (NOS-3) under oxidative stress were studied.

The results indicate a significant decrease in the concentration of MDA in the blood under the influence of afobazole in diabetic rats (from 8.65 ± 0.031 nmol / ml to 7.26 ± 0.061 nmol / ml (p <0.001) (see line 1, table 2). Analysis AOS activity showed a significant increase in SOD activity in blood serum and erythrocytes (from 1.45 ± 0.044 conventional units to 1.78 ± 0.148 standard units (p <0.05) and from 64.4 ± 1.53, respectively units to 74.6 ± 1.55 units (p <0.001)) (see line 4, table 2), and increased catalase and ceruloplasmin significantly decreased. However, it should be noted that the activity of catalase stayed up which is probably a manifestation of a cellular compensatory reaction.In the group of rats treated with afobazole, the concentration of total NO metabolites in blood serum statistically significantly increased from 32.54 ± 1.56 μmol to 48.7 ± 0.844 μmol (p <0.001) (cm line 2, table 2) A correlation analysis was performed to determine the efficacy of afobazole on lipid peroxidation and the activity of AOD enzymes. The data obtained in this group of diabetic rats treated with afobazole showed a direct significant correlation between ntsentratsiey MDA and catalase activity (r = + 0,60), and ceruloplasmin concentration (r = + 0,58) and feedback between the reduction of MDA concentration and increased SOD activity in serum (r = -0,61). There was an increase in the concentration of total NO metabolites and a correlation analysis showed the presence of a negative relationship with MDA (r = -0.57).

Thus, afobazole inhibits lipid peroxidation, corrects the relationship between AOZ enzymes and contributes to an increase in the concentration of total NO metabolites, although their level does not reach control values. The obtained data demonstrate the membrane protective properties of afobazole in vivo in diabetes in the experiment. Our studies showed for the first time that in vivo in diabetes mellitus in rats, afobazole, inhibiting CPO and restoring AOD, promotes an increase in eNOS expression and, accordingly, NO concentration.

A number of factors affect the expression of NOS-3 and the efficiency of NO formation:

- the availability of the substrate L-arginine

- the presence of an endogenous inhibitor of the NOS-3 enzyme - ADMA.

- condition of coenzymes: NADPH ₂ , TTBP, etc.

- the effect of oxidized LDL, causing atherogenic changes in the vascular wall.

Changes in cholesterol metabolism were studied: concentrations of cholesterol, cholesterol, cholesterol, LDL, TAG during treatment with afobazole in rats with diabetes. Analysis of the content of total cholesterol in blood serum and its distribution in lipoproteins of various densities showed that during treatment there is a statistically significant decrease in the concentration of cholesterol from 3.896 ± 0.161 mmol / L to 2.493 ± 0.04 mmol / L, p <0.001, an increase in HDL cholesterol from 0.556 ± 0.012 mmol / L to 0.601 ± 0.003 mmol / L, p <0.01 and a significant decrease in LDL from 3.124 ± 0.15 mmol / L to 1.706 ± 0.044 mmol / L, p <0.001 (see Fig. one). At the same time, the concentration of TAG in serum decreased from 0.476 ± 0.018 mmol / L to 0.41 ± 0.019 mmol / L, p <0.02 (see Fig. 1).

To clarify the relationship between the concentration of NO and the lipoprotein spectrum of blood serum, a correlation analysis was performed and a negative relationship was found between these indicators (r = -0.69; r = -0.67; r = -0.64; r = -0.71 ) Consequently, a decrease in total cholesterol and cholesterol of LDL under conditions of inhibition of lipid peroxidation contributed to an increase in the bioavailability of NO. To clarify the effect of afobazole on the availability of L-arginine substrate for NOS-3 in another research variant, afobazole was administered against the background of daily administration of L-arginine at a dose of 10 mg / kg body weight for 3 weeks to diabetic rats. At the end of the experiment, the concentration of total NO metabolites and oxidative stress indices were determined in blood serum. The results showed an increase in the NO concentration from 32.54 ± 1.56 μmol with ESD to 42.27 ± 0.893 μmol (p <0.01) with ESD + arginine and up to 50.54 ± 0.472 (p <0.001) (see line 2, table 3) on the background of the introduction of arginine with afobazole in these research options. A comparative analysis of data in rats with ESD against the background of L-arginine and the combination of L-arginine with afobazole showed a more significant decrease in the concentration of MDA and an increase in the concentration of NO against the background of the combined administration of L-arginine and afobazole (see table 3).

These data confirm that afobazole increases the bioavailability of the L-arginine substrate to its enzyme and, accordingly, the concentration of total NO metabolites. It can be assumed that afobazole in combination with L-arginine also affects the enzyme NOS-3 itself. To clarify this issue, we examined the expression of NOS-3 against the background of the combined administration of L-arginine and afobazole. The data showed an increase in the expression level of NOS-3 against the background of the combined use of L-arginine and afobazole.

In another variant of the experiments, afobazole was administered against the background of the L-NAME-inhibitor of the NOS-3 enzyme; the control was studies against the background of the administration of one L-NAME inhibitor. In intact and diabetic rats, the concentration of MDA, NO, and NOS-3 expression were determined in these variants. The data showed that the NO concentration decreased significantly 3 weeks after the administration of L-NAME from 51.069 ± 0.50 μmol to 33.13 ± 0.595 μmol (p <0.001) (see line 2, table 2), which could be caused by inhibition expression of eNOS. In another research option, with the administration of afobazole against the background of L-NAME, the concentration of total NO metabolites increased from 30.74 ± 0.567 μmol to 40.23 ± 0.62 μmol, p <0.001, while the MDA level in the blood decreased from 9.186 ± 0.009 nmol / ml to 8.01 ± 0.053 nmol / ml, p <0.001 (see lines 1, 2, table 2).

To confirm the involvement of NOS-3, the level of NO expression of the producing enzyme in aortic extracts was studied in diabetic rats (see table 1). The experimental data confirmed for the first time that the anxiolytic, afobazole, by inhibiting LPO, increases the concentration of NO and undoubtedly has a stimulating effect on the expression of NOS-3, whereas against the background of L-NAME, the level of expression of NOS-3 in aortic extracts was significantly reduced.

Correction of endothelial dysfunction, indicators of oxidative stress and NO metabolism was accompanied by an increase in the average and systolic blood flow velocities in the vessels of the microvasculature, a decrease in the density of the vascular wall - the Gosling index (PI) and specific peripheral vascular resistance - the Purselo index (RI) (see table 5, 6).

These hemodynamic changes indicate an increase in perfusion processes due to a decrease in vascular resistance - the rheographic index and an increase in the average and systolic blood flow velocities against the background of the administration of afobazole. To confirm the effectiveness of the influence of afobazole on hemodynamics in the microvasculature, a correlation analysis was performed between the NO concentration and blood flow velocity, respectively, of the average, systolic and diastolic (r = + 0.51; r = + 0.56; r = + 0.57; p <0.001 ) The table shows that between the increase in the concentration of NO and the processes of microcirculation revealed a positive strong correlation.

Blood flow studies in the main arterial vessels (BA) showed a decrease in mean (M), systolic (S) and diastolic (D) blood flow velocities during the administration of afobazole (see tables 4, 5, 6). The nature of the hemodynamic changes in the renal arteries showed a similar picture, characterized by a decrease in the average (M) and systolic (S) blood flow velocities during treatment with afobazole. The rheographic data revealed an increase in the Gosling index (PI), therefore, of the elastic-elastic properties (density) of the vascular wall, as well as the Purselo index (RI), which reflects regional peripheral vascular resistance against afobazole. In HPB, there is a decrease in average (M), systolic (S) and diastolic (D) blood flow velocities, as well as a decrease in rheographic indices: PI - Gosling index and RI - Purselo index (see tables 5, 6). These data indicate that afobazole, having an antioxidant effect, increases the concentration of total NO metabolites and the expression level of NO - producing enzyme - eNOS in the vascular endothelium. These changes in NO metabolism contribute to the restoration of endothelial function and reduce the level of hyperperfusion in the main arterial vessels. In contrast, the restoration of the function of regulators of vascular tone in the microcirculatory link contributes to an increase in perfusion - fluid exchange.

Using the proposed method will allow, in comparison with the prototype, to increase the effectiveness of treatment for the correction of experimental diabetes mellitus under conditions of oxidative stress, which characterizes its effect on the main pathogenetic link of developing angiopathy in alloxan diabetes in experimental animals, to increase reproducibility, convenience, accessibility, safety, and low experimental cost and efficacy in rats with experimental diabetes mellitus.

Table 1 “A method for the correction of oxidative stress ...” Change in the level of eNOS expression under the influence of afobazole and its combination with L-arginine and L-NAME L-name ESD + L-name arginine ESD + Arginine ESD + L-name + Afobazole ESD + Arginine + Afobazole M 0.567 0,305 1.05 0.65 0.753 1.51 m 0,088 0.003 0,087 0.104 0.199 0.12 P ₁ 0.01 0.001 - - 0.001 P ₂ 0.001 0.01 0.05 0.001 P ₃ 0.01 - 0.01 P ₄ - 0.001 P ₅ 0.01 0.01 Note: P ₁ - reliability relative to L-name; P ₂ - reliability relative to ESD + L-name; P ₂ - reliability relative to L-arginine; P ₄ - reliability relative to ESD + L-arginine; P ₅ - reliability relative to ESD + afobazole

table 2 “A method for the correction of oxidative stress ...” Changes in the parameters of the POL-AOS system against the background of the inhibitor of the NOS-3 enzyme - NG-nitroarginine methyl ester (L-NAME) in ESD Groups indicators N ESD L-name ESD + L-name ESD + Afobazole ESD + L-name + Afobazole MDA, nmol / ml M ± m 6.87 ± 0.044 8.65 ± 0.031 7.213 ± 0.012 9.186 ± 0.009 7.26 ± 0.061 8.01 ± 0.053 P ₁ 0.001 0.001 0.001 0.001 0.001 P ₂ 0.001 0.001 0.001 0.001 P ₃ 0.001 - 0.001 P ₄ 0.001 0.001 P ₅ 0.001 NO, μmol M ± m 51.069 ± 0.5 32.54 ± 1.56 33.13 ± 0.595 30.74 ± 0.567 48.7 ± 0.844 40.23 ± 0.62 P ₁ 0.001 0.001 0.001 0.02 0.001 P ₂ - - 0.001 0.001 P ₃ 0.01 0.001 0.001 P ₄ 0.001 0.001 P ₅ 0.001 catalase, mkat / l M ± m 225.56 ± 25.57 345.33 ± 3.16 310.03 ± 2.054 382.14 ± 1.58 307.6 ± 3.35 338.15 ± 1.84 P ₁ 0.001 0.01 0.001 0.01 0.001 P ₂ 0.001 0.001 0.001 0.05 P ₃ 0.001 - 0.001 P ₄ 0.001 0.001 P ₅ 0.001 SOD, units M ± m 88.2 ± 1.07 64.4 ± 1.53 82.1 ± 1.24 61.5 ± 2.63 74.6 ± 1.55 70.1 ± 1.51 P ₁ 0.001 0.01 0.001 0.001 0.001 P ₂ 0.001 - 0.001 0.01 P ₃ 0.001 0.01 0.001 P ₄ 0.001 0.01 P ₅ 0.05 ceruloplasmin, mg / l M ± m 338.66 ± 6.365 467.6 ± 8.945 360.2 ± 6.697 497.1 ± 5.25 394.2 ± 4.86 430.1 ± 5.08 P ₁ 0.001 0.02 0.001 0.001 0.001 P ₂ 0.001 0.01 0.001 0.01 P ₃ 0.001 0.001 0.001 P ₄ 0.001 0.001 P ₅ 0.001

Table 3 “A method for the correction of oxidative stress ...” Changes in indicators in the POL-AOS system against the background of L-arginine substrate in ESD Groups indicators N ESD arginine ESD + Arginine ESD + Afobazole ESD + Arginine + Afobazole MDA, nmol / ml M ± m 6.87 ± 0.044 8.65 ± 0.031 6.8 ± 0.017 7.88 ± 0.021 7.26 ± 0.061 7.03 ± 0.014 P ₁ 0.001 - 0.001 0.001 0.01 P ₂ 0.001 0.001 0.001 0.001 P ₃ 0.001 0.001 0.001 P ₄ 0.001 0.001 P ₅ 0.01 NO, μmol M ± m 51.069 ± 0.5 32.54 ± 1.56 53.25 ± 0.412 42.27 ± 0.893 48.7 ± 0.80 50.54 ± 0.472 P ₁ 0.001 0.01 0.001 0.02 - P ₂ 0.001 0.001 0.001 0.001 P ₃ 0.001 0.001 0.001 P ₄ 0.001 0.001 P ₅ 0.05 catalase, mkat / l M ± m 225.56 ± 25.57 345.33 ± 3.16 221.72 ± 1,056 320.2 ± 2.08 307.6 ± 3.35 283.64 ± 1.42 P ₁ 0.001 - 0.01 0.01 0.05 P ₂ 0.001 0.001 0.001 0.001 P ₃ 0.001 0.001 0.001 P ₄ 0.01 0.001 P ₅ 0.001 SOD, units M ± m 88.2 ± 1.07 64.4 ± 1.53 88.8 ± 1.031 70.3 ± 1,096 74.6 ± 1.55 82.5 ± 1.276 P ₁ 0.001 - 0.001 0.001 0.01 P ₂ 0.001 0.01 0.001 0.001 P ₃ 0.001 0.001 0.001 P ₄ 0.05 0.001 P ₅ 0.001 ceruloplasmin, mg / l M ± m 338.66 ± 6.365 467.6 ± 8.945 336.3 ± 1.43 419.8 ± 6.806 394.2 ± 4.86 351.4 ± 2.676 P ₁ 0.001 - 0.001 0.001 - P ₂ 0.001 0.001 0.001 0.001 P ₃ 0.001 0.001 0.001 P ₄ 0.01 0.001 P ₅ 0.001

Table 4 “A method for the correction of oxidative stress ...” The nature of the changes in hemodynamic parameters is normal, against the background of an inhibitor of the NOS-3 enzyme — NG-nitroarginine methyl ester (L-NAME) and L-arginine substrate in intact rats

$$\frac{T\mathrm{about}h\mathrm{to}\mathrm{but}l\mathrm{about}\mathrm{to}\mathrm{but}c\mathrm{and}\mathrm{and}}{\begin{array}{l}P\mathrm{about}\mathrm{to}\mathrm{but}s\mathrm{but}tel\mathrm{and}\\ gem\mathrm{about}d\mathrm{and}n\mathrm{but}m\mathrm{and}\mathrm{to}\mathrm{and}\end{array}}$$

Perfusion BA npv Pa left PA right the control M, cm / s 2.518 ± 0.076 13.468 ± 0.473 9,420 ± 0,440 5,052 ± 0,304 4.377 ± 0.402 S, cm / s 11.338 ± 0.264 38,8341,223 15,868 ± 1,754 20.019 ± 0.880 19.59 ± 1.020 D, cm / s 6.335 ± 0.168 2.083 ± 0.232 1.223 ± 0.137 4.638 ± 0.415 5.651 ± 0.521 PI, cm / s 7.668 ± 0.250 2.814 ± 0.155 1,692 ± 0,121 5.036 ± 0.297 4.830 ± 0.467 GD, mmHg 0.042 ± 0.001 0.548 ± 0.020 0.118 ± 0.008 0.148 ± 0.012 0.27 ± 0.011 RI, conv. 1.490 ± 0.036 0.948 ± 0.011 1,042 ± 0,008 1.191 ± 0.029 1.24 ± 0.044 L-arginine M, cm / s 2.62 ± 0.007 13.12 ± 0.0087 9.21 ± 0.011 4.92 ± 0.0097 4.22 ± 0.0086 S, cm / s 11.48 ± 0.01 37.9 ± 0.0097 14.75 ± 0.011 19.76 ± 0.0063 19.16 ± 0.01 D, cm / s 6.41 ± 0.009 1.91 ± 0.0086 1.13 ± 0.0073 4.75 ± 0.011 5.73 ± 0.013 PI, cm / s 7.5 ± 0.009 2.9 ± 0.0068 1.52 ± 0.0083 5.18 ± 0.0077 4.91 ± 0.012 GD, mmHg 0.047 ± 0.0028 0.48 ± 0.0058 0.08 ± 0.0068 0.11 ± 0.0089 0.1 ± 0.0061 RI, conv. 1.38 ± 0.0068 0.98 ± 0.0089 0.91 ± 0.0088 1.21 ± 0.0063 1.26 ± 0.01 L-NAME M, cm / s 2.42 ± 0.008 13.79 ± 0.011 9.56 ± 0.009 5.1 ± 0.089 4.45 ± 0.01 S, cm / s 11.05 ± 0.0097 39.02 ± 0.012 16.54 ± 0.012 20.84 ± 0.01 19.63 ± 0.009 D, cm / s 6.27 ± 0.011 2.16 ± 0.008 1.43 ± 0.008 4.51 ± 0.008 5.6 ± 0.008 PI, cm / s 8.05 ± 0.008 2.74 ± 0.01 1.71 ± 0.008 4.92 ± 0.008 4.79 ± 0.008 GD, mmHg 0.041 ± 0.001 0.58 ± 0.011 0.12 ± 0.009 0.154 ± 0.001 0.13 ± 0.007 RI, conv. 1.51 ± 0.008 0.92 ± 0.011 1.08 ± 0.008 1.18 ± 0.008 1.23 ± 0.01

Table 5 “A method for the correction of oxidative stress ...” The nature of hemodynamic changes in ESD, against an inhibitor of the NOS-3 enzyme — NG-nitroarginine methyl ester (L-NAME) and L-arginine substrate

$$\frac{T\mathrm{about}h\mathrm{to}\mathrm{but}l\mathrm{about}\mathrm{to}\mathrm{but}c\mathrm{and}\mathrm{and}}{\begin{array}{l}P\mathrm{about}\mathrm{to}\mathrm{but}s\mathrm{but}tel\mathrm{and}\\ gem\mathrm{about}d\mathrm{and}n\mathrm{but}m\mathrm{and}\mathrm{to}\mathrm{and}\end{array}}$$

Perfusion BA npv Pa left PA right ESD M, cm / s 2.137 ± 0.064 15.710 ± 0.518 9.871 ± 0.426 5,308 ± 0,245 4.946 ± 0.276 ^PP ) ^PP ) S, cm / s 10.48 ± 0.165 40.292 ± 0.855 18.204 ± 0.544 22.59 ± 0.50 20.025 ± 0.485 ^PP ) ^P ) D, cm / s 6.25 ± 0.220 2.95 ± 0.372 2.162 ± 0.219 4.09 ± 0.303 5,495 ± 0,378 ^PP ) ^PP ) ^PP ) PI, cm / s 9,504 ± 0,231 2.804 ± 0.149 1,793 ± 0,092 3,746 ± 0,259 3.973 ± 0.282 ^{PP) PP} ) GD, mmHg 0.04 ± 0.001 0.651 ± 0.018 0.124 ± 0.008 0.2 ± 0.01 0.15 ± 0.008 ¹ ) RI, conv. 1.572 ± 0.03 0.932 ± 0.010 1.12 ± 0.013 1,145 ± 0,021 1,207 ± 0,029 ESD + L-Arginine M, cm / s 2.28 ± 0.0085 15.03 ± 0.013 9.74 ± 0.015 5.27 ± 0.008 4.88 ± 0.0098 ³³³³ ) ² ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) S, cm / s 10.78 ± 0.012 39.7 ± 0.0098 17.89 ± 0.013 21.84 ± 0.016 19.99 ± 0.013 ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) D, cm / s 6.29 ± 0.0097 2.81 ± 0.027 2.02 ± 0.022 4.16 ± 0.019 5.54 ± 0.018 ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) PI, cm / s 8.64 ± 0.014 2.807 ± 0.018 1.75 ± 0.02 3.96 ± 0.013 4.09 ± 0.011 ³³³³ ) ²²² ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) GD, mmHg 0.05 ± 0.0011 0.61 ± 0.012 0.12 ± 0.0006 0.18 ± 0.009 0.148 ± 0.001 ²²²² ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) RI, conv. 1.53 ± 0.011 0.939 ± 0.0012 1.09 ± 0.0097 1.158 ± 0.001 1.214 ± 0.001 ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ESD + L-NAME M, cm / s 2.05 ± 0.012 15.98 ± 0.018 9.93 ± 0.011 5.38 ± 0.01 5.02 ± 0.01 ⁴⁴⁴⁴ ) ²²² ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) S, cm / s 10.07 ± 0.017 41.16 ± 0.012 18.35 ± 0.01 22.83 ± 0.012 21.08 ± 0.014 ⁴⁴⁴⁴ ) ²²² ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ² ) D, cm / s 6.02 ± 0.014 3.07 ± 0.01 2.26 ± 0.008 3.92 ± 0.01 5.35 ± 0.011 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) PI, cm / s 9.74 ± 0.015 2.78 ± 0.014 1.84 ± 0.012 3.47 ± 0.008 3.81 ± 0.013 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) GD, mmHg 0.031 ± 0.0011 0.69 ± 0.009 0.131 ± 0.001 0.23 ± 0.006 0.168 ± 0.001 ⁴⁴⁴⁴ ) ²²²² ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) RI, conv. 1.68 ± 0.017 0.942 ± 0.001 1.18 ± 0.008 1.117 ± 0.001 1.18 ± 0.008 ⁴⁴⁴⁴ ) ²²² ) ⁴ ) ⁴⁴⁴⁴ ) ²²²² ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) Note: P _i - relative to the norm; P ₂ - relative to ESD; P ₃ - relative to L-arginine; P ₄ - relative to L-name

Table 6 "A method for the correction of oxidative stress ..." The nature of the changes in hemodynamic parameters in ESD against the background of corrective therapy with afobazole and its combination with an NOS-3 inhibitor - NG-nitroarginine methyl ester (L-NAME) and L-arginine substrate

$$\frac{T\mathrm{about}h\mathrm{to}\mathrm{but}l\mathrm{about}\mathrm{to}\mathrm{but}c\mathrm{and}\mathrm{and}}{\begin{array}{l}P\mathrm{about}\mathrm{to}\mathrm{but}s\mathrm{but}tel\mathrm{and}\\ gem\mathrm{about}d\mathrm{and}n\mathrm{but}m\mathrm{and}\mathrm{to}\mathrm{and}\end{array}}$$

Perfusion BA npv Pa left PA right ESD + Afobazole M, cm / s 2.43 ± 0.072 14.5 ± 0.23 9.61 ± 0.37 5.1 ± 0.28 4.61 ± 0.24 ²²² ) ² ) S, cm / s 11.22 ± 0.137 39.08 ± 1.14 16.69 ± 0.85 20.87 ± 0.46 19.92 ± 0.96 ²²² ) ²² ) D, cm / s 6.29 ± 0.16 2.14 ± 0.031 1.59 ± 0.18 4.31 ± 0.32 5.51 ± 0.39 ² ) ² ) PI, cm / s 8.04 ± 0.28 2.81 ± 0.131 1,708 ± 0,11 4.64 ± 0.19 4.17 ± 0.26 ²²²² ) ²²² ) GD, mmHg 0.042 ± 0.001 0.59 ± 0.02 0.121 ± 0.009 0.16 ± 0.012 0.14 ± 0.01 ² ) ²² ) RI, conv. 1.51 ± 0.01 0.941 ± 0.02 1,063 ± 0,017 1.16 ± 0.022 1.22 ± 0.027 ² ) ²²² ) ESD + L-Arginine + Afobazole M, cm / s 2.44 ± 0.027 13.71 ± 0.105 9.47 ± 0.04 5.086 ± 0.034 4.46 ± 0.04 ³³³³ ) ¹ ) ³³³³ ) ³³³³ ) ³³³³ ) S, cm / s 11.25 ± 0.101 38.92 ± 0.08 16.1 ± 0.03 20.23 ± 0.055 19.69 ± 0.048 ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) D, cm / s 6.21 ± 0.028 2.17 ± 0.051 1.4 ± 0.035 4.57 ± 0.034 5.6 ± 0.03 ³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ³³³ ) PI, cm / s 7.91 ± 0.043 2.81 ± 0.062 1.7 ± 0.032 4.97 ± 0.029 4.62 ± 0.036 ³³³³ ) ³³³³ ) ³³³³ ) GD, mmHg 0.04 ± 0.002 0.52 ± 0.013 0.119 ± 0.001 0.15 ± 0.002 0.13 ± 0.007 ³³³³ ) ³³³³ ) ¹ ) ³³³ ) ³³ ) RI, conv. 1.5 ± 0.013 0.944 ± 0.001 1,052 ± 0,001 1.191 ± 0.001 1.234 ± 0.002 ³³³ ) ³³³³ ) ³³³³ ) ³³³³ ) ESD + L-NAME + Afobazole M, cm / s 2.34 ± 0.01 15.01 ± 0.011 9.668 ± 0.009 5.178 ± 0.098 4,471 ± 0,01 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ¹ ) ⁴⁴⁴⁴ ) ⁴ ) ⁴⁴⁴⁴ ) S, cm / s 10.97 ± 0.007 40.72 ± 0.009 17.13 ± 0.008 21.62 ± 0.009 19.83 ± 0.016 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) D, cm / s 6.22 ± 0.011 2.63 ± 0.009 1.809 ± 0.009 4.34 ± 0.009 5.573 ± 0.016 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ¹¹¹¹ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) PI, cm / s 8.17 ± 0.01 2.71 ± 0.007 1,744 ± 0,007 4,682 ± 0,009 4.46 ± 0.011 ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) GD, mmHg 0.38 ± 0.01 0.61 ± 0.008 0.123 ± 0.001 0.169 ± 0.009 0.14 ± 0.009 ⁴⁴⁴⁴ ) ¹¹¹¹ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴ ) RI, conv. 1.59 ± 0.007 0.91 ± 0.01 1,082 ± 0,006 1.171 ± 0.009 1.221 ± 0.012 ⁴⁴⁴⁴ ) ¹¹¹¹ ) ⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴⁴ ) ⁴⁴⁴ ) Note: P ₁ - relative to ESD + afobazole; P ₂ - relative to ESD; P ₃ - relatively - ESD + L-arginine; P ₄ - relative to ESD + L-NAME

Claims (1)

A method for the correction of oxidative stress and disturbance of the NO producing function of the endothelium in the case of vascular complications of diabetes mellitus in an experiment, comprising a preliminary single administration of a fasting rat intraperitoneally with a 5% aqueous solution of alloxan in a dose of 15 mg / kg of animal weight with the subsequent administration of a drug, characterized in that Afobazole is used as a drug, which is administered under conditions of oxidative stress after an increase in the level of glucose in rat blood, at least twice subcutaneously at 10 mg / kg of body weight once a day for 30 days at the background of daily administration of L-arginine, 10 mg / kg body weight or amid N ^G -nitroarginin methyl ester (L-NAME) - NOS-3 enzyme inhibitor at a dose of 25 mg / kg of animal weight.

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