RU2367451C1 - Regeneration stimulator for cardiac hystiocytes and their endocellular structures and methods of application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: stimulation of physiological or reparative regeneration of cardiac hystiocytes and their endocellular structures is enabled by applying swine adrenal cortex extract. Stimulation of physiological regeneration of cardiac hystiocytes and their nuclei is enabled by 5-week course of swine adrenal cortex extract introduction, every second day in single doses 0.5 ml. Stimulation of or reparative regeneration of cardiac hystiocyte mitochondria in hypobaric hypoxy medium is enabled by double application of swine adrenal cortex extract day before and 30 minutes prior to hypoxy.

EFFECT: effective physiological or reparative regeneration of cardiac hystiocytes.

3 cl, 2 tbl, 6 dwg

Description

The present invention relates to medicine, namely to cardiology, and can be used to stimulate the physiological and reparative regeneration of cardiomyocytes and their intracellular structures (nuclei, mitochondria).

In the future (after conducting appropriate clinical studies), a possible area of use of the proposed group of inventions may be the treatment of dystrophic processes in the muscle of the heart, loss of part of the contractile myocardium and heart failure.

The problem of myocardial metabolism is the most discussed issue in modern cardiology. The prevalence of heart failure (HF) in European countries ranges from 1.5 to 4.0%. Studies in eight regions of Russia (V.Yu. Mareev et al. 2003) showed that the occurrence of such a criterion as shortness of breath plus the presence of any cardiovascular disease in the population was 9.7%. When the criterion was tightened, the occurrence of a triad of symptoms - shortness of breath, fatigue and palpitations - was 5.5%, which is about 2.5 times more than in Western Europe. But if you evaluate even more stringent criteria - shortness of breath, fatigue, palpitations and swelling, i.e. signs of clinically pronounced heart failure - the third or fourth functional class (Ш-1У ФК), then such patients 2.3%.

An assessment of the influence of nosology on the formation of heart failure showed that the primary diagnosis in the vast majority of cases (78%) was a chronic form of coronary heart disease (CHD). But if we consider the relative risk, then it turns out that arterial hypertension (AH) due to prevalence comes in first place. In second place ischemic heart disease, diabetes comes in third place. If we consider patients with severe HF (Sh-1U FC), those who, in addition to shortness of breath, palpitations, fatigue, also have edema (they are only 2.3%), then AH and IHD as risk factors remain the undisputed leaders, and diabetes definitely goes to third place. If we consider the value of the additional role of the population risk of heart failure - the estimated number of cases of severe heart failure (Ш-1УФК) in patients with a particular disease will have heart failure in 80 patients with AH out of 1000 (8%), 5.5% with IHD and 1, 3% with diabetes.

At any stage of the development of heart failure, there are impaired myocardial metabolism. Even with a slight decrease in blood flow, metabolic changes develop that are associated with a decrease in the formation of adenosine triphosphates, impaired oxygen utilization, and an increase in the production of lactate and hydrogen ions.

In chronic heart failure (CHF), the metabolic syndrome includes disorders of carbohydrate and protein metabolism, metabolic disorders of leptin and waste syndrome (L.I. Olbinskaya, 2003). Carbohydrate metabolism disorders are associated with insulin resistance and reflex hyperinsulinemia. This leads to the fact that glucose tolerance is impaired and subsequently diabetes mellitus, usually the second type, can develop. Among the reasons, activation of the sympathoadrenal system is in the first place, and activation of the cytokine system, changes in the leptin system, endothelial dysfunction with impaired production of nitric oxide and impaired formation of bradykinin, as well as morphofunctional changes in organs and tissues, in particular in the pancreas, come first.

Disturbances of protein metabolism are expressed in increased catabolism, protein loss as a result of increased activity of the sympathoadrenal system, increased formation of leptin, activation of the cytokine system and impaired renal function. On the other hand, disturbances in protein metabolism are associated with impaired absorption and protein synthesis, in particular with impaired absorption in the intestine during congestion. Protein metabolism disorders are manifested by progressive weight loss and asthenia, protein-free edema appears, not associated with heart failure. A marked decrease in exercise tolerance is observed, and dysproteinemia is observed in parallel. The content of albumin in plasma decreases sharply, anemia, impaired liver, kidney and immunodeficiency conditions develop.

Leptin is a peptide hormone that is close in structure to pro-inflammatory cytokines, is secreted by adipose tissue cells and circulates in the blood in a free and bound state. It leads to a decrease in food intake, and a decrease in food intake increases leptin activity. An increase in the activity of the sympathoadrenal system also increases the activity of leptin, which can increase energy expenditure, increasing the rate of metabolic processes. Leptin has receptors in the lungs, kidneys, liver, pancreas, adrenal glands and muscles. And the degree of metabolic disturbance, its aggressive effect, depends on the state of these receptors.

The criteria for "waste syndrome" is an unintentional loss of more than 70% of body weight over a period of more than six months, a body mass index of less than 24 kg / m ² , malnutrition and muscle atrophy, atrophy of the temporal muscles, decreased muscle strength. Among the reasons in the first place is the activation of the pro-inflammatory cytokine system, also the activation of the sympathoadrenal system and the associated increase in metabolism, hyperlipidemia, especially leptin hyperresistance, malabsorption in the intestines, malnutrition and concomitant anorexia.

In the treatment of chronic heart failure, a large place is given to drugs that affect tissue metabolism: a decrease in the activity of the sympathoadrenal system, correction of disorders in the nitric oxide system, an effect on the bradykinin system and a decrease in the activity of the cytokine system, as well as the effect on leptin metabolism.

Among the tools that are used to correct impaired carbohydrate metabolism in patients with CHF are maxonidine preparations (physiotens in a daily dose of no more than 1-2 mg), acarbose, which slow down the absorption of carbohydrates from the small intestine by inhibiting the enzyme a-glucosidase.

Violations of protein metabolism are corrected by inhibition of catabolism, which is achieved by using beta-blockers, as well as receptor blockers for tumor necrosis factor-TNF-a (etanercept, remicade). Replenishment of protein losses and violation of the synthetic functions of the liver is carried out using nutritional support (nutrilan, etc.), parenteral administration of albumin. The inclusion of besoprolol contributes to a decrease in TNF-a activity. Impaired leptin metabolism is achieved by the appointment of beta-blockers and angiotensin-converting enzyme (ACE) inhibitors.

Since the "waste syndrome" is associated with an increase in the activity of the sympathoadrenal system and the renin-angiotensi-aldosterone system (RAAS), drugs that interrupt this process are used. These are beta-blockers (carvediol, metoprolol, bisoprolol), ACE inhibitors (perindopril), antioxidants and drugs that improve tissue respiration (hypoxene). To suppress the activity of TNF-a, pentoxifyline (trental), TNF-a receptor antagonists (etanercept) are also recommended.

It is noteworthy that the use of all antihypertensive and enzyme inhibitory drugs for hypertension and coronary heart disease is accompanied by side effects, presented in various combinations and varying degrees of severity. For example, the use of captopril (an ACE inhibitor) may be accompanied by loss of appetite, nausea, vomiting, headache, drowsiness, ataxia, and disturbance of accommodation. There are leukopenia, thrombocytopenia, agranulocytosis, hepatitis, allergic reactions in the form of skin rash, dermatitis, edema, lymphadenopathy, interstitial nephritis, galactorrhea, hypo- or hypertension, gynecomastia.

The use of concor (beta-selective adrenergic blocker) can cause fatigue, dizziness, sensation of flushing, headaches, sleep disturbance, depression, hallucinations, decreased tearing, conjunctivitis, orthostatic hypotension, bradycardia, impaired atrioventricular conduction, in some with increased heart failure the development of peripheral edema, diarrhea, constipation, nausea, abdominal pain, muscle weakness, cramps, itching, excessive sweating, psoriasis-like rashes or Silene existing psoriasis, impaired glucose tolerance, decreased potency.

Cordaflex (calcium channel blocker) can lead to severe arterial hypotension, peripheral edema, tachycardia, rarely - increased angina attacks, paresthesia in the extremities, tremor, sleep disturbance, drowsiness, minor transient visual disturbances, hypertrophic gingivitis, diarrhea, constipation, intrahepatic increased activity of hepatic transaminases, thrombocytopenia, leukopenia, rarely anemia, cause exanthema, skin itching, hyperemia of the face and skin of the upper body, fever, hyperhydro , Myalgia, gynecomastia, decreased libido etc..

In recent years, approaches to myocardial protection have been developed. This is a strategy to suppress fatty acid metabolism and stimulate glucose metabolism. It is achieved by the ingestion of enzymes that carry out the oxidation of fatty acids in mitochondria (mainly trimetazidine); inhibition of enzymes responsible for the transport of fatty acids through mitochondrial membranes (etomoxin); inhibition of the biosynthesis of the carrier of fatty acids through mitochondrial membranes (carnitine). Under conditions of oxygen deficiency, there is a violation of the utilization of fatty acids and an increase in their content.

The most studied specific modulator of fatty acid metabolism is trimetazidine, which prevents the increase in fatty acid oxidation during reperfusion. Today, the mechanism of action of trimetazidine has been established, leading to a decrease in the oxidation of fatty acids and stimulation of glucose oxidation. The result of the cardioprotective effect of trimetazidine is to optimize myocardial function under ischemic conditions by reducing the production of protons and limiting the intracellular accumulation of sodium and calcium. This is an acceleration of the renewal of membrane phospholipids and the protection of membranes from the damaging effects of long chain acyl derivatives.

The effect of trimetazidine has been studied both in the form of monotherapy and combination therapy, including in severe heart failure. 60 mg trimetazidine therapy for two months was accompanied by a significant increase in the ejection fraction and a decrease in the final diastolic volume.

According to Shlyakhto, E.V. (2003), who had been treating patients with heart failure with trimetazidine for three months, the functional class of heart failure decreased from 2.9 to 2.27 compared with the control, and an increase in the ejection fraction was observed. The effects of trimetazidine occurred without significant changes in hemodynamics, without changing blood pressure, the number of heart contractions, realized at the level of the myocardium.

The effectiveness of modern treatment of cardiovascular diseases, accompanied by the loss of part of a workable myocardium (coronary heart disease, myocardial infarction, heart failure), is limited to 30-35% reduction in the risk of death. Further progress in this direction is associated with the development of new approaches to drug treatment, in particular regenerative or cell therapy (Yu.N. Belenkov, F.T. Ageev, V.Yu. Mareev et al., 2003).

The idea of regeneration changed as the opportunities for understanding biological processes expanded, especially with the introduction of electron microscopy. L.V. Polezhaev (1968) referred to regeneration as “restoration of an organism, organ, tissue, cells or part of cells after their damage”. At the same time, A.A. Voitkevich (1968) wrote that “regeneration is the formation of new cellular and subcellular components of an organ”. D.S.Sarkisov (1970) classifies intracellular regenerative and hyperplastic processes as the main forms of regenerative reaction. In his opinion, if the patterns of the regenerative reaction were studied experimentally under conditions close to clinical and not having little in common with them, then you would easily see that with numerous pathological processes in various internal organs the most important, most common and constantly occurring are dystrophic changes in cells and their subsequent disappearance in cases of recovery, i.e. first of all, they would encounter destructive and regenerative processes concentrated inside the cells. Mitotic activity, i.e. cell reproduction, here recedes into the background, since it is not so much about the death of cells, but about one degree or another of their damage, and then restoration. (Regeneration and its clinical significance, 1970, p. 64.) Reparative intracellular regeneration can be expressed not only in the normalization of the structure of dystrophically altered cells, but also in compensatory intensification of the rhythm of intracellular physiological regeneration of ultrastructures. The next form of intracellular reparative regeneration is compensatory hyperplasia of ultrastructures in one cell with the death of another. Finally, another form of reparative intracellular regeneration is compensatory hyperplasia of ultrastructures in case of organ hyperfunction.

Intracellular reparative regeneration is usually observed with diffuse and focal dystrophic changes in muscle fibers that occur with parenchymal and interstitial myocarditis of various etiologies, various toxic effects, hypoxic conditions, nutritional disorders, etc., and in pure form it happens exactly when dystrophic changes do not reach necrotic, i.e. when there is no death of muscle fibers. If the latter also takes place, then intracellular compensatory hyperplasia of ultrastructures joins to one degree or another to intracellular reparative regeneration. Physical activity is accompanied by essentially the same changes in the ultrastructures of the muscle cells of the heart that occur during various pathological processes: swelling of mitochondria, enlightenment of their matrix, partial or significant decrease in the number of their internal partitions, expansion of the tubules of the sarcoplasmic reticulum. Physiological regeneration in extreme situations is approaching reparative regeneration.

The intracellular form of the regenerative reaction is characteristic of the myocardium and is the most appropriate way of material support of various modes of the heart. It is noteworthy that after even a significant volume of the myocardium has decreased, the weight of the heart will soon be restored due to intracellular compensatory hyperplasia of the ultrastructures of the remaining muscle. In organs, which are mainly characterized by an intracellular regenerative reaction (which include primarily the brain and muscle of the heart), structural (nodular) parenchyma restructuring does not occur during the disease.

When analyzing the causes and mechanisms of decompensation of a hypertrophied heart, D.S. Sarkisov suggests keeping in mind three main premises that:

1) myocardial hypertrophy is a special form of a regenerative reaction, as complete in the sense of providing compensation for impaired functions as others, but in contrast to them, concentrated mainly intramuscular fibers;

2) myocardial hypertrophy, in principle, can reach very high degrees and provide the necessary level of cardiac activity for a long time, however, as a rule, this does not happen and the decompensation of the latter occurs long before the myocardium has exhausted its reserve capabilities;

3) factors lying outside the muscle fiber play a crucial role in such a premature onset of heart failure. These include neuroendocrine effects, anatomical changes in blood vessels (atherosclerosis) and the development of cardiosclerosis.

It should be noted that in neurology (as in cardiology with respect to cardiomyocytes) it is generally accepted that brain neurons are not capable of regeneration. However, in recent years there has been an interest in the proliferation of new neurons from precursors in the pancreatic structures of the brain (dentate gyrus and hippocampus), their migration to other regions, which are more pronounced at an early age, but, decreasing, persist throughout life. Adult mammalian dentate gyrus contains precursor nerve cells with multipotential development and self-renewal. The origin and maturation of nerve precursors are determined in accordance with the composition and level of trophic factors in their microenvironment (Kosaka N. et al, 2006).

As for cell therapy, the most advanced methods are currently transplanting autologous myoblasts, as well as stem cells (SC) of bone marrow directly into damaged heart tissue. Skeletal muscle myoblasts are not stem cells. From the very beginning, being highly differentiated cells, myoblasts practically lose their ability to divide and do not self-reproduce (with the exception of 5% of satellite muscle cells). However, after special treatment, the ability of myoblasts to mitosis is restored, at least in vitro. Until 2003, more than 50 of these patients were treated in Europe and the USA. Initially, in all cases, patients showed improvement, increased myocardial contractility. Subsequently, along with clinical improvement, some of the operated on began to experience episodes of life-threatening ventricular arrhythmia associated with this intervention. They began to explain this phenomenon by the formation of transplantation of myoblasts of muscle cell colonies that were not associated with the surrounding tissue and without the corresponding electrical infrastructure.

At the same time (from 2001 to 2003), bone marrow stem cell transplantation was performed in more than 100 patients in Europe, Japan and the USA. Stem cells in an amount of 2 million per infusion were introduced through a catheter into the ischemic zones. There was a general improvement, an increase in the contractility of the myocardium, which is especially noticeable in the segments in which the injections were made, the areas of hypoperfusion were reduced. However, many unresolved issues remain, in particular, the causes of the mass death of transplanted cells, which cellular and genetic mechanisms regulate these processes.

Thus, among the known means there are no such that would directly affect the regeneration of cardiomyocytes and their intracellular structures.

The objective of the invention is the development of tools and methods for stimulating the regeneration of cardiomyocytes and their intracellular structures (nuclei, mitochondria).

The task is achieved by using extracts of pork or fetal adrenal cortex as a means of stimulating the physiological and reparative regeneration of cardiomyocytes and their intracellular structures.

The task is also achieved by a method of stimulating the physiological regeneration of cardiomyocytes and their nuclei, DISTINCTING in that they inject pork or fetal adrenal cortex extract in a single dose of 0.5 ml per 100 g of body weight every other day, with a general course of 5 weeks.

The objective is also achieved by a method of stimulating the reparative regeneration of mitochondria of cardiomyocytes under conditions of hypobaric hypoxia, characterized by the fact that they inject pork or fetal adrenal cortex extract in a single dose of 0.5 ml the day before and 30 minutes before exposure to hypobaric hypoxia.

The possibility of using extracts of pork or fetal adrenal glands as a means of stimulating the regeneration of cardiomyocytes and their intracellular structures is shown in the present invention for the first time. Previously, extracts of pork and fetal adrenal cortex were used for other purposes, namely: to stimulate reparative processes in a pathologically altered liver (RU No. 2185835, C2 07.27.2002); for the correction of acute renal failure in toxic hepatorenal syndrome (RU No. 2290940, C2 10.01.2007); to normalize the energy processes in the tissues of the body under hypoxia (RU No. 2190416, C2 10.10.2002).

In modern approaches to understanding HF, previously established ideas about the most important value of neuroendocrine mechanisms that cause various functional changes in the myocardium and through oxidative stress, ischemia, gene expression induce apoptosis are preserved.

In light of these data, the anabolic effects of minor concentrations of cortacosteroid contained in extracts of the fetal and porcine adrenal cortex on myocardium in experimental animals under hypobaric hypoxia and with long-term (for 5 weeks) parenteral administration are noteworthy.

As you know, the adrenal glands, along with the placenta, play an exceptional role in ensuring intensive intrauterine growth. In the first 5 months, the body length of the fetus is equal to the age in months squared, and in the next 5 months, it is the age in months multiplied by 5. So the body never grows intensively. This is facilitated by the combination of individual hormones, the presence in an increased concentration of dehydroepiandrosterone, which is a weak androgen and a precursor of testosterone and estrogens, which are synthesized from it at the tissue level. Peak blood concentrations of dehydroepiandrosterone reach 25 years, and after 40 years, gradually decreases by 95% by approximately 85 years of age. There are prerequisites for linking longevity and a lower likelihood of death from cardiovascular disease with a high availability of this hormone. Recently, the ability of dehydroepiandrosterone to suppress pro-inflammatory cytokines, such as interleukin-6, tumor necrosis factor-alpha, which increase with the aging of the body and occupy an important place in the pathogenesis of heart failure, has recently been discovered. In addition, dehydroepiandrosterone increases the body's ability to “convert food into energy and burn extra fat” (LeowattanaW / 2001).

Experimental studies on growing rats (Patel MA et all, 2006) showed that at a dose of 1 mg / kg for 5 weeks, dehydroepiandrosterone did not change the gain in body weight, but increased brain mass in postpartum life, stimulated oxidative processes in brain mitochondria, increasing the content of cytochromes (3) and b, dehydrogenase activity and ATPase activity.

Thus, adrenal cortex extracts containing a gamma of hormone-active compounds that have paracrine effects on metabolic processes in tissues can be useful in dystrophic processes in the heart muscle, loss of part of the contractile myocardium and heart failure.

The proposed method was developed in two series of experiments on growing animals. In the first series, the experiment was conducted on 25 growing outbred male rats with a body weight of 99.8 + -3.7 g at the beginning of the experiment and a body length of 12.44 + -0.58 cm. At the end of the experiment, the animals had a weight of 181.7+ -12.7 g and a body length of 14.0 cm. Animals were divided into 5 groups, of which 5 animals were intact (1 control group), 5 animals received a 10% solution of ethanol in physiological sodium chloride solution (2 control group) , 5 animals received hydrocortisone (10 mg / kg) based on a 10% solution of ethyl alcohol (group 3); 5 animals received an extract of the pork adrenal cortex (group 4) and 5 animals received an extract of the bark of fetal adrenal glands taken at autopsy of human fetuses who died in childbirth (group 5).

In another series of studies (18 rats), animals underwent hypobaric hypoxia with a slow rise (at 1000 m per minute) to a height of 10,000 m, staying at that height for 40 minutes and subsequently the same slow lowering (at 1000 m per minute) to normal atmospheric pressure. One day and 30 minutes before hypobaric hypoxia, 3 groups of animals were injected subcutaneously in a volume of 0.5 ml with preparations: fetal adrenal cortex extract, pork adrenal cortex extract, hydrocortisone (10 mg / kg) based on isotonic sodium chloride solution. As a control (1 group), intact (1 group) were used; animals that underwent hypobaric hypoxia without prior administration of drugs (group 2); animals with the preliminary introduction of ethyl alcohol on the basis of physiological solution of sodium chloride amounted to group 3. After returning, the animals were decapitated and their heart was taken for electron microscopy.

Adrenal cortex extracts were prepared according to the principle of organ preparations; 1 ml of extract was obtained from 3.0 g of tissue. Preparations in a volume of 0.5 ml were administered every other day for 5 weeks. The method of radioimmunological analysis in extracts of the adrenal cortex determined the concentrations of hydrocortisone, dehydroepiandrosterone and progesterone. Hydrocortisone — 65 nmol, dehydroepiandrosterone — 0.83 nmol, and progesterone — 1.1 nmol, were contained in 0.5 ml of pork adrenal cortex extract. In 0.5 ml of extract of the fetal adrenal cortex contained hydrocortisone - 5.0 nmol, dehydroepiandrosterone - 1.15 nmol and progesterone - 0.45 nmol. In 0.5 ml of the hydrocortisone solution administered to the animals, 2.6 μmol of the preparation was contained.

After 16 injections of the preparations, the animals were beaten every other day, the heart was paraffinized, and the sections were stained with hematoxylin and eosin. Microscopic measurements were carried out at a magnification of 7 × 90, and using a scale, two core diameters (large and small) and the transverse diameter of the cardiomyocyte were determined. The value of the scale division was determined using a micrometer; one scale division was 16.6 mkm.

When calculating the area of the nucleus, we used the formula

S mkm ² = 3.14 · 0.12 · (a / 2 · 16.6) · (b / 2 · 16.6), where 0.12 is the correction coefficient for the optical medium of the microscope, and is the large diameter, b - small diameter, 16.6 - the value of one division of the scale. The diameters of the cell nucleus were determined by the formula:

d1, d2mkm = 3.14 · 0.12 · a (or b) · 16.6. When measuring mitochondrial diameters, the correction factor (0.12) was excluded from the formula. The increase obtained by electronic photography in the computer was multiplied by 2.5 times.

Cell sizes previously calculated in this way were comparable with the normative data cited by Yu.M. Zhabotinsky in the monograph Normal and Pathological Morphology of the Neuron, 1965, and the sizes of mitochondria of cardiomyocytes with the normative data cited in the histology textbook for students of medical institutes 1983 (Yu. I. Afanasyev, p. 340).

The quantitative indicators of the proposed methods of stimulating regeneration are significant.

Single dose of administered extracts of pork and fetal adrenal cortex

(0.5 ml per 100 g of body weight) does not significantly change the physiological concentrations of hormones in the body and is at least 30 times lower than those used in medical practice in relation to hydrocortisone, which avoids the negative effects of large doses of hormones. As shown by our previous studies, the pork and fetal adrenal cortex extracts administered at such a dose had a positive effect on the regenerative and energy processes in other tissues.

Twice administration of adrenal cortex extracts is due to the fact that a greater stimulus of the body was expected to re-administer the drug, which is also used for vaccination. The administration of adrenal cortex extracts 30 minutes before hypobaric hypoxia was determined by the sufficiency of such a time for absorption of the drug into the blood after subcutaneous administration of it (T.T. Podvigina et al., 2002), which was important for animals exposed to hypoxic stress.

Research data are presented in tables and electronic photographs.

Figure 1 presents a photograph of electron microscopy of the heart of an intact animal (magnification 6 thousand × 2.5).

Figure 2 presents a photograph of electron microscopy of the heart of an animal after hypobaric hypoxia (magnification 13 thousand × 2.5).

Figure 3 presents a photograph of electron microscopy of the heart of an animal after hypobaric hypoxia with preliminary administration of an extract of the fetal adrenal cortex (magnification 6 thousand × 2.5).

Figure 4 presents a photograph of electron microscopy of the heart of an animal after hypobaric hypoxia with preliminary administration of an extract of pork adrenal cortex (an increase of 6 thousand × 2.5).

Figure 5 presents a photograph of electron microscopy of the heart of an animal after hypobaric hypoxia with the preliminary introduction of hydrocortisone. (increase: 7 thousand × 2.5).

Figure 6 presents a photograph of electron microscopy of the heart of an animal after hypobaric hypoxia with the preliminary introduction of a 10% solution of ethyl alcohol in physiological NaCl solution (magnification: 6 thousand × 2.5).

As studies show, in comparison with intact animals (Fig. 1) with hypobaric hypoxia without drug protection (Fig. 2), significant edema of cardiomyocyte structures is observed, swelling and ruptures of mitochondria, fetal adrenal glands (Fig. 3) hypobaric hypoxia proceeded with a small edema of cardiomyocyte , mitochondria are represented in an increased amount, of different sizes and preserved structure, cristae are well represented, and there are no mitochondrial ruptures.

In animals treated with pork adrenal cortex extract (Fig. 4), before the experiment with hypobaric hypoxia, the electron microscopic picture of cardiomyocyte did not differ much from that after administration of the fetal adrenal extract. Intracellular edema, expansion of the endoplasmic reticulum, mitochondria of various sizes are moderately expressed, crista is well represented, there are no ruptures of mitochondria.

Noteworthy is the fundamentally different picture of an electronic photograph of a cardiomyocyte in animals, which were injected with hydrocartisone at a dose of 10 mg / kg weight before climbing to a height (Fig. 5). Mitochondria are enlarged, the pattern of their cristae and myofibrils is smeared, the borders of mitochondria are emphasized (possibly due to a decrease in the synthesis of hexoseaminoglycans that are hydrophilic, and this can partially explain the anti-inflammatory effect when they are used).

It should be noted that a similar picture is presented by electron microscopy in animals that received, instead of adrenal cortex extracts, a 10% alcohol solution in physiological saline (Fig. 6). Ethyl alcohol apparently easily penetrates the mitochondria and, having an osmotic effect, led to swelling of the mitochondria and dehydration of the surrounding structures.

These data suggest that corticosteroids contained in minor concentrations of adrenal cortex extracts mitigate the adverse effects of both hypoxia and ethyl alcohol.

Table 1 draws attention to the increase in both diameters and the area of mitochondria in the group of animals that were injected with hydrocortisone; mitochondrial rounding is also indicated by a decrease in d2 / d1 (1.18). With the introduction of extracts of the fetal adrenal cortex, the length of the mitochondria was longer than the diameter (d2 / d1 = 1.43); with the introduction of extracts of pork adrenal cortex, the ratio of the length to the mitochondrial diameter was slightly smaller, which reflected some rounding and, accordingly, the mitochondrial area was larger (1.389 + -0.505 μm ² ). It should be emphasized that the area of mitochondria after administration of extracts of the fetal adrenal cortex was less increased in comparison with the control, which, in our opinion, reflects the softness of their anabolic effect.

As shown in table 2, after prolonged (5-week) administration of adrenal cortex extracts, the diameter of the cardiomyocyte was significantly increased only in animals treated with adrenal cortex extracts, and to a greater extent after administration of extracts of the fetal adrenal cortex in comparison with pork (p = 0.005) . The core area of the cardiomyocyte was also larger in animals treated with adrenal cortex extracts (p1-4.5 = 0.000), there was a tendency to a larger increase in the core area after administration of the fetal adrenal cortex extract compared to pork (p4-5 = 0.093). The rounding of the core of the cardiomyocyte was presented more after the administration of the extract of pork adrenal glands (d2 / d1 = 3.92), and the lengthening of the core after hydrocortisone (d2 / d1 = 4.8) and the alcohol solution (d2 / d1 = 4.92).

The conducted experimental studies confirm the protective role of minor concentrations of corticosteroids contained in extracts of the fetal and pork adrenal cortex on the heart muscle under hypoxia in contrast to ethyl alcohol and hydrocortisone, presented in therapeutic dosages.

With prolonged use of extracts of the adrenal cortex, cardiomyocytes and their nuclei increase in size, which is associated with the trophic effect of minor concentrations of hormones (in contrast to pharmaceutical concentrations of hydrocortisone) on metabolic processes in cells that ensure their regeneration. The inhibitory effects of dehydroepiandrosterone, progesterone and hydrocortisone on the activity of nuclear transcription factor B (NFkappaB), which has a pro-inflammatory effect through the activation of IL-1,6 and TNF and other cytokines, may also contribute to the anabolic effect of minor concentrations of corticosteroids contained in adrenal cortex extracts. At the same time, low concentrations of dehydroepiandrosterone, progesterone and hydrocortisone suppress the activity of pro-inflammatory cytokines and inhibit the activity of proteasome 26, eliminating the excessive loss of intracellular proteins, in contrast to high concentrations of hydrocortisone, which stimulate gluconeogenesis at the cost of protein waste. It should be noted that the catabolic effect of excess maternal hydrocortisone on the fetus during fetal development is inhibited by the placenta, in which the activity of 11-beta hydroxysteroid dehydrogenase is increased, which inactivates the activity of hydrocortisone, turning it into inactive cortisone, which prevents growth and development retardation (Michael AE et al. 2003); at the same time, violations of the formation and intake of hydrocortisone in the fetus inhibit the growth and differentiation of its organs, especially the lungs, which leads to respiratory distress syndrome in immature infants requiring hydrocortisone in the perinatal period (G.A. Lodygensky et.al. 2005).

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Claims (3)

1. The use of extracts of pork adrenal cortex as a means of stimulating the physiological and reparative regeneration of cardiomyocytes and their intracellular structures 2. A method of stimulating the physiological regeneration of cardiomyocytes and their nuclei, characterized in that the extract of porcine adrenal cortex is introduced in a single dose of 0.5 ml per 100 g of body weight every other day, with a total course of 5 weeks. 3. A method of stimulating the reparative regeneration of mitochondria of cardiomyocytes under conditions of hypobaric hypoxia, characterized in that an extract of pork adrenal cortex is introduced in a single dose of 0.5 ml the day before and 30 minutes before exposure to hypobaric hypoxia.

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