engleski

Olive Leaves Polyphenols Improve Antioxidative Status in Diabetic Rat Liver

Oxidative stress (OS) is considered as one of the major risk factors for the onset and progression of diabetes. Some of the consequences of oxidative activity in diabetes development are the promotion of insulin resistance (IR), β-cell dysfunction, diminished glucose tolerance, and mitochondrial dysfunction [1]. The elevated levels of OS in diabetic animals are due to autoxidation of glucose, protein glycation, lipid peroxidation, and low activities of antioxidant enzymes [2]. Olive leaf is a rich source of phenolic compounds that are biologically active and have better antioxidant activity, anti-inflammatory, and radical scavenging effects. The aim of this study was to evaluate the effects of olive leaf extract on the antioxidant activity in the liver tissue in diabetic rats. Male Wistar rats (same age, approximately the same weight) were used in this study. They were divided into six groups of 6 animals in each group as follows: group C – healthy control, group DM – streptozotocin (SZT)-induced diabetes, group TI – insulin-treated diabetic rats, groups TOL1, TOL2 and TOL3 –diabetic rats treated with OLP extract in dose-dependent manner (0.5 g/kg, 0.77 g/kg, 1 g/kg, respectively). SZT - induced diabetes was administrated intraperitoneally (i.p.) by a single dose (60 mg/kg). OLP extracts were injected (i.p.) in diabetic animals daily, over a period of 10 days. Insulin was injected subcutaneously (sub-cut.) in diabetic animals daily, over a period of 10 days in the dose 10 U/kg. Whole liver protein extracts were prepared by tissue homogenization with ceramic beads in 0.1 M PBS (pH 7.4). Sequential extraction of proteins was used for isolation of the mitochondrial and cytosol fraction by differential centrifugation method using Potter Elvehjem homogenizer [3]. Mitochondrial and cytosolic fractions were used further for SDS-PAGE, Western blot and enzymatic assays. BCA protein assay method was used for determination of the protein content in homogenates and subcellular fractions. Antioxidant activity of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) were determined in whole protein extracts using commercial kits. The SOD activity was determined in both mitochondrial and cytosolic fraction, while GPX activity was determined in the cytosolic fraction. SDS-PAGE and Western blot were used for determination of SOD1, GPX1 and CAT in cytosol fraction, while SOD2 was determined in the mitochondrial fraction. Data were expressed as mean±SD. Differences between groups were assessed by a nonparametric Kruskal–Wallis median, overall median test. Differences with P<0.05 were considered to be statistically significant. Impaired antioxidant status was involved in oxidative stress in the liver in diabetes and associated with lower activity and suppression of main antioxidative enzymes. The highest activity was found in SOD, then GPX, and the lowest, CAT in all examined groups. Elevated levels of glutamate were found in the liver. With the protein fractionation, antioxidant enzymes were localized in the cell. SOD showed the highest activity in the mitochondrial fraction (as MnSOD or SOD2), while GPX was determined only in the cytosolic fraction after sequential protein extraction of the liver. A higher dose of OLPs treatments increased the activity of total SOD and increased the expression of mitochondrial SOD. However, the activity of cytosolic SOD (as [Cu-Zn] or SOD1) increased in a dose-dependent manner. Insulin therapy normalized the activities and protein expression similar to OLP treatment in the group TOL2. Thus, diabetes-associated reductions in antioxidant enzymes can be ameliorated by insulin and/or antioxidant therapy.

diabetes ; liver ; SOD ; GPX ; L-glutamate ; sequentional extraction

This work has been supported in part by the University of Rijeka under the project code uniri-prirod-18-46, University of Rijeka Foundation