BACKGROUND:Brain-derived neurotrophic factor (BDNF) mediates opiate dependence phenomenon. In the brain of morphine dependent animals BDNF level is controlled transcriptionally, however, post-transcriptional mechanisms of BDNF regulation in this context remain unknown. Regulation of mRNA by binding of specific proteins to the 3'-untranslated region (3'-UTR) is one of such mechanisms. Among RNA-binding proteins neuronal Hu antigen D (HuD) is the best characterized positive regulator of BDNF, however its involvement in opiate dependence remains obscure. We suggested that HuD binding to the BDNF 3'-UTR may be linked to changes in BDNF expression induced by morphine. The aim of this study was to investigate potential association of HuD with BDNF 3'-UTR in relation to BDNF expression (Exon- and 3'-UTR-specific mRNA variants and protein level) in the frontal cortex and midbrain of male Wistar rats after chronic morphine intoxication and spontaneous withdrawal in dependent animals.RESULTS:After chronic morphine intoxication but not during morphine withdrawal HuD binding to the long BDNF 3'-UTR in the frontal cortex decreased as compared with the corresponding control group, however after intoxication BDNF expression did not change. The level of BDNF Exon I as well as mature BDNF polypeptide increased in the frontal cortex upon morphine withdrawal, while no changes in HuD binding could be detected.CONCLUSION:Thus, contrary to the assumption, HuD-BDNF 3'-UTR interaction and BDNF expression in the frontal cortex differentially change in a manner dependent on the context of morphine action.

Opiate reinforcement is considered as a stimulus inducing addiction, however underlying neurobiological mechanisms remain obscure. According to the literature, BDNF (brain-derived neurotrophic factor) in the lateral ventral tegmental area (VTA) could modulate morphine reinforcement, but the role of BDNF in the midline VTA has not been studied yet. The aim of the study was to investigate the effect of a single intra-midline VTA injection on the acquisition and expression of morphine conditioned place preference (CPP). CPP procedure was composed of eight conditioning sessions (one session per day): morphine (i.p., 10 mg/kg) and saline injections were paired to the compartments and counterbalanced. Recombinant human BDNF (0.75 ug) or phosphate-buffered saline (PBS) as a vehicle were injected once into the midline VTA one day before or after conditioning. According to the CPP test rats spent more time in the morphine-paired compartments a scompared to the saline-paired compartments (p < 0.05). After a single BDNF injection into the midline VTA before conditioning, but not after conditioning, differences in time spent in morphine- and saline-paired compartments did not reach significance (p > 0.05). Thus, taking into account limitations of the results, we suggest that BDNF in the midline VTA may block morphine reinforcement.

Разработан скрининг-тест для определения множественно-модифицированных липопротеинов низкой плотности (ммЛПНП) в сыворотке крови для рутинных исследований. Инкубация сыворотки в буфере, содержащем 9,1% поливинилпирролидона 35000 (ПВП-35) приводит к полной агрегации ммЛПНП. Патогенность выделенных ммЛПНП подтверждена в тестах связывания системы комплемента и литической активности in vitro , а также в тесте сосудистой дистонии у крыс in vivo . Предлагаемый способ отличается высокой производительностью и низкой себестоимостью. Метод включает 2 операции - смешивание сыворотки с раствором 10% ПВП-35 в 96-луночных иммунологических планшетах, инкубация в течение 10 мин при комнатной температуре и турбидиметрия при 450 нм на ИФА-ридере. Проведены исследования взаимосвязи концентрации ммЛПНП в сыворотке крови и толщины комплекса интима-медиа по данным ультразвукового дуплексного сканирования сонных артерий у 78 человек с низким сердечно-сосудистым (SCORE <1%) и низким кардио-метаболическим рисками (CMDS 0-1). Из всех фракций липидного спектра крови ммЛПНП показали самый высокий коэффициент корреляции с показателями субклинического атеросклероза, что подтверждает высокий атерогенный потенциал ммЛПНП. A screening test was developed to determine multiply-modified low-density lipoprotein (mmLDL) in serum for routine studies. Incubation of serum in a buffer containing 9.1% polyvinylpyrrolidone 35,000 (PVP-35) results in complete aggregation of mmLDL. Pathogenicity of isolated mmLDL was confirmed during binding tests of the complement system and lytic activity in vitro , and also in vascular dystonia test with rats in vivo . The proposed method is characterized by high productivity, low cost, includes 2 operations - mixing serum with a solution of 10% PVP-35 in 96-well immunoassay plates, incubating for 10 min at room temperature and turbidimetry at 450 nm on an ELISA reader. A study was made to determine relationship between concentration of mmLDL in the blood serum and thickness of the intima-media complex from data of ultrasound duplex scanning of carotid arteries with 78 people with low cardiovascular risk (SCORE <1%) and low cardiovascular risk (CMDS 0-1). Of all fractions of the lipid spectrum of the blood, mmLDL showed the highest correlation coefficient with the indices of subclinical atherosclerosis, which confirms high atherogenic potential of mmLDL.

Цель: В связи с ролью оксидативного стресса в патогенезе ишемических повреждений головного мозга изучить влияние препарата мелаксена (химический аналог гормона мелатонина) на активность антиоксидантных ферментов и некоторых ферментов окислительного метаболизма, способных лимитировать свободнорадикальные процессы при ишемии. Методика. В качестве объекта исследования использовали самцов белых лабораторных крыс. Индуцирование ишемии головного мозга у животных опытных групп осуществляли путем 30-минутной окклюзии общих сонных артерий, реперфузии достигали снятием окклюзоров. в головном мозге и сыворотке крови крыс изучали активность глутатионпероксидазы, глутатионредуктазы и глутатионтрансферазы, содержание восстановленного глутатиона, активность глюкозо-6-фосфатдегидрогеназы и НАДФ-изоцитратдегидрогеназы, способных выступать в роли поставщиков НАДФН для работы глутатионовой антиоксидантной системы. Активность ферментов и концентрацию восстановленного глутатиона определяли спектрофотометрически. Результаты. При действии мелаксена выявлено восстановление активности ферментов и уровня восстановленного глутатиона до значений близких к таковым у ложнооперированных животных. Полученные результаты могут быть объяснены с точки зрения торможения свободнорадикальных процессов за счет реализации антиоксидантных и нейропротекторных свойств мелатонина на фоне развития оксидативного стресса в условиях нарушения мозгового кровообращения, что приводит к снижению степени мобилизации антиоксидантной системы и некоторых ферментов окислительного метаболизма, которые, по-видимому, при реализации адаптивного ответа действуют как единая система. Заключение. Полученные результаты могут служить обоснованием дальнейшего исследования возможности применения мелатонин-корригирующих средств для фармакологической коррекции изменений метаболизма при развитии патологий подобного рода. Due to the involvement of oxidative stress in brain ischemic damage and to antioxidant properties of the hormone melatonin, it was relevant to study the effect of melatonin on antioxidant enzymes and some enzymes of oxidative metabolism that limit free radical processes in pathology. The aim was to study the effect of melaxen, a melatonin analogue, on activities of glutathione peroxidase, glutathione reductase, and glutathione transferase, content of reduced glutathione, and activities of glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase, which can supply NADPH to the glutathione antioxidant system, in the brain and blood serum of rats with cerebral ischemia/reperfusion. Methods. White male rats were used in the study. Cerebral ischemia was induced by 30-min occlusion of common carotid arteries; reperfusion was induced by removing the occlusion. Activities of enzymes and concentration of reduced glutathione were measured spectrophotometrically. Results. Melaxen reversed the increased enzyme activities and the reduced glutathione level induced by the pathological conditions returning them to the control values. This can be explained by inhibition of free radical processes under the action of the antioxidant melatonin and its neuroprotective effect in oxidative stress associated with disorders of cerebral circulation. The result is decreased mobilization of the antioxidant system and some enzymes of oxidative metabolism, which act as a single system in the adaptive response. Conclusion. The study justified further investigation of the possibility for using melatonin-correcting agents for pharmacological correction of metabolic changes in such pathologies.

A review of recent data on the role of the multifunctional enzyme, associated with high density lipoproteins - paraoxonase 1 (PON1) in maintaining healthy endothelial function by detoxifying both oxidized low density lipoproteins and homocysteine thiolactone. The additional contribution to the protection of the endothelium against damage makes organophosphatase activity of PON1 involved in the detoxification products of tobacco smoke. The reduction of antioxidant activity of PON1 promotes the differentiation of monocytes into macrophages and the development of inflammation. The reduction of thiolactonase activity of PON1 is accompanied by a decrease of methionine re-synthesis from homocysteine causing DNA- hypomethylation and alteratioin of the expression patterns of pro- and anti-atherogenic genes. Global hypomethylation of the genome is regarded as one of the three most important mechanisms of the increased risk of somatic complications of alcoholism. The accumulation of homocysteine thiolactone serving agonist of glutamate receptors and antagonist of dopamine receptors is a prerequisite to increased alcohol abuse. Clinical observations focusing on gene polymorphisms of PON indicate that three different genotypes of polymorphism PON1Q192R have unequal degrees atheroprotective properties.

Brain-derived neurotrophic factor (BDNF) is involved in the formation of dependence on opiates. BDNF mRNA expression is altered during opiate dependence; however, the biological basis of this phenomenon has been insufficiently studied. At the posttranscriptional level, microRNAs may potentially regulate BDNF expression. Short non-coding microRNAs bind in a complementary manner, mostly to the 3'-noncoding area (3'-UTR) of mRNA and thus initiate degradation of a target by the RNA-induced silencing complex (RISC). In the present study, the potential contribution of microRNAs to the regulation of the BDNF mRNA expression was evaluated in the brain of rats with morphine dependence after spontaneous withdrawal. In order to form dependence in rats, morphine was injected twice a day in increasing doses of 10᾿00 mg/kg for 6 days. Expression of BDNF mRNA and microRNA, which may potentially regulate BDNF, was studied in brain regions 40 h after spontaneous morphine withdrawal using the real-time PCR method. Interaction of the Argonaute 2 protein (Ago-2), a component of RISC, with 3'-UTR BDNF mRNA was evaluated using RNA immunoprecipitation. We found that morphine withdrawal was followed by an increase in the BDNF exon I-containing mRNA in the frontal cortex and midbrain. In the frontal cortex, the increased BDNF mRNA level observed in abstinence was associated with a decrease in the miR-206 microRNA and Ago-2 bound to 3'-UTR BDNF mRNA contents. In the midbrain the abstinence condition was accompanied by a decrease in the miR-382 level, whereas Ago-2 binding to 3'-UTR BDNF mRNA remained unchanged. We believe that the weakening of miR-206-mediated degradation of BDNF mRNA may be responsible for the elevated BDNF expression in the frontal cortex in the conditions of morphine dependence.

Nitric oxide (NO) mediates pharmacological effects of opiates including dependence and abstinence. Modulation of NO synthesis during the induction phase of morphine dependence affects manifestations of morphine withdrawal syndrome, though little is known about mechanisms underlying this phenomenon. Neurotrophic and growth factors are involved in neuronal adaptation during opiate dependence. NO-dependent modulation of morphine dependence may be mediated by changes in expression and activity of neurotrophic and/or growth factors in the brain. Here, we studied the effects of NO synthesis inhibition during the induction phase of morphine dependence on the expression of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF1) as well as their receptors in rat brain regions after spontaneous morphine withdrawal in dependent animals. Morphine dependence in rats was induced within 6 days by 12 injections of morphine in increasing doses (10–100 mg/kg), and NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) (10 mg/kg) was given 1 h before each morphine injection. The expression of the BDNF, GDNF, NGF, IGF1, and their receptors in the frontal cortex, striatum, hippocampus, and midbrain was assessed 40 h after morphine withdrawal. L-NAME treatment during morphine intoxication resulted in an aggravation of the spontaneous morphine withdrawal severity. Morphine withdrawal was accompanied by upregulation of BDNF, IGF1, and their receptors TrkB and IGF1R, respectively, on the mRNA level in the frontal cortex, and only BDNF in hippocampus and midbrain. L-NAME administration during morphine intoxication decreased abstinence-induced upregulation of these mRNAs in the frontal cortex, hippocampus and midbrain. L-NAME prevented from abstinence-induced elevation of mature but not pro-form of BDNF polypeptide in the frontal cortex. While morphine abstinence did not affect TrkB protein levels as well as its phosphorylation status, inhibition of NO synthesis decreased levels of phosphorylated TrkB after withdrawal. Thus, NO signaling during induction of dependence may be involved in the mechanisms of BDNF expression and processing at abstinence, thereby affecting signaling through TrkB in the frontal cortex.

We conducted a comparative study of content proinflammatory cytokines, biomarkers of inflammatory process, biochemical indicators of congestive heart failure (CHF) and hemodynamic parameters in patients with alcoholic cardiomyopathy (ACMP) and ischemic heart disease (IHD) with various NYHA classes. We examined 62 men with ACMP (n = 45) and IHD (n = 17) and NYHA class III-IV CHF. Patients of both groups had lowered ejection fraction (EF), dilated cardiac chambers, and increased left ventricular (LV) myocardial mass index (MMI). Relative LV wall thickness was within normal limits but in the ACMP group it was significantly lower than in IHD group what corresponded to the eccentric type of myocardial hypertrophy. Higher NYHA class was associated with lower EF and larger end diastolic and end systolic LV dimensions. In ACMP it was also associated with larger dimension of the right ventricle while in IHD--with substantially larger (by 30%) dimension of atria. Substantial amount of endotoxin found in blood plasma of patients with IHD corresponded to the conception of increased intestinal permeability of in CHF. Alcohol abuse was an aggravating factor of endotoxin transmission and its concentration in patients with ACMP was 3 times higher than in patients with IHD. Patients with ACMP had substantially elevated blood concentrations of interleukins (IL) 6, 8, 12, tumor necrosis factor α (TNF-α), and its soluble receptor s-TNF-R; they also had twofold elevation of C-reactive protein concentration. ACMP was associated with manifold rise of blood content of brain natriuretic peptide (BNP). Patients with IHD also had elevated blood concentrations of IL 6, 8 and 12 but their values were 1.5-2 times lower than ACMP group. Blood content of TNF-α and s-TNF-R in IHD group was within normal limits. Higher NYHA class in ACMP patients was associated with higher concentrations of IL 6 and 8, TNF-a, and BNP. In both groups of patients contents of IL-12, s-TNF-R, TGF-1β and factors of acute phase of inflammation did not reflect severity of CHF. Functional insufficiency of myocardium in IHD patients was best characterized by blood content of IL-6 while in ACMP patients--of BNP.

In the serum of patients with alcoholism with varying degrees of severity of liver fibrosis were studied the content markers of fibrosis, endothelial dysfunction and proinflammatory cytokines. Concentration in blood indicators of fibrogenesis--collagen type 4, hyaluronic acid, TIMP-1, TIMP-2, YKL-40 and MMP-2 is considerably increased at the 4 degree of fibrosis and moderately increased at low and zero degrees of liver fibrosis. Similar results were obtained in respect of proinflammatory cytokines Il-6, IL-8, IL-12/p70 and IL-12/p40. The magnitude of endothelial dysfunc- tion, calculated based on its content in the blood markers--VEGF-A, MCP-1, s-VCAM, s-ICAM and endothelin, was maximal at 4 degrees of fibrosis and less pronounced at low degrees of fibrosis. Correlations between of the average degree of fibrosis, endothelial dysfunction, and blood levels of proinflammatory cytokines were installed. Close relation between the immune cells releasing stimulators of inflammation and fibrogenesis, perisinusoidal fat cells producing collagen, and endothelium secreting vasoconstrictors in the pathogenesis of alcoholic liver fibrosis and cirrhosis was installed.

Brain-derived neurotrophic factor (BDNF) is believed to play a crucial role in the mechanisms underlying opiate dependence; however, little is known about specific features and mechanisms regulating its expression in the brain under these conditions. The aim of this study was to investigate the effects of acute morphine intoxication and withdrawal from chronic intoxication on expression of BDNF exon I-, II-, IV-, VI- and IX-containing transcripts in the rat frontal cortex and midbrain. We also have studied whether alterations of BDNF exon-specific transcripts are accompanied by changes in association of well-known transcriptional regulators of BDNF gene-phosphorylated (active form) cAMP response element binding protein (pCreb1) and methyl-CpG binding protein 2 (MeCP2) with corresponding regulatory regions of the BDNF gene. Acute morphine intoxication did not affect levels of BDNF exons in brain regions, while spontaneous morphine withdrawal in dependent rats was accompanied by an elevation of the BDNF exon I-containing mRNAs both in the frontal cortex and midbrain. During spontaneous morphine withdrawal, increased associations of pCreb1 were found with promoter of exon I in the frontal cortex and promoters of exon I, IV and VI in the midbrain. The association of MeCP2 with BDNF promoters during spontaneous morphine withdrawal did not change. Thus, BDNF exon-specific transcripts are differentially expressed in brain regions during spontaneous morphine withdrawal in dependent rats and pCreb1 may be at least partially responsible for these alterations.

A method for determining atherogenicity of the immune complexes containing multiple modified low-density lipoprotein (mmLDL) in complement fixation test has been found. In the proposed method, the precipitate immune complexes containing mmLDL (IC mmLDL) was prepared from human serum by treating it with buffer (8.3% of th PEG 3350 and 3.3% PVP 12600 th in the ratio 1: 1.2) for 10 min at 23 °C. IC mmLDL aggregates were separated by centrifugation at 3100g for 10 min at 23 °C. The precipitate IC mmLDL was dissolved in buffer without PEG and PVP, cholesterol content and the degree of binding of guinea pig complement were measured. Atherogenicity of the IC mmLDL was registered as the ratio of the degree of complement binding to cholesterol in the the immune complexes.

Parameters reflecting oxidative stress (OS) have been studied in 37 patients with alcoholic liver disease (ALD) during admission to the hospital and 2 weeks after the beginning of therapy. The patients were divided into 3 groups: alcoholic hepatitis (AH), alcoholic cirrhosis with hepatic insufficiency (the group C by the Child-Paquet scale) and terminal stage patients (subsequently died). All patients were characterized by a significant increase in plasma products of lipid peroxidation (conjugated diene and malondialdehyde) and a decrease of the ceruloplasmin level. The coefficient C OS significantly exceeded normal values both on admission and after the 2-week course of traditional therapy. This suggests an important role of the OS with ALD.

Цель работы - оценка роли каскада окиси азота (NO)/циклический гуанозинмонофосфат (цГМФ) в механизмах формирования зависимости от морфина. Для формирования физической зависимости морфин вводили крысам интраперитонеально дважды в день в течение шести суток в возрастающих дозах - от 10 до 100 мг/кг. При оценке роли каскада NO/цГМФ в формировании зависимости ингибитор NO-синтазы метиловый эфир L-N G -нитроаргинина (L-NAME) вводили интраперитонеально в дозе 10 мг/кг за 1 ч перед каждой инъекцией морфина. Введение L-NAME при интоксикации морфином приводило к усилению проявлений спонтанного синдрома отмены, что сопровождалось более выраженным снижением уровня цГМФ в среднем мозге и стриатуме. Предполагается, что регионспецифическое снижение активности сигнального каскада NO/цГМФ в головном мозге может являться одним из механизмов, определяющих формирование опийной зависимости.

AIM:To estimate the contribution of immuno-inflammatory changes to the formation of clinical and hemodynamic features in alcoholic patients with chronic heart failure (CHF).SUBJECTS AND METHODS:Forty-five males with CHF in the presence of alcohol-induced heart damage (AIHD) who had been admitted to therapeutic units for decompensated heart failure were examined. A control group consisted of 20 men with the CHF severity comparable with the NYHA classification in the presence of prior myocardial infarction. All the patients underwent examination of the immune-inflammatory status--the cytokines: interleukin (IL) 6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1, endotoxin, cellular immune parameters, and cardiac structure and function by echocardiography.RESULTS:The patients with CHF in the presence of AIHD, as compared to those with ischemic cardiomyopathy, showed the higher levels of inflammatory cytokines (IL-6, TNF-alpha, IL-12, and endotoxin) and cell-mediated immunity changes (the smaller count of suppressor T cells, natural killer cells, and a shift of the T-helper/T-suppressor ratio towards the T-helper population). The magnitude of these changes correlated with the severity of CHF and cardiac morphofunctional changes.CONCLUSION:The relationship of immuno-inflammatory changes to the severity of CHF and the morphofunctional state of the heart irrespective of the etiology of heart failure demonstrated the role of immune inflammation in its pathogenesis particularly in alcoholic patients who were found to have more marked immuno-inflammatory changes than in those with ischemic cardiomyopathy.

We present in this review contemporary views on pathogenesis of alcoholic cardiomyopathy. Alcoholic cardiomyopathy has features of dilated cardiomyopathy and is manifested by increased volume and hypertrophy of the left ventricle, diminished contractile capacity, and when decompensated - by lowering of cardiac output. Pathogenic action of alcohol on cardiomyocytes leads to activation of apoptosis, dysfunction of intracellular organelles, alterations of the system of myofilaments, disorder of intracellular homeostasis of calcium. Ethanol metabolite acetaldehyde, products of minor pathway of catecholamine metabolism, changes in the endocannabinoid system, and activation of processes of lipid peroxidation all contribute to the myocardial damage. The basis of pathogenesis of alcoholic cardiomyopathy constitute proliferation of microperoxisomes and disbalance between acyloxidase and catalase leading to accumulation of hydrogen peroxide inside myocytes.