Досліджено вплив флуренізиду (антибіотик протимікробної, протитуберку-льозної, антихламідійної, імуномодулювальної, антиоксидантної, гепатопротек-торної, протизапальної, противірусної дії) на інтенсивність процесів пероксидного окиснення ліпідів у зародків в'юна Misgurnus fossilis L. на етапі розвитку 2, 16, 64, 256 та 1024 бластомерів. Виявлено, що флуренізид у всіх досліджуваних концентра ціях (0,01; 0,05; 0,15; 1; 5; 15 мМ) призводить до значного підвищення вмісту пер-винних продуктів ліпопероксидації (гідропероксидів) на стадії 16 і 1024 бластоме-рів і до зниження -на стадії 256 бластомерів. Встановлено, що вміст вторинних продуктів ліпопероксидації (ТБКпозитивних продуктів) підвищується за впливу антибіотика у концентраціях 0,01÷1 мМ і знижується за концентрації 15 мМ. З'ясо вано, що зародки в'юна є найчутливішими до дії флуренізиду на етапі розвитку 16 бластомерів, коли вміст гідропероксидів є максимальним. Найменш чутливою стадією розвитку зародкових клітин до екзогенних чинників є стадія 8го поділу (256 бластомерів).Ключові слова: зародки в'юна, флуренізид, пероксидне окиснення ліпідів, гідропероксиди, ТБКпозитивні продукти. ВСТУППід час розвитку патологічних процесів стрімко зростає інтенсивність ліпопе-роксидації, що робить її універсальним методом "розпізнавання" пошкодження клі-тинних мембран. Продукти пероксидного окиснення ліпідів (ПОЛ) порушують структурну цілісність мембран клітини, їхню осмотичну резистентність і електрич-ний потенціал, окиснюють тіолові сполуки і SHгрупи білків, ушкоджують структуру білків, амінокислот і т. п. [1,3]. Актуальність дослідження процесів ПОЛ обумовле-на важливою патогенетичною роллю вільнорадикального окиснення як потужного фактора мембранодеструкції.Первинні продукти ПОЛ (гідропероксиди ліпідів) є речовинами нестійкими, які досить швидко руйнуються з утворенням вторинних продуктів ліпопероксидації. Серед них найвідоміший -малоновий діальдегід (МДА), накопичення якого в орга-Biol. Stud. 2016: 10(1); 53-60 •
The influence of histamine and quercetin, as well as their combined effect on the content of histamine in whole blood, the content of the superoxide anion radical, sialic acids, sulfhydryl groups, the activity of catalase, glutathione peroxidase, and glutathione transferase in rat erythrocytes was studied. It was established that quercetin at a concentration of 0.1 mM causes an increase in the content of histamine in the whole blood of rats, while all other studied concentrations cause a significant decrease in the content of biogenic amine. Adding histamine to whole blood at a concentration of 1 μM leads to a decrease in the content of endogenous histamine in the blood, while at a concentration of 10 μM, it causes an increase in the content of this biogenic amine. The combined effect of exogenous histamine and quercetin mainly leads to an increase in the amount of endogenous histamine in the whole blood of rats. In rat erythrocytes, quercetin causes the generation of the superoxide anion radical. An increase in the superoxide anion radical content occurs under the influence of histamine at a concentration of 0.1; 1 and 10 μM, while under the action of biogenic amine 0.01 μM, the amount of the studied product decreases. The combined effect of histamine and quercetin intensifies the formation of superoxide anion radical in erythrocytes, in addition to the effect of flavonoid in therapeutic concentration. Adding quercetin and histamine to whole blood causes an increase in the content of sialic acids. Such an effect was also found under the combined action of histamine at a concentration of 0.01 µM and quercetin at a concentration of 0.1; 0.5; 3; 5 mM. The combined effect of histamine at a concentration of 10 µM and quercetin at a concentration of 5 mM leads to a decrease in the content of sialic acids in erythrocytes. Addition of quercetin to whole blood causes an increase in the content of sulfhydryl groups, except for a concentration of 5 mM, at which the content of this indicator decreases. Histamine in a concentration of 0.01; 0.1 μM leads to an increase in the content of SH-groups, and at a concentration of 1 μM – to a decrease. Histamine against the background of exposure to quercetin leads to an increase in the content of sulfhydryl groups. The degree of influence of histamine and quercetin, as well as their combined effect, is the same on the content of endogenous histamine in the blood, sialic acids, sulfhydryl groups in erythrocytes. The independent effect of histamine and quercetin causes a weak effect on the indicated indicators of blood and erythrocytes. The combined effect of histamine and quercetin on the content of the studied indicators has a more pronounced effect. The most significant effect of the combined effect of histamine and quercetin was found on the content of the superoxide anion radical and sialic acids. Quercetin in a concentration of 0.1; 3; 5 mM leads to a decrease in catalase activity, while the studied flavonoid, with a concentration of 0.3; 1 mM, leads to an increase in the activity of the studied enzyme. Histamine at a concentration of 0.1 and 10 µM activates catalase, while biogenic amine (at a concentration of 0.01 and 1 µM) reduces the activity of the enzyme. The combined action of histamine and quercetin leads to an increase in the activity of catalase in hemolysates of erythrocytes of rats. Quercetin only at a concentration of 5 mM, histamine at a concentration of 0.1; 1; 10 μM enhances the activity of glutathione peroxidase. Simultaneous addition to the blood of histamine at a concentration of 10 μM and quercetin at a concentration of 3 and 5 mM, as well as the combined effect of histamine at a concentration of 0.01 μM and quercetin at a concentration of 0.1; 0.5; 3; 5 mM results in the intensification of glutathione peroxidase. Quercetin at a concentration of 0.1 and 0.3 mM does not change the activity of glutathione-S-transferase, while the studied flavonoid (at a concentration of 0.5; 1; 3; 5 mM) causes a significant increase in the activity of the studied enzyme. Histamine in a concentration of 0.01; 0.1; 1 and 10 μM significantly dose-dependently activate glutathione-S-transferase. The combined action of histamine, at a concentration of 10 µM, and quercetin, at a concentration of 0.1; 0.5; 3; 5 mM, leads to a decrease in enzyme activity in rat erythrocyte hemolysates compared to samples to which only histamine was added, but glutathione-S-transferase values did not reach control limits. Compared to the control, the activity of glutathione-S-transferase increases under the simultaneous action of histamine and quercetin. Simultaneous exposure to histamine at a concentration of 0.01 µM and quercetin causes a significant increase in the activity of glutathione-S-transferase. Histamine has a significant effect on catalase activity in rat erythrocytes, while the combined administration of quercetin and histamine into the blood has a powerful effect on the work of glutathione peroxidase and glutathione-S-transferase.
The effect of histamine and quercetin, as well as their combined effect on the activity of superoxide dismutase, catalase and the content of reduced glutathione in the blood plasma of rats was studied. It was found that the addition to the blood of quercetin at a concentration of 0.1; 0.3; 0.5; 1.0; 5.0 mM causes an increase in superoxide dismutase activity. It was found that histamine at concentrations of .01 and 0.1 μm leads to a decrease in superoxide dismutase activity by 31 and 17 %, respectively. Whereas the biogenic amine in the lowest and highest concentrations does not change the activity of superoxide dismutase in plasma. At simultaneous introduction into blood of histamine in the maximum concentration (10.0 μm) and quercetin in concentration of 0,1; 0.5; 3.0 mM normalizes the activity of superoxide dismutase. And only the combined action of histamine of this concentration and quercetin at a concentration of 5.0 mM reduces the activity of the enzyme by 21 %. Histamine at a concentration of 0.01 μm and the simultaneous action of quercetin at a concentration of 0.1; 0.5; 3.0; 5.0 mM increases the activity of superoxide dismutase, which indicates the generation of reactive oxygen species, in particular the superoxide anion radical. It was found that the addition of whole concentrations of quercetin to whole blood causes a decrease in plasma catalase activity. The combined action of quercetin and histamine causes a decrease in catalase activity. It was found that the addition to the blood of quercetin at a concentration of 0.1; 0.3; 0.5; 1.0 mM causes a decrease in the content of reduced glutathione. Quercetin at a concentration of 3.0 and 5.0 mM causes an increase in reduced glutathione by 27 and 14 %, respectively, compared to the reference plasma. Histamine at concentrations of 10.0, 1.0 and 0.01 μm leads to an increase in the amount of reduced glutathione by 24, 26 and 19 %, respectively. And at a concentration of 0.1 μm, the biogenic amine reduces the GSH content by 39 %. With simultaneous introduction into the blood of histamine at a concentration of 10.0 μm and quercetin at a concentration of 0.1; 0.5; 3.0 mM there is an increase in the content of reduced glutathione. And only at a concentration of 5.0 mM quercetin on the background of the action of histamine (10.0 μm), the content of reduced glutathione is slightly reduced. However, with the combined action of histamine at a minimum concentration (0.01 μM) and quercetin at a concentration of 0.1; 0.5; 3.0 and 5.0 mM there is a decrease in the content of reduced glutathione. After performing a dispersion analysis, it was found that the state of the antioxidant system, both enzymatic and non-enzymatic, is significantly affected by histamine. The smallest, but significant share of the effect is on the action of quercetin in the blood plasma of rats. The indirect and significant effect of the simultaneous action of histamine and quercetin on the antioxidant system of rat plasma was revealed.
It is known that flurenizide is a newly synthesized drug with pronounced antimicrobial, immunomodulatory functions. However, its effect on the functional properties of cells, in particular germ cells, is unknown. The aim of this study was to determine the presence of sialic acids as the final components of glycoconjugates, the main macroergic compound - ATP, to evaluate the generation of O2¯ in embryos of lobster (Misgurnus fossilis L.) under the influence of the antibiotic flurenizide. Studies have been performed on embryos of Misgurnus fossilis L. After fertilization, the zygotes were placed in Petri dishes with solutions of flurenizide in concentrations of 0.01; 0.05; 0.15; 1.0; 5.0; 15.0 mM, where allowed to develop. At the development stages, 2 blastomeres, 16 blastomeres, 64 blastomeres, VIII (256 blastomeres) and X divisions (1024 blastomeres) were sampled. In parallel, control studies were performed, where flureniside was not added to the samples. The content of sialic acids, ATP, superoxide anion radical was determined in the selected samples. We found that flureniside at the lowest concentration of 0.01 mM does not cause changes in the content of superoxide anion radical during early embryogenesis. The antibiotic in the maximum concentration causes changes in the content of free radical from the stage of development of 16 blastomeres to 1024 blastomeres, and from the stage of 64 blastomeres there is an increase in its number. Probably flurenizide at a concentration of 15.0 mM is the most reactive. It is known that flurenizide has antioxidant properties, but in its structure there are groups that have a toxic effect, which is most pronounced when exposed to high concentrations. In general, at the stage of development of 16 blastomeres there is a decrease in the amount of О2¯ under the influence of the studied antibiotic. At this time, the content of this free radical in the control increases, compared with other stages of development. Flureniside leads to an increase in the content of superoxide anion radical at development stage 2, 64 blastomeres and is particularly pronounced at concentrations from 0.05 to 15.0 mM at development stages 256 and 1024 blastomeres. 1024 blastomeres are the 10th stage of separation, where desynchronization occurs and the mitotic index decreases. Probably, the changes that take place at this stage of development are related to this. The antibiotic causes an increase in the content of sialic acids in the first stage of crushing (2 blastomeres). In step 16 of the blastomere, flureniside in low concentrations leads to a decrease in the content of sialic acids. However, already at the stage of separation of 64 and 256 blastomeres, the test substance in all concentrations causes a predominant decrease in the amount of sialic acids. At the last stage of synchronous crushing (1024 blastomeres) flurenizide in the maximum investigated concentration (15.0 mM) causes a significant accumulation of sialic acid content. Flureniside at concentrations of 0.15, 5.0 and 15.0 mМ causes a decrease in ATP content in loach embryos at the stage of development of 256 blastomeres by 28, 67 and 38 %, respectively. An increase in ATP content by 33 % occurs under the influence of flurenizide at a concentration of 1.0 mM. The ATP content also increases at the stage of development of embryos of 1024 blastomeres under the influence of flurenizide of all studied concentrations. Analysis of variance has shown that a significant contribution to the growth of ATP, sialic acid and superoxide anion radical, during the early embryogenesis of lobster embryos, is made by the factor of embryo development, while the factor of flureniside has a smaller contribution. Therefore, studies have shown that flurenizide causes increased generation of superoxide anion radical, decreased sialic acid content, as well as changes in ATP content in embryonic cells during early embryogenesis.
The content of products of lipoperoxidation and oxidative modification of proteins in rat blood plasma under the action of quercetin and histamine was investigated. Quercetin was used at concentrations of 0.1; 0.3; 0.5; 1; 3; 5 mm, and histoma - 0.01; 0.1; 1; 10 μm. It was found that quercetin in the blood plasma of rats causes a slight increase in the content of hydroperoxides and TBA-positive products, except the concentration of 0.5 mm, at which the content of secondary products is reduced and the content of primary remains within the control. Histamine at concentrations of 0.01; 0.1; 1; 10 µM leads to an increase in the content of hydroperoxides and a decrease in the number of TBK-positive products. The combined effect of histamine and quercetin causes a significant increase in primary lipoperoxidation products, whereas TBK-positive lipid peroxidation products decrease. Quercetin in the concentration range of 0.1÷1 mM intensifies the accumulation of carbonyl groups of proteins of basic and neutral nature, while flavonoid at concentrations of 3.5 mM – slows down. Histamine at all tested concentrations leads to an increase in the content of carbonyl groups of proteins, except the concentration of 0.1 μm. Against the background of high concentration histamine, quercetin reduces the intensity of oxidative modification of proteins. The simultaneous action of low concentration histamine and quercetin at a concentration of 0.5 and 5 mm leads to the accumulation of carbonyl groups of proteins of a neutral nature, as well as to the main only when exposed to quercetin at a concentration of 5 mm. According to the analysis of variance, it was found that quercetin has a maximum effect on the accumulation of TBA-positive products and carbonyl groups of proteins of basic character. Histamine is strongly influenced by the accumulation of lipid hydroperoxides. Quercetin at concentrations of 0.1; 0.3; 0.5; 1; 3 mm determines between the indices of free radical processes under study, in the blood plasma of rats, a strong interconnectedness, and a bioflavonoid at a concentration of 5 mm leads to the formation of interconnections of average strength. Histamine at concentrations of 10; 1; 0.1 µM causes a close correlation of the mean strength between the individual indices of free radical oxidation. Histamine at a concentration of 0.01 μm causes a significant increase in the correlation between the parameters of sex and oxidative modification of proteins. The combined effect of quercetin at a concentration of 0.1 mm and histamine at a concentration of 0.01 μm causes a change in the nature of the correlation dependence, which becomes strong between most of the studied parameters.
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