Experimental hyperthermia: expression of proteins involved in the regulation of ovarian corpus luteum apoptosis in the acute and recovery periods

Background.Heat shock effects can initiate apoptosis of oocytes and corpus luteum cells in mammalian ovaries. During folliculogenesis, follicular apoptosis is regulated by Bcl-2 and BAX proteins which are key effectors of granular cell death. Mechanisms of disruption of the ovarian corpus luteum development under heat stress remain largely unclear.Aim of the research: to identify the expression features of anti-apoptotic Bad and proapoptotic Bcl-2 proteins in the rat ovarian luteocytes in the acute (by day 3) and recovery (by days 7 and 14) periods after a single exposure of experimental hyperthermia (EH) (rectal temperature 43.5 °C).Materials and methods. The expression of Bad and Bcl-2 was determined immunohistochemically using an indirect two-stage streptavidin-biotin method.Results. On day 3 after EH, the expression areas of both Bad and Bcl-2 increased 2-fold, but the ratio of Bcl-2/Bad areas did not change, indicating that the intensity of apoptosis along the mitochondrial pathway in luteocytes in the acute period was  maintained within physiological values. On day  7, the Bad and Bcl-2 expression areas remained at the level of day 3, but the Bcl-2/Bad index decreased, indicating the activation of the apoptosis internal pathway in the ovarian corpus luteum cells. By day 14, the protein expression areas decreased (Bad – by 1.7 times, Bcl-2 – by 3.2 times) compared to the acute period, and the Bcl-2/Bad index decreased by 2 times compared to the control and the acute period group.Conclusion. The observed predominance of proapoptotic Bad protein over antiapoptotic Bcl-2 in luteocytes on day 14 after EH indicates the anti-apoptotic protection violation, which leads to the apoptosis mitochondrial pathway activation of  the latter. A decrease in Bcl-2 expression can be regarded as a manifestation of the defective luteocytes removal mechanism and the body’s desire to normalize the ovarian-uterine cycle disrupted by high temperature exposure

Effect of a sorbent composition based on aluminum oxide and polydimethylsiloxane on the reproductive system of <i>db/db</i> female mice with genetically determined obesity and type 2 diabetes mellitus

Metabolic syndrome, obesity, type 2 diabetes mellitus are characterized by the accumulation of toxic metabolic products in the internal environment of the organism. The development of innovative medicines based on a sorbent matrix modified with  biologically active molecules remains relevant. The sorbent composition from aluminum oxide and polydimethylsiloxane is considered promising. The aim of the study. To investigate the effect of the sorbent composition from aluminum oxide and polydimethylsiloxane on the uterus and ovaries of db/db  mice with obesity and type 2 diabetes mellitus. Materials and methods. The sorbent composition (0.665 g/kg in 200 μl of distilled water) was administered to 14-week-old animals through an intragastric tube once a day for 7 days. The comparison groups were female rats injected with placebo (daily intragastrical administration of 200 µl of water for 7 days) and intact animals. Digital images of light-optical preparations stained with hematoxylin and eosin were processed using Image-Pro Plus 4.1 software. In the ovaries, the numerical density of primordial, primary, secondary follicles and corpus luteum was determined. The width of the uterus layers, the diameters of the blood and lymphatic vessels, the width of the interstitial fissures in both organs were measured. The statistical significance of differences was determined using the Mann – Whitney test. Results. In the myometrium and endometrium of the uterus of db/db mice, dilatation of arteries, veins, lymphatic vessels and edema were noted due to the accumulation of tissue fluid in the interstitium layers. There were no tertiary follicles in the ovaries. The introduction of the sorbent composition contributed to a decrease in the diameters of arteries, veins, lymphatic vessels of the uterus, a decrease in edema in both organs due to the narrowing of the prelymphatic slits, and stimulated an increase in the numerical density of secondary follicles. Conclusion. A corrective effect of the sorbent composition of aluminum oxide and  polydimethylsiloxane on prelymphatic slits, blood and lymphatic vessels in the uterus and ovaries in db/db mice with obesity and type 2 diabetes mellitus was revealed

noxin, a novel stress-induced gene involved in cell cycle and apoptosis

We describe a novel stress-induced gene, noxin, and a knockout mouse line with an inactivated noxin gene. The noxin gene does not have sequelogs in the genome and encodes a highly serine-rich protein with predicted phosphorylation sites for ATM, Akt, and DNA-dependent protein kinase kinases; nuclear localization signals; and a Zn finger domain. noxin mRNA and protein levels are under tight control by the cell cycle. noxin, identified as a nitric oxide-inducible gene, is strongly induced by a wide range of stress signals: gamma- and UV irradiation, hydrogen peroxide, adriamycin, and cytokines. This induction is dependent on p53. Noxin accumulates in the nucleus in response to stress and, when ectopically expressed, Noxin arrests the cell cycle at G1; although it also induces p53, the cell cycle arrest function of Noxin is independent of p53 activity. noxin knockout mice are viable and fertile; however, they have an enlarged heart, several altered hematopoietic parameters, and a decreased number of spermatids. Importantly, loss or downregulation of Noxin leads to increased cell death. Our results suggest that Noxin may be a component of the cell defense system: it is activated by various stress stimuli, helps cells to withdraw from cycling, and opposes apoptosis

The interactions between inflammation and insulin resistance: prospects of immunoregulation as a potential approach for the type 2 diabetes mellitus treatment

In the modern world the prevalence of obesity and type 2 diabetes mellitus (T2DM) significantly increases. In this light the risks of obesity-associated complications also grow up. The crucial linkage between obesity and its complications is inflammation, which is a convenient target for potential anti-diabetic therapy. There are some anti-inflammatory therapy strategies: action on secreted cytokines, circulating lipids or intracellular signaling cascades. Canakinumab (antibody to IL-1b receptor) and colchicine (IL-6 secretion blocker) have the most balanced anti-diabetic and cardioprotective action among cytokine anti-inflammatory therapy. Lipid-lowering therapy is very diverse, but bempedoic acid nowadays has the best combination of anti-inflammatory and cardioprotective effects. Salicylate is an inhibitor of IKK-dependent inflammatory signaling cascade and significantly lowers glycated hemoglobin and C-reactive protein levels among obese patients. The future of anti-inflammatory T2DM therapy can be related with anti-inflammatory cytokines (IL-4, IL-37), chimeric engineered cytokines (IC7Fc), novel inhibitors of inflammatory and cytokines signaling cascades (imatinib, CC90001) and cell-based therapy (mesenchymal stem cells). In summary, despite on the limitations of current clinical trials, anti-inflammatory drugs have a potential to become a part of modern combined T2DM therapy with anti-diabetic and cardioprotective properties. Novel findings in potential anti-inflammatory T2DM therapy have great perspectives in protection against T2DM and related complication prevention

The interactions between inflammation and insulin resistance: molecular mechanisms in insulin-producing and insulin-dependent tissues

In the modern world the prevalence of obesity and type 2 diabetes mellitus (T2DM) significantly increases. In this light the risks of obesity-associated complications also grow up. The crucial linkage between obesity and its metabolic and cardiovascular complications is inflammatory process. The mechanism of this linkage is similar in pancreas and insulin-dependent tissues both on cells, cell-to-cell communication and signaling pathway levels: the catalysts are different lipids (cholesterol, free fatty acids, triglycerides), which are able to activate Toll-like receptors of innate immunity and inflammation. Nextly, IKK- and JNK-dependent cascades activate the secretion of inflammatory cytokines TNFa, IL-1b, IL-6 and others, which act by paracrine and autocrine manner and support inflammation both in local and systemic levels. Thus, insulin-producing and insulin-dependent tissues, which are involved in T2DM pathogenesis, through the inflammatory process integrate in pathogenic and self-maintaining cycle, which leads to the suppression of insulin secretion, pancreatic β-cell failure and the development of insulin-dependent tissues insulin resistance

Apoptosis in the liver of male <em>db/db</em> mice during the development of obesity and type 2 diabetes

Obesity and diabetes mellitus are known to lead to the development of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). The mechanisms of programmed cell death are actively involved in maintaining cellular homeostasis along development of NAFLD. Proteins of the BCL-2 family are key regulators of physiological and pathological apoptosis. Homozygous males of BKS.Cg-Dock7mLeprdb/+/+/J mice (db/db mice) are characterized by progressive obesity and the development of type 2 diabetes mellitus (DM2) with severe hyperglycemia at 4–8 weeks and organ lesions at 8–10 weeks of age. The aim of this research was to study the expression of molecular cell regulators of apoptosis in liver cells of db/db mice males at different stages of obesity and diabetes development (at the age of 10 and 18 weeks). Immunohistochemical analysis (using the indirect avidin-biotin peroxidase method) and morphometric evaluation of the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in liver cells of studied animals at different stages of obesity and DM2 were carried out. An excess of the value of the Bcl-2 protein staining area over the Bad protein staining area was revealed in the liver of 10-week-old animals. The Bcl-2/Bad expression area ratio in 10-week-old animals was twice as high as in 18-week-old animals, which indicates the presence of conditions for blocking apoptosis in the liver of younger db/db mice. At the 18th week of life, db/db mice displayed an almost threefold increase in the expression area of the Bad protein against the background of an unchanged expression of the Bcl-2 protein. The decrease in the Bcl-2/Bad staining area ratio in 18-week-old animals was due to the increase in the Bad expression area, which indicates the absence of antiapoptotic cell protection and creates conditions for activation of the mitochondrial pathway of apoptosis in the liver of male db/db mice with pronounced signs of obesity and DM2

The expression of apoptosis-regulating proteins Bcl-2 and Bad in liver cells of C57Bl/6 mice under light-induced functional pinealectomy and after correction with melatonin

The presence of humans and animals under long-term continuous lighting leads to a suppression of melatonin synthesis, that is, to light-induced functional pinealectomy (LIFP), and the development of desynchronosis. To create LIFP, C57Bl/6 mice were kept under 24-hour lighting (24hL) for 14 days. The animals in the control group were kept under standard lighting conditions. In the next series of experiments, mice with LIFP received daily intragastrically either melatonin (1 mg/kg body weight in 200 μl of distilled water) or 200 μl of water as a placebo. The comparison group consisted of intact animals that received placebo under standard lighting conditions. Immunohistochemical analysis (using an indirect avidin-biotin peroxidase method) revealed the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in sinusoid liver cells (a heterogeneous population consisting of the endotheliocytes, Kupffer cells, Ito cells, and Pit cells) and in individual hepatocytes. The Bad expression area in the liver of LIFP mice increased 4 times against a background of the unchanged Bcl-2 expression area. Changes in the brightness (a parameter inversely proportional to the marker concentration) of Bad and Bcl-2 areas did not reach significance. Our results indicate a weakening of the antiapoptotic protection of liver cells of LIFP animals, which creates conditions for activation of the “mitochondrial branch” of apoptosis. Melatonin treatment of LIFP mice resulted in a 3.3-fold increase in Bcl-2 expression area and a 2.7 % decrease in Bcl-2 region brightness compared with the experimental untreated group. Bad protein parameters were unreliable. Thus, melatonin treatment of animals cancels the effect of LIFP, restoring the Bcl-2 expression area and increasing this protein concentration, which indicates an increase in antiapoptotic protection and creates conditions for blocking the development of the “mitochondrial branch” of apoptosis in liver cells

СТРУКТУРНЫЕ ИЗМЕНЕНИЯ В ТИМУСЕ В АНАБОЛИЧЕСКОЙ ФАЗЕ ПОСЛЕ ВОЗДЕЙСТВИЯ ЭКСПЕРИМЕНТАЛЬНОЙ ГИПЕРТЕРМИИ

In the anabolic phase of postoperational period, restoration of the structure of the blood-thymic thymus barrier, reduced edema of perivascular and interstitial spaces in in the thymus barrier, and emergence of a large number of epithelial cells with signs of unmodified structural organization are observed, indicating weakening of the destructive processes and increasing drainage of interstitial spaces in the body.В анаболической фазе постгипертермического периода происходит восстановление структуры гематотимического барьера тимуса, снижение отечности периваскулярных и интерстициальных пространств в нем, появление большого количества эпителиальных клеток с признаками неизмененной структурной организации, что свидетельствует об ослаблении деструктивных процессов и об усилении дренажа межклеточных пространств в органе

ВЛИЯНИЕ ЭКСПЕРИМЕНТАЛЬНОГО АЛИМЕНТАРНОГО ОЖИРЕНИЯ НА СТРУКТУРУ ПЕЧЕНИ КРЫС ЛИНИИ ВИСТАР

At the present time obesity is an important social and medical problem. The liver is a unique organ where all metabolic ways cross and principle exchange processes run. Therefore the aim of research was to reveal and estimate the character of structural changes in the liver of Vistar line rats in the model of alimentary obesity. The objectives of the research are to carry out morphometric and light optics examination of the liver, to analyze the morphometric data in the structure and cell composition of the organ on the light optics level at experimental obesity of alimentary etiology. The experiment involved 2-month old sexually mature femalerats of Vistar line with the initial body weight 180–200 g. There were two groups of animals in the experiment: control (intact rats receiving standard laboratory food diet) and experimental (receiving the model of standard laboratory food diet added to by edible fats of animal origin during 3 months). The animals were slaughtered under etaminal anesthesia (40 mg per 1 kg of animal body weight) by decapitation. For the morphometric and light optics examinations (microscope LEICA DM 750, camera LEICA ICC 50 HD) histologic preparations were fixed in 10% buffered formalin. The morphometric examination of liver preparations was carried out with 1000-folded magnification in the cuts of 5mcm thick and stained with Mayer's hematoxylin and eosin using the method of applying point morphometric grids (a grid of 256 points). Relative areas of sinusoid networks, nuclei, and cytoplasm of hepatocytes, numerical densities of sinusoid cells, hepatocytes and binucleated parenchyma cells were determined; nuclear-cytoplasm ratio, the ratio of numerical density of sinusoid cells to the numerical density of all hepatocytes were calculated; the part of diplokaryocytes was calculated from the total number of hepatocytes, Vizotto coefficient was used to calculate the ratio of sinusoid network area to the area of parenchyma of all the hepatocytes. The experimental alimentary obesity is established to result in adipose dystrophy in liver parenchyma and simultaneous stimulation of hepatocytes functional activity. Structural changes in the parenchyma cells are concomitant with functional tension of capillary-connective tissue structures expressed by disorders in blood circulation and lymph flow in the organ.Ожирение является в настоящее время важной социальной и медицинской проблемой. Печень представляет собой уникальный орган, где перекрещиваются все метаболические пути и  осуществляются ключевые обменные процессы. Поэтому целью исследования было выявить и оценить характер структурных изменений в печени крыс линии Вистар в модели алиментарного ожирения. Задачи исследования: провести морфометрическое и светооптическое исследование печени, анализ морфометрических данных структуры и клеточного состава органа на светооптическом уровне при экспериментальном ожирении алиментарной этиологии. В эксперименте использовались половозрелые крысы-самки линии Вистар с исходной массой тела 180–200 г в возрасте 2 месяцев. Было выделено две группы животных: контрольная группа (интактные крысы, получавшие стандартный лабораторный пищевой рацион) и группа, животным которой создавалась модель алиментарного ожирения путем добавления к стандартному лабораторному рациону пищевых жиров животного происхождения в течение 3 месяцев. Животных забивали под этаминаловым наркозом (40 мг на 1 кг массы тела животного) путем декапитации. Для морфометрического и светооптического исследований (микроскоп LEICA DM 750, камера LEICA ICC 50 HD) гистологические препараты фиксировали в 10%-м забуференном формалине. Морфометрическое исследование препаратов печени проводили при увеличении в 1000 раз на срезах толщиной 5 мкм, окрашенных гематоксилином Майера и эозином, используя метод наложения точечных морфометрических сеток (cетка 256 точек). Определяли относительные площади сети синусоидов, ядер и цитоплазмы гепатоцитов, численные плотности синусоидных клеток, гепатоцитов и двуядерных паренхиматозных клеток; рассчитывали ядерно-цитоплазматическое отношение, отношение численной плотности синусоидных клеток к численной плотности всех гепатоцитов, вычисляли долю диплокариоцитов от общего числа гепатоцитов, рассчитывали коэффициент Vizotto – отношение площади сети синусоидов к  площади паренхимы всех гепатоцитов. Установлено, что экспериментальное алиментарное ожирение приводит к  развитию в  паренхиме печени жировой дистрофии и  одновременно стимулирует функциональную активность гепатоцитов. Структурные изменения в паренхиматозных клетках сопровождаются функциональным напряжением капилляро-соединительнотканных структур, выраженными нарушениями кровообращения и лимфотока в органе

МОРФОМЕТРИЧЕСКОЕ ИССЛЕДОВАНИЕ ПЕЧЕНИ КРЫС ВИСТАР С МОДЕЛЬЮ АЛИМЕНТАРНОГО ОЖИРЕНИЯ

Experimental alimentary obesity leads to significant structural changes in the liver of rats. Structural changes in the parenchymal cells are accompanied by functional strain in the capillary-connective tissue structures, and by disorders in the blood circulation and lymph drainage in the liver.Экспериментальное алиментарное ожирение приводит к значительным структурным изменениям в печени крыс. Структурные изменения в паренхиматозных клетках сопровождаются функциональным напряжением капилляросоединительнотканных структур, нарушением кровообращения и лимфотока в печени