Diagnostic technique and method of endothelial dysfunction correction in vascular complications of alloxan diabetes in experiment

FIELD: medicine.

SUBSTANCE: endothelial dysfunction is diagnosed if observing the twofold increase of a rat's blood glucose level. The technique involves determining antioxidant system values, a concentration of total NO metabolites, eNOS activity, a micro- and macrohaemodynamic status, a MDA concentration and Na+ K+ ATP-ase activity in myocardial and hepatic cell membranes. The values variations enables stating the presence of endothelial dysfunction in alloxan diabetes. Such dysfunction is corrected by the subcutaneous introduction of the preparation Ubiquinone (Coenzyme Q10) 0.11 mcl/100 g of animal's weight once a day for 30 days.

EFFECT: more accurate and reliable diagnosis of the vascular disorders accompanying said type of diabetes, and effective correction of such disorders also provides reproducibility, ease, availability, safety and low cost of performing the experiment.

2 cl, 2 tbl, 6 dwg, 1 ex

 

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

To protect the unsaturated fatty acids of the phospholipids of cell membranes from lipid peroxidation (LPO) in the body there are biological endogenous lipophilic antioxidants: coenzyme Q, and α-tocopherol (vitamin E). Coenzyme Q10occupies a Central place in the antioxidant system. Drug ubiquinone (synonym coenzyme Q10) is a fat-soluble vitamin-like compound from the class of benzoquinoines, actively participates in the activity of all organs and systems in the human body, as the electron carrier in the respiratory chain of mitochondria - the "factory" of energy substrates (ATP) (see Morton R.A., G.M. Wilson, J.S. Lowe et al., 1957). Its essential role in bioenergetics installed within the chemiosmotic theory of energy transport Nobel laureate Peter Mitchell in 1978

Coenzyme Q10it is synthesized in the body from the amino acid tyrosine, with the participation of vitamins In2In3In6In12with folic and Pantothenic acids, some minerals. The constant presence of this compound in human cells at physiological concentrations is necessary. Set the go, that deficiency of coenzyme Q1025% is a prerequisite for the development of pathological conditions, and the deficit by 75% incompatible with life. Being a component of the respiratory chain, involved in one-electron and two-electron transfer, it is found in high concentrations in heart muscle, due to aerobic metabolism in the cardiomyocyte (see Littarru G.P., But L. Folkers K., 1972). With age (after 30 years) the content of ubiquinone in the myocardium is reduced to 1.5-2 times.

Over the last 10-20 years have accumulated information in the literature on effectiveness ubiquinone-coenzyme Q10in the patients with cardiovascular diseases (see Shabalin A.V., Nikitin P., 1999). It is used in patients with congestive heart failure of various etiologies (see kapelko VI, Route E.K., 2002), in coronary artery disease. In rats with streptozotocin-induced diabetes the content of coenzyme Q10in liver mitochondria and the heart is reduced and increased education of flow (MDA) (see J. Kucharska, Braunova Z., Ulicna O., 2000).

Studies of changes in mitochondrial bioenergetics in isolated renal cortical mitochondria in rats with streptozotocin diabetes showed a reduction process of oxidative phosphorylation, decreased activity of the third complex chain of electron transfer. Under these conditions, isolated is by mitochondria produced increased amounts of superoxide (O 2-and showed manifestations of oxidative lesions. This caused post-translational modifications of mitochondrial proteins of eNOS, which plays a pathogenetic role in the damage of the vascular endothelium and functions of visceral organs of oxidative stress in chronic stages of diabetes (see Rosca .G, Mustata .G., Kinter M.L. et al., 2005). These sources indicate that antioxidants (AO), acting selectively on mitochondria, have a corrective effect.

However, the drug ubiquinone-coenzyme Q10not used for correction of endothelial dysfunction and vascular complications of diabetes.

Therefore, it is highly important research activity indicators the FLOOR, the antioxidant system (AOS), the concentration of NO in the blood and cells of the visceral organs, as well as the development of possible ways of correction of violations with the use of mitochondria oriented antioxidant ubiquinone-coenzyme Q10, in experimental diabetes mellitus (EDS). However, it should be noted that in the available literature there are no data to study the effects of ubiquinone-coenzyme Q10biochemical markers of endothelial dysfunction and condition of the visceral organs of the liver and heart in experimental alloxan diabetes.

Closest to the claimed technical re the structure is a method of diagnosis and treatment of nitroaromatic with alloxan diabetes in the experiment, including the modeling of disease and determining the level of glucose in the blood of rats after two to four weeks, as well as the correction method, comprising the introduction of a medicinal product, under conditions of oxidative stress associated with diabetes, four weeks after modeling the disease in a dose of up to 100 g wet weight rats once a day for 30 days (see patent No. 2372898, publ. 20.11.2009,).

The disadvantages of the prototype are narrow Arsenal distribution, the lack of data on the diagnosis and correction of endothelial dysfunction vascular complications alloxan diabetes in experimental models of diabetes mellitus type -1, not high reliability and accuracy assessment of the presence of endothelial dysfunction vascular complications alloxan diabetes.

The technical result is to increase the accuracy and reliability of diagnostics and correction of experimental alloxan diabetes in a state of oxidative stress, repeatability, convenience, availability, security and low cost of experiments on animals.

The technical result is achieved in that in the method for the diagnosis of endothelial dysfunction and vascular complications alloxan diabetes in the experiment, including the modeling of disease and determining the level of glucose is in the blood of rats, according to the invention, when increasing the level of blood glucose at least twice, against oxidative stress determine the indices of the antioxidant system, the total concentration of NO metabolites, eNOS activity and status of micro - and microhemodynamic, the concentration of MDA and the activity of Na+ K+ ATP-ases in the membranes of myocardial cells and the liver and the change of these indicators is judged on the presence of endothelial dysfunction vascular complications alloxan diabetes in animals.

The technical result is also achieved by a method of correction of endothelial dysfunction vascular complications alloxan diabetes in the experiment, including the diagnosis of these complications before and after the introduction of the medicinal product, under conditions of oxidative stress, related to the development of diabetes after modeling the disease in a dose of up to 100 g wet weight rats once a day for 30 days, according to the invention, endothelial dysfunction in vascular complications of diabetes establish a method according to claim 1, and in the quality of drug use ubiquinone-coenzyme Q10that is administered to rats in the amount of 0.11 μl/100 g wet weight.

This method will allow to expand the understanding of the pathophysiological mechanisms of development of angiopathy in chronic alloxan diabetes, predlozhenie angiopathy with alloxan diabetes in experimental animals, increase reproducibility, convenience, accessibility, safety and low cost of experiments on animals.

The essence of the proposed method is confirmed graphically, and by tables, in which figure 1 shows the dynamics of GENDER indicators in erythrocytes, liver and myocardium in the standard, with DPS and treatment ubiquinone-coenzyme Q10, figure 2 - changes in the activity of SOD in erythrocytes and serum in the standard, with DPS and treatment ubiquinone-coenzyme Q10in figure 3 - performance of catalase activity and the concentration of ceruloplasmin in the blood plasma in the standard, with DPS and treatment ubiquinone-coenzyme Q10, figure 4 - indicators of the activity of Na+, K+- ATP-ases in the liver and myocardium in the standard, with DPS and treatment ubiquinone-coenzyme Q10, figure 5 is the total amount of NO metabolites in the standard, with DPS and treatment ubiquinone-coenzyme Q10, figure 6 - the nature of the changes of flow in rats in the standard, with DPS and treatment ubiquinone-coenzyme Q10.

Table 1 - dynamics of indicators microhemodynamic in experimental diabetes mellitus in table 2 - dynamics of indicators microhemodynamic in experimental diabetes mellitus on the background of corrective therapy ubiquinone-coenzyme Q10.

A method for the diagnosis and correction of endothelial dysfuncti the vascular complications of diabetes in the experiment is as follows.

To defeat insulinogenic β-cells of the islets of Langerhans experimental diabetes (alloxan) caused by intraperitoneal administration of 5% aqueous solution of alloxan (synthesized in the laboratory of the Department of pathobiochemistry, IBMI WITZ RAS and RNO-a) at the dose of 15 mg/kg of body weight of the animal on an empty stomach, on the background of the 24-48 hour fast with free access to water. Within 48-72 hours of fasting took blood from the tail (microcaecilia) and was determined by the glucose glucose oxydase method (test sets). With the increase of blood sugar in 2 times determined the indices of the antioxidant system, the state of micro - and microhemodynamic, as well as the total concentration of NO metabolites and the activity of eNOS in conditions of oxidative stress, and the change of these indicators, judged the presence of endothelial dysfunction vascular complications of diabetes mellitus in animals.

For the correction of violations FLOOR, AOC, indicators of diabetic angiopathy during the month of rats with experimental diabetes mellitus subcutaneously every 14-21 days after the introduction of a 5% aqueous solution of alloxan introduced ubiquinone-coenzyme Q10at a dose of 0.11 μl/100 g body weight.

On expiry of the period of the experiment studied perfusion at different points locations tissues (fluid exchange) - testing sensor 10 MHz, operating on the principle of "blind" Doppler at narcotizes the bathrooms animals. Then the rats were killed under hypentelium anesthesia; took blood from the heart to use as anticoagulant of 2.8% solution of EDTA to determine the concentration of malondialdehyde (MDA) and serum to determine the activity of SOD, catalase, ceruloplasmin, and NO. Extracted liver, heart to determine the tissue homogenates activity of Na+, K+- ATP-ases, intensity FLOOR. In the endothelium of the aorta was determined by the activity of eNOS.

Example. Studies were performed on rats male Wistar rats weighing 190-210 grams found on the standard diet with free access to water and natural light regime in the spring season. Experimental diabetes mellitus characterized by insulin deficiency caused by intraperitoneal administration of 5% aqueous solution of alloxan at a dose of 15 mg/kg of body weight on an empty stomach in the background 24-48 hour fast with free access to water. Within 48-72 hours of fasting took blood from the tail (microcaecilia) and was determined by the glucose glucose oxydase method (test sets). With the increase of blood sugar in 2 times, determined indicators of the antioxidant system, the state of micro - and microhemodynamic, as well as the total concentration of NO metabolites and the activity of eNOS in conditions of oxidative stress, and the change of these indicators (see figure 1-6), judged cash is chii endothelial dysfunction and vascular complications of diabetes mellitus in animals.

For the correction of violations FLOOR, AOC, flow was introduced through 14-21 day to rats with experimental diabetes mellitus in the month subcutaneously ubiquinone-coenzyme Q10at a dose of 0.11 μl/100 g body weight.

After a period of introduction of the investigated indicators, flow, GENDER, antioxidant activity of Na+, K+- ATP-ases in the myocardium and liver tissue.

In rats with DPS develops oxidative stress, which leads to reduced production of NO by endothelial cells, or accelerated decay, turning into peroxynitrite. The data showed that in rats with EDS, the total concentration of NO metabolites were statistically significantly reduced compared with control animals, which was accompanied by the predominance of vasoconstrictor influences (see figure 5). This reflected increased vascular tone and impaired microcirculation, as evidenced by hemodynamic data presented in this table. These perfusion of tissues showed that when DPS all locate points revealed a decrease in the average velocity of flow (M) due to the reduction of systolic blood flow velocity (S), with increasing diastolic blood flow velocity (D). Geographical indicators are characterized by higher values of the index Gosling, reflecting the increase in the elastic properties (density) of the vascular wall and decrease the pressure gradient in the microcirculatory vessels. The index Pursel (eographically index RI), which reflects the specific peripheral vascular resistance (UPSS), in animals with DM rats was statistically significantly higher than that in the control (see Fig.6). These results confirms the prevalence vasoconstrictor effects in the microvasculature due according to our data, the decrease in the content of metabolites of nitric oxide in the blood (with 51,07±0,53 to 32,54±1,42) in diabetic rats. The result of endothelial dysfunction impaired flow in experimental animals in the chronic phase alloxan diabetes is accompanied by a statistically significant reduction in the activity of an ion-transporting enzyme, Na+, K+- ATP-ases in tissue homogenates of liver and myocardium (see figure 4). For the correction of excess process FLOOR, disturbances of antioxidant defense system and activity of Na+, K+- ATP-ases in liver tissue and cardiac muscle in rats with DPS during the month was introduced ubiquinone-coenzyme Q10at a dose of 0.11 μl/100 g body weight. The drug was accompanied by a significant decrease in the concentration of MDA in erythrocytes in the tissue homogenates of the liver and myocardium (see figure 1). On the background of the introduction of the drug increased the activity of antioxidant enzymes. The activity of SOD in serum and erythrocytes almost recovered to ur is VNA control (see 2), and catalase - statistically significantly decreased, but has not reached the same level as the control animals (see figure 3). Conducted correlation analysis to determine the effectiveness of antioxidants on peroxidation processes and antioxidant defence enzymes. Shows the presence of a strong direct correlation between the concentration of MDA and catalase activity (r=+0.64, p<0.001), and a strong negative relation between the level of decrease in the concentration of MDA and increased the activity of SOD (r= - 0.54; p=0.03). Introduction to animals with DPS drug Ubiquinone compositum was accompanied by increased activity of the Na, K - ATP-ASE in the cortex and the medulla renal tissue, hepatocytes and cardiomyocytes. This contributed to the normalization of the lipid microenvironment of the enzyme results in ingibirovaniya FLOOR in the membranes of cells of the visceral organs, increasing the efficiency of oxygen in the chain of electron transport and energy production. Drug - ubiquinone-coenzyme Q10positive influence on the functional activity of vascular endothelium, has increased the concentration of NO metabolites in the serum of animals with DPS during the month and the increase of SOD activity (r= - 0.54; p=0.03). Introduction to animals with DPS drug Ubiquinone compositum was accompanied by increased activity of the Na, K - ATP-ASE in the hepatic and myocardial parodentium is. This contributed to the normalization of the lipid microenvironment of the enzyme as a result of inhibition FLOOR in the membranes of cells of the visceral organs, increasing the efficiency of oxygen in the chain of electron transport and energy production. Drug - ubiquinone-coenzyme Q10positive influence on the functional activity of vascular endothelium, has increased the concentration of NO metabolites in the serum of animals with DPS, within one month treated with this drug. Correction of endothelial dysfunction drug ubiquinone-coenzyme Q10that was accompanied by a statistically significant decrease in specific peripheral vascular resistance and elastic properties of the vessels of the microvasculature and increase perfusion, because of the increasing average and systolic flow velocity (table 2). Correlation analysis showed a negative correlation of medium strength between the concentration of NO metabolites and velocity of blood flow (r=-0.41; p=0.03).

The use of the proposed method will allow for a comparison with the prototype to improve the accuracy, the reliability of the diagnostic concentration of MDA in the liver cells and heart, AOS activity according to SOD in erythrocytes and serum catalase and ceruloplasmin in plasma concentration of total metabolites of NO and activity of eNOS, as well as to increase the power of the treatment with ubiquinone-coenzyme Q 10, reproducibility, convenience, accessibility, safety and low cost of experiments on animals.

Table 1 and 2
Dynamics of indicators of microhemodynamic normal, in experimental diabetes and in the background of corrective therapy ubiquinone-coenzyme Q10.(M±M)
Point locationMSDPIGDRI
The average blood flow velocitySystolic blood flow velocityDiastolic blood flow velocityIndex GoslingThe pressure gradientGeographical index
cm/scm/scm/swith the/s mm HgWed
CONTROL
BA13,46838,8342,0832,8140,5480,948
±0,473±1,223±0,232±0,155±0,020±0,011
NIP9,42015,8681,2231,692amount of 0.1181,042
±0,440±1,754±0,137±0,121±0,008±0,008
PA left5,05220,0194,6385,0360,1481,191
±0,304±0,880±0,415±0,297±0,012±0,029
PA right4,37719,5995,6514,830to 0.1271,24
±0,402±1,020±0,521±0,467±0,011±0,044
ON the BACKGROUND of EXPERIMENTAL DIABETES mellitus
BA15,7101111)40,2922,951111)2,8040,6511111)0,932
±0,518±0,855±0,372±0,149±0,018±0,010
NIP9,87118,2042,1621111)1,7930,1241,121111
±0,426±0,544±0,219±0,092±0,008±0,013
PA left5,30822,5911) 4.09 to1111)3,7461111)0,21111)1,145
±0,245±0,500±0,303±0,259±0,010±0,021
PA right4,94620,0255,4953,9730,151)1,207
±0,276±0,485±0,378±0,282±0,008±0,029
ON the BACKGROUND of CORRECTIVE THERAPY UBIQUINONE-COENZYME Q10.
BA14.07222)38,92.172 2,810,57222)0,937
±0,36±1,31±0,14±0,09±0,023±0,01
NIPto 9.5716,922)1,222)1,7140,1191,572222)
±0,029±0,72±0,11±0,086±0,009±0,009
PA left5,1420,8222)4,474,75222)0,1622) 1,18
±0,27±0,32±0,39±0,28±0,013±0,034
PA right4,5619,785,584,5122)0,1321,23
±0,35±0,62±0,46±0,35±0,009±0,031
Note: BA - abdominal aorta, LEL - lower Vena cava, PA - renal artery (right and left)1111) - p<0,001;111) - p<0,01;11) - R<0,02;1) - p<0.05 relative accuracy control2222) - p<0,001;222) - p<0,01;22) - R<0,02;2) - p<0.05 relative accuracy of experimental diabetes.

1. Method for the diagnosis of endothelial dysfunction in vascular complications is the second alloxan diabetes in the experiment, including the modeling of disease and determining the level of glucose in the blood of rats, characterized in that with the increase of glucose level in blood of rats, at least twice, against oxidative stress determine the indices of the antioxidant system, the total concentration of NO metabolites, the activity of eNOS, the state of micro - and microhemodynamic, the concentration of MDA and the activity of Na+ K+ ATP-ases in the membranes of myocardial cells and the liver, and the change of these indicators is judged on the presence of endothelial dysfunction in vascular complications alloxan diabetes in rats.

2. Method of correction of endothelial dysfunction in vascular complications in the face of alloxan diabetes in the experiment, including the diagnosis of these complications before and after the introduction of the medicinal product, under conditions of oxidative stress, in doses up to 100 g wet weight rats once a day for 30 days, characterized in that the endothelial dysfunction in vascular complications of diabetes establish a method according to claim 1, and in the quality of drug use ubiquinone-coenzyme Q10that is administered to rats in the amount of 0.11 μl/100 g wet weight.



 

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FIELD: medicine.

SUBSTANCE: acne surface is pre-disinfected in the aseptic environment with 2% chlorhexidine or 96° ethanol to prepare 3-4 imprinted smears by attaching a sterile slide to a pathological centre with covering an apparently health skin area and applying a pathological acne secretion on the slide with using a tampon. The prepared imprinted smears are air dried, fixed in 96° ethanol or above an alcoholic lamp and stained in methylene blue by Romanowsky-Giemsa and Gram wherein fuchsin is replaced by neutral red. If the cytogram shows no inflammatory reaction by the 6-7th day from the beginning of treatment: observing single neutrophilic leukocytes with no destruction, fibroblasts and polyblasts (8.87% and 21.8%, respectively), the absence of microflora and the presence of epithelisation process - a plenty of epithelium cells, the absence of slime, the therapy is assessed to be effective.

EFFECT: more accurate assessment of controlling clinical effectiveness in acne that enables well-timed therapeutic correction.

2 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to traumatology and orthopedics, and can be applied for optimisation of knitting of extremity bones in case of fractures, unknitted fractures or false joints. For this purpose realised is stable osteosynthesis, which includes reposition of bone fragments and their stable immobilisation. After that on 8 - 10 day after operation into the area of bone defect introduced is nitroglycerine in dose to 10 mg, diluted in 2 ml of physiological solution. Immobilisation of bones of damaged segment is preserved until their complete consolidation.

EFFECT: method ensures maximal vasodilatation effect in the zone of injury and due to it acceleration of terms of fracture consolidation and their consequences.

10 dwg, 2 ex

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