European science review № 7–8 2015 July–August «East West» Association for Advanced Studies and Higher Education GmbH Vienna 2015 European Sciences review Scientific journal № 7–8 2015 (July–August) ISSN 2310-5577 Editor-in-chief Lucas Koenig, Austria Consulting editors Uwe Eisenberg, Austria Minik Olsen, Sweden International editorial board Melinda Boros, Hungary Miroslavka Murkovič, Slovenia Jana Ilyna, Russia Suleyman Suleymanov, Uzbekistan Wu Pan, China Dragan Novak, Croatia Dirk Eggers, Germany Yashkova Tatiana, Russia Proofreading Kristin Theissen Cover design Andreas Vogel Additional design Stephan Friedman Editorial office European Science Review “East West” Association for Advanced Studies and Higher Education GmbH, Am Gestade 1 1010 Vienna, Austria Email: [email protected] Homepage: www.ew-a.org European Science Review is an international, German/English/Russian language, peer-reviewed journal. It is published bimonthly with circulation of 1000 copies. The decisive criterion for accepting a manuscript for publication is scientific quality. All research articles published in this journal have undergone a rigorous peer review. Based on initial screening by the editors, each paper is anonymized and reviewed by at least two anonymous referees. Recommending the articles for publishing, the reviewers confirm that in their opinion the submitted article contains important or new scientific results. Instructions for authors Full instructions for manuscript preparation and submission can be found through the “East West” Association GmbH home page at: http://www.ew-a.org. Material disclaimer The opinions expressed in the conference proceedings do not necessarily reflect those of the «East West» Association for Advanced Studies and Higher Education GmbH, the editor, the editorial board, or the organization to which the authors are affiliated. © «East West» Association for Advanced Studies and Higher Education GmbH All rights reserved; no part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission of the Publisher. Typeset in Berling by Ziegler Buchdruckerei, Linz, Austria. Printed by «East West» Association for Advanced Studies and Higher Education GmbH, Vienna, Austria on acid-free paper. Protective effect of salvifolin on liver mitochondrial function in rats with experimental diabetes Section 1. Biology Pozilov Mamurjon Komiljonovich, Junior research, A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, E‑mail: [email protected] Asrarov Muzaffar Islamovich, Professor, A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, E‑mail: [email protected] Urmanova Gulbakhor Urunboevna, Senior lecturer Tashkent pediatric medical Institute Eshbakova Komila Alibekovna, Senior research, S.Yu. Yunusov Institute of Chemistry of Plant Substances, Academy of Sciences of Uzbekistan Protective effect of salvifolin on liver mitochondrial function in rats with experimental diabetes Abstract: The influence of diterpenoid salvifolin on mitochondrial function was investigated. It was shown that in streptozotocin-induced diabetes damaged functional systems of rat liver mitochondria: respiration and oxidative phosphorylation, mitochondrial permeability transition pore and ATP -dependent potassium channel. Pharma- cotherapy with salvifolin (intraperitoneally in dose of 3,5 mg/kg body weight) for 8 days has a protective effect on mitochondria in experimental diabetes, correction membrane disorders. Keywords: diabetes, mitochondria, respiration, oxidation phosphorylation, permeability transition pore, salvifolin. Introduction potassium channel of the mitochondria (mitoKATF channel) Studying the mechanisms of damage to cellular struc- in diabetes, as well as modulators of their data available. It was tures and functions in various pathologies and ways to treat found that open ATP-dependent potassium channel (mito- these injuries with pharmacological agents is a priority of KATP channel) protects the heart from ischemic contractures modern biomedical research. Despite the variety of treat- and improve its post ischemic functional recovery [4, 2463– ments for diabetes remains popular search for new pharma- 2469; 5, 51–57], play an important cardioprotective role dur- cological agents and their “targets”. In the cell, such “targets” ing all phases of the ischemia-reperfusion myocardial injury are the mitochondrial membrane and localized in their struc- [6, 1–21]. The literature also little information on potassium ture, firstly mitochondrial respiratory chain, megapora (mito- channel modulators of plant origin, having the property of chondrial permeability transition pore, mPTP) [1, 101–127]. lowering blood sugar. The formation of reactive oxygen species in cells and excessive Clerodane-type salvifolin isolated from the Pulicaria salvi‑ activation of free radical oxidation processes underlie the de- ifolia, having a hypoglycemic effect, it normalizes the metabol- velopment of diabetes [2, 1405–1423; 3, 1986–2001]. It is ic processes of the body in experimental diabetes [7, 86–91; 8, also known that the mitochondria is disrupted the functioning 161–163]. However, salvifolin influence of on mitochondrial of the citric acid cycle, there is a partial uncoupling of oxida- function is not investigated and therefore the aim was to study tion and phosphorylation. These processes are considered as the effect of salvifolin on respiration and oxidative phosphory- a universal mechanism that combines the basic biochemical lation, state of mPTP and mitoKATP channel in the rat liver pathways of the toxic effect of hyperglycemia on the body. mitochondria of streptozotocin (STZ)-induced diabetic rats. The development of experimental diabetes also involves Methods ATP-dependent potassium channels in the plasma mem- For screening and detailed study of the mechanism of ac- brane-localized β-cells of the pancreas are opened. To date, tion of pharmacological agents are widely used various experi- known inhibitors and activators of potassium channels. How- mental models of diabetes caused by administration of alloxan ever, in the literature on the role and status of ATP-dependent and STZ al., cytotoxic activity on β-cells of the pancreas. We 3 Section 1. Biology have in this study used an experimental model of diabetes in- tochondria corresponded to 3 mg/ml of the reaction medium duced by STZ. ADP (200 µM) was added as a respiratory stimulant. Calculat- Experiments were performed on 90 white mongrel male ed the rate of mitochondrial respiration in different metabolic rats weighing 180–200 g The animals were divided into three states: V3 –respiration rate after making ADP, V4 — respiration groups: I group — intact, II group — the animals with experi- rate after spending listed ADP. The indices characterizing pair mental diabetes, which once were injected intraperitoneally of oxidation and phosphorylation in mitochondria: respira- with an STZ (50 mg/kg body weight intraperitoneally in a tory control (RC) ratio (RC=V3/V4) and the coefficient of 0,1 mol/L citrate buffer, pH 4,5) (control) and III group — phosphorylation of ADP/O. Mitochondrial respiration rate in STZ-induced diabetes + salvifolin (intraperitoneally dose of different metabolic states are expressed in nanograms of con- 3,5 mg/kg body weight) for 8 days starting from 12 days after sumed oxygen atoms per 1 minute per 1 mg of mitochondrial administration of STZ and reaching a predetermined level of protein. The respiratory control and ADF/O ratio was cal- hyperglycemia. Blood glucose was determined using glucose culated according to the method of Chance [12, 409–427]. oxidase method set «Glucose — enzymatic-colorimetric The rate of swelling of the mitochondria was studied us- test» (Cypress diagnostic, Belgium). ing a photometer LMF-69. Mitochondrial swelling induced 2+ The content of mitochondrial protein was determined by the activation KATP Mg channel was recorded using a change in Lowry method in the modification of the Peterson [9, 346–356]. light scattering at a wavelength of 540 nm. Mitochondria were Mitochondria isolated from rat liver by differential centrifu- added to the standard incubation medium of the following gation according to Schneider [10, 619–635]. Nuclei and cellular composition: 125 mM KCl, 10 mM Hepes, 5 mM succinate, fragments were removed by centrifugation at 600 g for 7 min- 1 mM MgCl2, 2,5 mM K2HPO4, 2,5 mM KH2PO4, rotenone utes in a centrifuge. The mitochondria are pelleted at 10000 g for 1 µM/ml, oligomycin 1 µ/ml, pH 7,4. 15 minutes at the same temperature. The mitochondrial pellet Static analysis of data was performed using the program was washed twice in the isolation EDTA-free medium. features Origine 7.5 (Microcal Software Inc., Northampton mPTP condition assessed by the speed of Cа 2+ -depen- MA). The data were expressed as means ± S. E. M. Paired Stu- dent swelling of mitochondria, the mitochondrial suspension dent’s t-test was used for estimation of significance; minimum recording light scattering at 540 nm. Experiments at room accepted level of significance was p < 0.05. temperature 25 °C in a medium containing 200 mM sucrose, Results and discussion 20 µM EGTA, 5 mM succinate, 2 µM rotenone, 1 µg/ml oli- Figure 1 shows the results of a study of respiratory and gomycin, 20 mM Tris, 20 mM HEPES, and 1 mM KH2PO4, phosphorylating activity of rat liver mitochondria from STZ-