Терапевтическая эффективность Ронколейкина при экспериментальной гипергликемии

Abstract

На модели аллоксанового диабета у крыс изучено влияние Тиоцетама (500 мг/кг) и рекомбинантного интерлейкина-2 (Ронколейкин, 0,01 мг/кг) на показатели системы глутатиона, энергетического метаболизма и окислительной модификации белков. Установлено, что развитие гипергликемии у экспериментальных животных сопровождалось изменением системы глутатиона (повышением уровня окисленного глутатиона и снижением уровня восстановленного глутатиона, а также активности глутатионпероксидазы и глутатионредуктазы), энергетическим дефицитом и повышением уровней маркеров окислительной модификации белков – альдегидфенилгидразонов и кетонфенилгидразонов. Показано, что курсовое введение Тиоцетама и Ронколейкина способствовало нормализации активности глутатионпероксидазы и глутатионредуктазы, стабилизации уровней макроэргических фосфатов (АТФ, АДФ, АМФ) и показателей окислительной модификации белков; максимальное влияние на изученные параметры отмечено у Ронколейкина. На моделі алоксанового діабету у щурів вивчено вплив тіоцетаму і рекомбінантного інтерлейкіну-2 (Ронколейкін) (0,01 мг/кг) на показники системи глутатіону, енергетичного метаболізму та окиснювальної модифікації білків (ОМБ). Встановлено, що розвиток гіперглікемії у експериментальних тварин супроводжувався дестабілізацією системи глутатіону (підвищенням рівнів окиснених форм глутатіону на тлі різкого зниження їх відновлених форм та активності глутатіонпероксидази і глутатіонредуктази), енергетичним дефіцитом і підвищенням рівнів маркерів ОМБ – АФГ і КФГ. Виявлено, що курсове введення тіоцетаму і Ронколейкіну сприяло нормалізації активності глутатіонпероксидази і глутатіонредуктази, стабілізації рівнів макроергічних фосфатів (АТФ, АДФ, АМФ) та показників ОМБ; максимальна активність відзначена у Ронколейкіну. Aims. Diabetes mellitus (DM) is one of 7 main mortality causes in most countries of the world and is third among immediate causes of death after cardiovascular and oncological diseases. Nowadays, a body of evidence is amassed in the whole world, as to the fact that effective DM control determines life expectancy of the patients and their performance capability, as well as may keep development of the related complications to a minimum. It is proved, that the most important biological role for antioxidant system is in reduction oxidative reactions, in which thiol groups are easily oxidized, generally to form disulfide groupings, and again regenerated under their reductive splitting. Based on such transformations, a reversible thiol-disulfide system (TDS) is formed. TDS intermediates exhibit transport properties to nitrogen oxide (NO), thus increasing its bioavailability. Besides, many thiols – glutathione, cysteine, methionine – are able to considerably limit cytotoxicity of NO and its derivatives, thus multiplying neuron chances to survive in case of ischemia. Agents, providing both for damaging effect and cell viability system in the ischemia/hypoxia area, include cytokines – intercellular communication transmitters in health and disease, which establish communication signal network between cells of the immune system and cells of other organs and tissues. According to present day ideas, nature of immune response and peculiarities of development of the pathophysiological changes in ischemic/hypoxic tissue disorders depends on preemptive activation of the T-lymphocyte subpopulations, their synthesis of cytokines of various types and formation of "cytokine cascade". Primary goal of the effective DM therapy is to block interdependent mechanisms of DM progression – vascular, metabolic events and oxidant stress phenomenon, for which reason increasingly greater attention is paid to medications with antioxidant effect. Agents, providing both for damaging effect and cell viability system in the ischemia/hypoxia area, include cytokines. Therefore, application of cytokine preparations may become effective perspective link in the complex therapy of port-ischemic complications in DM. The aim of the study was to investigate the influence of thiocetam (500 mg/kg) and recombinant interleukin-2 (Roncoleukin) (0.01 mg/kg) on the performance of the glutathione system, energy metabolism and protein oxidative modification products in experimental diabetes. Methods. Experiments were performed in 40 Wiatar rats. Experimental diabetes was simulated by means of single subcutaneous administration of alloxan monohydrate water solution (Sigma, USA) – 150 mg/kg as 5% acetate buffer solution with pH 4.5. Brain tissue fragments from mesencephalic artery region, homogenized in liquid nitrogen, were used as materials for biochemical researches. This solution was administered after preliminary 24-hour food deprivation with allowed access to water. In order to generate full and stable diabetes, the animals were put on a standard diet for 11 days. Blood glucose level was determined on the 11th day after alloxan administration using glucose meter Optium Omega (Abbot Diabetes Care Inc., USA). For further researches, only animals with high glucose level (> 11 mmol/l) were used. Cytosolic fraction was separated by differential centrifugation (15,000 g) at the temperature of +4oС on phosphate buffer 0.15 М with pH 7.8. Protein-free extract was obtained by adding exact amount of brain tissue homogenate into perchloric acid (0.6 M) followed by neutalization by 5.0 M of potassium carbonate. To study thiolsulfide system activity in the rat brain tissue homogenate, levels of reduced and oxidized thiols and glutathione, and activity of glutathione peroxidase (GP) and glutathione reductase (GR) were determined. Content of total SH-groups were measured by spectrophotometry. Concentration of reduced and oxidized glutathione was measured by fluorometry. Activity of TDS enzymes – GP and GR – was measured by spectrophotometry. Content of methionine and cysteine in the homogenate was measured by chromatography. Also, using biochemical methods, content of protein oxidative modification products was measured in the brain homogenate by levels of aldehyde (APhH) and carboxyle (KphH) products. Statistical data processing was carried out by means of Statistica 6.0 software package; comparative group analysis was performed by means of one-way analysis of variance ANOVA using Newman–Keuls test for multiple comparisons. Differences with р<0.05 were considered statistically significant. Results. It was found that the development of hyperglycemia in experimental animals was accompanied by destabilization of the glutathione system (increased levels of oxidized glutathione and a sharp decrease in its activity and reduced forms of glutathione peroxidase and glutathione reductase), energy shortages and rising levels of markers protein oxidative modification products – aldehyde (APhH) and carboxyle (KphH) products. The course administration of thiocetam and Roncoleukin contributed to the normalization of activity of glutathione peroxidase and glutathione reductase, to stabilization of the levels of energy phosphates (ATP, ADP, AMP) and protein oxidative modification products figures; the maximum activity was observed for Roncoleukin. Conclusion. The therapeutic efficacy of Roncoleukin in experimental hyperglycemia is manifested in the stabilization of the glutathione system and energy balance, the prevention of oxidative and nitrosylating stress, and exceeds that of Thiocetam.