[Effect of NF-kappaB activation inhibitor curcumin on the oogenesis and follicular cell death in immune ovarian failure in mice].

In experiments on CBA mice, we studied the influence of an inhibitor of nuclear transcription factor kappaB activation curcumin, obtained from Curcuma longa, on the meiotic maturation of oocytes and apoptotic and necrotic death of follicular cells at immune ovary failure induced by immunization of animals with allogenic ovarian extracts. NF-kappaB plays a pivotal role in the induction of genes encoding pro-inflammatory factors (cytokines, adhesion molecules, inducible NO-synthase and cyclooxygenase) and in regulation of cell proliferation and death. It has been shown that immunization of mice increased the death of follicular cells through anapoptotic and necrotic pathways, which led to inflammatory response (according to blood leukogram and impairment the oocyte meiotic maturation at metaphase I and II). Intragastric administration of curcumin (Sigma, USA, 2 mg of the mouse weight, four times a week during the period of immunization) reduced the number of the follicular cells died through apoptotic and especially necrotic pathway. Curcumin attenuated an inflammatory response and improved the meiotic maturation of oocytes impaired under experimental immune ovarian failure in mice.

[Proliferation and death of liver mononuclear cells in immune lesions induced by concanavalin A and anti-liver antibodies in mice].

Two types of experimental liver failure in mice were investigated to study the immune mechanisms of liver disease: 1) T-cell-mediated injury induced by administration of concanavalin A (ConA) and 2) antibody-mediated injury induced by administration of anti-liver antibodies (ALA, gamma-globulin fraction of sera from rabbits immunized with liver tissue). It was established, that both types of liver injury were accompanied by the activation of immune processes in the liver, as shown by the increase of liver mononuclear cell proliferation, estimated using IPO-38 monoclonal antibodies. In contrast to ConA treatment, the immune activation under ALA-treatment was also associated with the increase in the percentage of plasma cells and small lymphocytes in liver mononuclear cells. At the same time, an increase in apoptotic and necrotic mononuclear cell death was more pronounced under ConA-treatment. This was accompanied by enhanced Fas receptor expression in these cells. Thus, it was shown that in case of T-cell mediated liver injury, the balance between cell proliferation and cell death in mononuclear liver cells was shifted toward the significant increase of apoptotic and necrotic cell death, particularly Fas-mediated apoptosis, while immune processes activation and cell proliferation were more pronounced in the case of antibodies-mediated injury.

[The protective effect of molsidomin in immune ovarian pathology in mice].

Experimental immune ovarian failure in CBA mice was induced by either administration of xenogenic anti-ovarian antibodies (scheme 1) or immunization with allogenic ovarian extracts (scheme 2). It was shown that both types of treatment impaired the meiotic maturation of oocytes: the number of cells at the stages of metaphase I and metaphase II decreased compared to the cells of control mice. In both schemes of experiments, impaired oogenesis was accompanied by reduction of percentage of viable follicular cells and by increase in the part of cells possessing morphological features of apoptosis. In contrast, the number of necrotic follicular cells increased in scheme 2 only. The donor of nitric oxide molsidomin (10 mg/ kg), when injected an hour before administration of xenogenic anti-ovarian antibodies or allogenic ovary extracts, improved the meiotic maturation of oocytes and favored follicular, lymph nodes and thymus cells survival by decreasing the number of apoptotic and necrotic cells.

[Effect of thromboxane B2 on morphological features of apoptosis in cultured rat hepatocytes exposed to hepatotropic substances].

Thromboxanes (TX) are known to have damaging effect on a liver but their influence on the cell death, in particular on hepatocyte apoptosis and its morphological features is not investigated enough. Cell death of the rat hepatocytes was investigated in primary culture by double vital staining with fluorescent nuclear stains Hoechst 33342 and propidium iodide and by electron microscopy. It was established that exogenous Tx B2 increases the amount of hepatocytes with early stages of apoptosis - the condensed chromatin and nucleus and cell size reduction. The changes in a percentage of hepatocytes with morphological features of the late stages of apoptosis - fragmented nuclei and division on apoptotic bodies were not revealed. Tx B2 intensified the carbon tetrachloride action on hepatocyte apoptotic death and increased chromatin condensation. Tx B2 application to hepatocytes injured by chenodeoxycholic acid significantly increased the amount of cells with a final stage ofapoptosis.

[Death of the ovarian follicular cells in mice with impaired oogenesis of the immune nature].

An impairment of the meiotic maturation of the oocytes has been shown in vitro for 2 types of immune damage of the ovaries in mice induced by xenogenic antiovarial antibodies and immunization with allogenic ovaria. Impairment of the oogenesis was followed by the follicular cell death, primarily by on the apoptic way, but under the immunization with allogenic ovary a necrotic way of their death was also activated.

[Effect of high-dispersed silica on the cultured rat hepatocytes].

Highdispersed silica (HDS) is an active substance of medicine "Siliks", which demonstrates high protein adsorption property and is used for hatching out toxic agents of protein origin. To discover new potential applications of this substance, it is necessary to study the direct effect of HDS on cell viability. We studied the effects of HDS in concentration 0,0001%, 0,001% and 0,01% on cultured rat hepatocytes at 4 and 24 hours. To estimate the number of alive vs. apoptotic and necrotic cells, fluorescent nucleic dyes Hoechst 33342 and propidium iodide were employed. Cells that underwent autophagic processes were estimated using a specific fluorescent autophagosome marker monodansylcadaverine. We show here that HDS has dose-dependent effect on cell death; the number of cells with apoptotic features increased after 4 hours and in greater extend after 24 hours of treatment with HDS. At the lowest concentration HSD did not significantly affect cell viability, and we observed decrease in postapoptotic necrosis in cell culture. The highest concentration of HSD dramatically increased cell death through both necrosis and apoptosis. At the same time, autophagic activity was suppressed by 0,01% HSD.

[Apoptotic, autophagic, and oncotic death of cardiomyocytes in anoxia-reoxygenation]. / Apoptotychna, autofahichna ta onkotychna zahybel' kardiomiotsytiv pry anoksiï-reoksyhenatsiï.

In experiments on isolated rat neonatal cardiomyocytes under anoxia-reoxygenation it was established the different types of cell death: non-programmed (oncotic) and programmed (apoptotic and autophagic). Propidium iodide and Hoechst 33342 staining revealed that alive, necrotic and apoptotic cell ratio after anoxia-reoxygenation was 77%, 14%, 9% respectively (86%, 9.6% and 4.4% in control). Electron microscopy of neonatal rat cardiomyocytes allows us to clarify the ultrastructural peculiarities of each type of cell death. Cytoplasm hydratation, swelling, cell membrane components vacuolization and sarcollemmal integrity alteration were typical for necrosis. The features of apoptosis include: cytoplasm osmiophylia, cell organelle destruction, chromatin condensation and nucleus fragmentation as well as blebs and apoptotic body formation. Autophagic cell death was characterized by the presence of large amount of different size vacuoles accompanied by significant cytoplasm osmiophylia, chromatin condensation or nucleus picnosis (nucleus fragmentation was not observed in these cells) and preservation of sarcollemmal integrity. The autophagic cardiomyocytes destruction was proved by specific monodansylcadaverine staining of vacuolar structures. We first showed the anoxia-reoxygenation-related autophagic cell death of neonatal rat cardiomyocytes. These results strongly suggest the necessity of autophagic cell death investigation in pathogenesis of cardiovascular diseases.

[Lipoxygenase inhibitors induce rat hepatocyte apoptosis in primary culture]. / Blokatory lipoksyhenaz indukuiut' apoptoz hepatotsytiv shchuriv u pervynnii kul'turi.

Arachidonic acid metabolites have been shown to have a wide range of effects on cell proliferation and viability. In this study, the effects of lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) on the viability of cultured rat hepatocytes (HC) were investigated. As a result, treatment with NDGA and CA for 4 h and 24 h decreased ALT release from HC and increased a number of apoptotic cells. Apoptosis inducing effects of general LO inhibitor NDGA were more pronounced, than those of 5-LO inhibitor CA. The results suggest that lipoxygenase pathway of arachidonic acid metabolism, in particular 5-LO, is essential regulator of hepatocyte survival and apoptosis.

[Effect of exogenous leukotrienes and lipoxygenase inhibitors on apoptosis and necrosis in cultured rat hepatocytes]. / Vplyv ekzohennykh leikotryieniv i blokatoriv lipoksyhenaz na apoptoz ta nekroz hepatotsytiv shchuriv u pervynnii kyl'turi.

Liver cell death by apoptosis and necrosis occurs upon the liver injury. Lipoxygenase pathway of arachidonic acid metabolism is known to regulate the viability and apoptosis in some cell types, but its role in hepatocyte cell death is not fully understood. We studied the influence of leukotrienes (LT) and lipoxygenase inhibitors on apoptosis and necrosis in rat hepatocyte primary culture by double staining with Hoechst 33342 and propidium iodide and electron microscopy. Treatment with general lipoxygenase inhibitor nordihydoguaiaretic acid and 5-lipoxygenase inhibitor caffeic acid (2. 10(-5) M) for 4 and 24 h induced hepatocyte apoptosis. LTB4 and LTC4 (10(-8) M) decreased the number of living cells and increased the number of necrotic cells. LTs exerted the same necrotic effect on hepatocytes, treated with lipoxygenase inhibitors. It is important that LTs decreased apoptosis induced by inhibitors treatment. These data suggest that lipoxygenase pathway of arachidonic acid metabolism is important regulator of hepatocytes viability and apoptosis The increase of lipoxygenase product formation, in particular LTs, may diminish apoptosis and increase necrosis in hepatocytes upon the liver injury.

[Effect of cultured mice hepatocytes on the proliferation of immunocompetent cells]. / Vplyv faktoriv, shcho vydiliaiutsia kultyvovanymy hepatotsytamy myshei, na proliferatsiiu imunokompetentnykh klityn.

Mice hepatocytes in primary cultures were treated during 2 h with normal rabbit antibodies (0.5 mg/ml), antiliver antibodies (0.5 mg/ml) and CCl4 (5 mM). Functional state of cells was studied according to DNA, RNA and protein synthesis. Cell-free supernatants were collected during 24 h after cells treatment. Spontaneous proliferation in vitro of syngeneic cells from the spleen, thymus, lymph node and bone marrow cultured with hepatocyte supernatants was investigated. Cultured intact hepatocytes were shown to produce factors that changed proliferation of the thymus and spleen cells. Hepatocyte supernatants demonstrated different effect on cells from different immune organs. Hepatocyte function alteration (minimal under normal antibodies treatment and maximal under CCl4 treatment) changed liver cells humoral influence on the immune cells.

[Changes of neutrophil functions resulting from intact and injured liver cells in mice]. / Zminy funktsionalnoi aktyvnosti neitrofiliv pid vplyvom faktoriv, shcho vydiliaiutsia intaktnymy ta urazhenymy klitynamy pechinky myshei.

Separately cultured parenchymatous and nonparenchymatous liver cells of mice produce factors to influence migration properties and oxygen-dependent metabolism of neutrophils. Cell-free supernatant of intact parenchymatous cells activates neutrophil chemotaxis only, while supernatant of intact nonparenchymatous cells stimulates oxygen-dependent reactions as well. Injure of the liver cells with 5 mM tetrachlormethane significantly increases neutrophil functional activation. Treatment of the liver cells with anti-liver antibodies (0.5 mg/ml) results in suppression of neutrophil activation. At the same time, supernatant of the liver cells treated with normal rabbit antibodies (0.5 mg/ml) possessed neutrophil stimulating properties, nonparenchymatous cell factors stimulating mainly the neutrophil oxygen-dependent metabolism.