РОЛЬ НЕТОЗА В ПАТОГЕНЕЗЕ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТИ1 Юринская М.М., 2 Сусликов А.В., 1 Винокуров М.Г. 1 Институт биофизики клетки Российской академии наук -обособленное подразделение ФГБУН «Федеральный исследовательский центр «Пущинский научный центр биологических исследований» Российской академии наук», Пущино, e-mail:

АДРЕНОБЛОКАТОРЫ ПРОПРАНОЛОЛ И ФЕНТОЛАМИН МОДУЛИРУЮТ LPS-ИНДУЦИРОВАННУЮ ВНУТРИКЛЕТОЧНУЮ СИГНАЛИЗАЦИЮ НЕЙТРОФИЛОВ ЧЕЛОВЕКА1,2 Юринская М.М., 1,2 Винокуров М.Г., 2 Сусликов А

КЛЕТОЧНАЯ ГИБЕЛЬ И ФУНКЦИОНАЛЬНАЯ АКТИВНОСТЬ НЕЙТРОФИЛОВ У БОЛЬНЫХ ХРОНИЧЕСКОЙ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТЬЮ ПРИ ЭНДОТОКСЕМИИ1,2 Юринская М.М., 1,2 Винокуров М

Lipopolysaccharides (LPS), components of the cell wall of Gram-negative bacteria, activate neutrophils that trigger pathological processes, including Gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellular glutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by lps and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

Lipopolysaccharides (LPS), components of the cell wall of gram-negative bacteria, activate neutrophils that trigger pathological processes, including gram-negative sepsis. LPS inhibit spontaneous apoptosis of neutrophils that leads to inflammation. In this work we tested the action of H2S donor (GYY4137) on the activation of human neutrophils by E. coli LPS. We estimated the changes in redox status (ROS level, intracellularglutathione, NO), apoptosis and mitochondrial potential of neutrophils under the LPS action in the presence and absence of GYY4137. GYY4137 reduces the ROS level, slightly reduces GSH, does not influence the NO level and has no apoptogenic effect. LPS induce the increasing of ROS level and inhibit spontaneous apoptosis of neutrophils. We found that GYY4137 prevents the growth of ROS caused by LPS and leads to a reduction of LPS-induced inhibition of neutrophil apoptosis. Thus the mechanism of GYY4137 protection against inflammation, triggered by bacterial infection, is concerned with the neutralization of LPS effect on neutrophils.

В различных заболеваниях млекопитающих большое значение имеют эндотоксины (ЭТ) грамотрицательных энтеробактерий, вызывающие увеличение генерации активных форм кислорода (ROS) полиморфноядерными лейкоцитами (ПМЯЛ). Индуцибельный белок теплового шока человека Бтш70 при внеклеточном применении снижает продукцию ROS полиморфноядерными лейкоцитами, индуцированную ЭТ. В работе установлено, что в механизме защиты ПМЯЛ белком Бтш70 от активации клеток ЭТ важная роль принадлежит клатринзависимому и рецепторзависимым путям эндоцитоза Бтш70.

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

The possibility of the effective delivery of lactoferrin to the cells of the innate immune system (neutrophils and monocytes) by means of biodegradable polyelectrolyte microcapsules has been shown. A study of the effect of the structural component of microcapsules, poly-L-arginine, has shown that the production of reactive oxygen species (ROS) was maximum at a concentration of the polyelectrolyte of 0.5 μg/mL. Increasing the polycation concentration significantly decreased the neutrophil viability and ROS production. The study of neutrophil apoptosis showed that hollow microcapsules containing no lactoferrin accelerated the apoptosis of phagocytes by 15–20%. Encapsulated lactoferrin inhibited the neutrophil apoptosis by 65–70%, whereas nonencapsulated lactoferrin decreased it by 35%. It was found that encapsulated lactoferrin reduced the production of TNF-α by THP-1 promonocytic cells to an approximately the same degree as nonencapsulated lactoferrin.

Microencapsulated heat shock proteins HSP 70 were studied in terms of their effects on neutrophil apoptosis, production of reactive oxygen species, and secretion of TNF-α by human neurtrophils and monocytes. Encapsulated HSP70 inhibited neutrophil apoptosis by 65% as compared to the effect of nonencapsulated HSP70; TNF-α production by the promonocytic THP-1 cells was similarly inhibited by the non-encapsulated and encapsulated HSP70. Thus, the polyelectrolyte micromolecules can be used as containers for effective delivery of HSP70 up to neutrophils and monocytes to correct the innate immunity functions.

The paper deals with the NMR spectra obtained using preparations of five different human biological body fluids. Characteristic metabolite signals of blood, urine, tears, saliva, and sweat spectra have been determined and classified. The biological body fluid samples were used for search and identification of biomarkers of cardiovascular disease. Absolute functional biomarkers for diseases such as coronary heart disease (CHD) have not been recognized even in the case acute myocardial infarction. A hypothesis explaining reasons of lack of such markers has been formulated. The results of comparative analysis of blood and urine samples from humans and some laboratory animals are given. Identify and analyze signals of metabolites of pathogenic microflora and their dynamics in the urine from patients with urogenital diseases have been determined and analyzed and characteristic biomarkers have been recognized.

The comparison of succinate dehydrogenase activity (SDH) was carried out in the presence of succinate (SUC) in commonly used millimolar ("substrate") concentrations that significantly exceeds the SUC content in tissues, and in micromolar concentrations really present in the organism, currently called "signaling" concentrations. This name reflects the function of SUC, a substrate of oxidation, as a signaling molecule regulating physiological processes. Regulatory signaling action of SUC occurs in significantly lower concentrations, at least 1000 times lower than "substrate" concentration. Signaling action is related to adrenergic regulation including SDH as its target. It is realized via specific SUC receptor, GPR91. Here the SDH activity was studied under different physiological conditions in rat and human using new cytobiochemical (CBCH) method, which sensitively reveals changes in the organism due to the preservation of the native state of mitochondrial network. SDH activity was measured in lymphocytes in blood smear by nitro blue tetrazolium (NBT) reduction due to oxidation of added and endogenous substrates. The high statistical validity is an additional advantage of the CBCH method, because every separate value is measured by videomicroscopic scanning of multitude of stained objects (mitochondria), 200-700 in one sample containing images of 30 lymphocytes of an individual. High sensitivity of CBCH method allowed detection of the SDH activity with SUC being in both millimolar and micromolar concentrations. Activation of SDH was revealed upon increase in adrenergic regulation in the norm (by adrenaline administration) and in acute phase of hypertension, followed by inhibition under arterial pressure stabilization by hypotensive preparations, which are adrenaline receptor blockers and internal regulators at chronic stages. The use of micromolar SUC concentrations reveals changes in SDH activity in the organism related to the differences in the physiological states more efficiently than the use of millimolar concentrations, which activate SDH and smooth over the development of inhibition under pathology progressing. While SDH activity measured at millimolar SUC concentrations was depressed, a significant rise in NBT reduction was observed in the presence of micromolar concentrations. This suggests that the effect of signal (but not substrate) concentrations can be realized through generation of superoxide and hydrogen peroxide. which possess vitally beneficial regulatory effects in micromolar concentrations in mitochondria.

Проведено сравнительное исследование активности сукцинатдегидрогеназы при добавлении янтарной кислоты в обычно используемых, сильно завышенных по сравнению с содержанием в тканях, миллимолярных, “субстратных” концентрациях (5 мМ), и реально существующих в организме микромолярных концентрациях (5–50 мкM), называемых в настоящее время “сигнальными”. Это название отражает вторую функцию субстрата окисления янтарной кислоты как сигнального вещества – регулятора физиологических процессов. Регуляторное, сигнальное действие янтарной кислоты проявляется в значительно более низких концентрациях, чем субстратное (не менее чем в 1000 раз). Сигнальное действие янтарной кислоты связано с адренергической регуляцией, включающей сукцинатдегидрогеназу как ее мишень. Оно реализуется через специфический рецептор янтарной кислоты GPR91. В данной работе определяли активность сукцинатдегидрогеназы крыс и человека новым цитобиохимическим методом, который выявляет изменения в организме благодаря сохранению нативного состояния митохондриальной сети. Активность сукцинатдегидрогеназы может быть измерена в лимфоцитах на мазке крови по восстановлению нитросинего тетразолия за счет окисления добавленных и эндогенных субстратов. Дополнительным достоинством метода является высокая статистическая значимость, так как каждая отдельная величина измеряется видеомикроскопическим сканированием множества (200–700) окрашенных объектов (митохондрий) в одном образце, содержащем изображения 30 лимфоцитов индивидуума. Высокая чувствительность цитобиохимического метода позволила измерить активность сукцинатдегидрогеназы в присутствии янтарной кислоты не только в миллимолярных, но и в микромолярных концентрациях. Выявлена активация сукцинатдегидрогеназы при усилении адренергической регуляции в норме (введением адреналина) и в острой фазе гипертонии с переходом к ингибированию при стабилизации артериального давления гипотензивными препаратами – блокаторами адренорецепторов и внутренними регуляторами при хроническом течении. Использование янтарной кислоты в микромолярных концентрациях позволяет с большей чувствительностью выявить изменения активности сукцинатдегидрогеназы, соответствующие различиям в физиологическом состоянии организма, чем при использовании субстратных концентраций, которые активируют фермент и маскируют развитие ингибирования при углублении патологии. При сниженной активности сукцинатдегидрогеназы, измеренной в присутствии янтарной кислоты в миллимолярной концентрации, наблюдается значительное повышение восстановления нитросинего тетразолия в присутствии микромолярных концентраций янтарной кислоты. Это явление указывает на то, что действие сигнальных концентраций, в отличие от субстратных, может реализоваться через образование в митохондриях супероксида и перекиси водорода, которые в микромолярных концентрациях обладают жизненно необходимыми благоприятными регуляторными эффектами.

Arginase activity in erythrocytes is higher in patients with arterial hypertension and atherosclerosis as compared with healthy patients. Therapy with either lisinopril alone or in combination with simvastatin for 3–6 months causes a decrease in the arginase activity to the control level. Both the monotherapy and the combination therapy increased the concentrations of N 2 − , N 3 − , and total N 2 − + N 3 − in the plasma of hypertensive patients. The N 2 − + N 3 − concentration in erythrocytes decreases in hypertensive patients but is completely restored after therapy with lisinopril alone or in combination with simvastatin. Thus, lisinopril and lisinopril plus simvastatin display a pronounced and equal normalizing effect on arginase activity in human erythrocytes, which is elevated in hypertension, as well as on the endothelial nitric oxide synthase activity, which is decreased in hypertension.

Statins and angiotensin-converting enzyme (ACE) inhibitors have beneficial impact on the serum cholesterol and blood pressure. It is supposed that statins and ACE inhibitors may modify the antioxidative status of erythrocytes. The study objective was to compare the effects of two treatments, lisinopril alone vs lisinopril plus simvastatin, on erythrocyte antioxidant enzyme activities. The study involved 32 patients with arterial hypertension, the initial serum total cholesterol, LDL-cholesterol and triglycerides within the normal range. Patients of two groups, each of 16 subjects, were treated with lisinopril (10 mg/day) or with lisinopril (10 mg/day) plus simvastatin (20 mg/day). Before and after 3 and 6 months of follow-up therapy, activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GLR) in purified erythrocytes were determined. In all patients, significantly higher catalase activity (by 79.3-106.5%, p<0.0001) and significantly lower GPx activity (by 20.7-30.6%, p<0.001) were observed after therapy as compared to the baselines. Just the same results were obtained in both groups (lisinopril and lisinopril + simvastatin), after both periods (3 and 6 month) of treatments. SOD activity was increased only in the lisinopril group and only after 6 months (p=0.0345). No changes of GLR reductase activity were seen under all conditions indicated. Thus, the lisinopril monotherapy and combined lisinopril plus simvastatin therapy exhibit specific, pronounced and equipotent effects on antioxidant enzymes in human erythrocytes. Administration of lisinopril or lisinopril plus simvastatin may protect erythrocytes and other tissues from oxidative damage.