Published online Dec 15, 2019. doi: 10.4239/wjd.v10.i12.546
Peer-review started: July 26, 2019
First decision: August 19, 2019
Revised: October 20, 2019
Accepted: October 27, 2019
Article in press: October 27, 2019
Published online: December 15, 2019
All treatments of the experiment were consistent with the requirements of the European legislation on the protection of animals (Directive 2010/63/EU).
The laboratory rats were divided into four groups: Group 1 (n = 20), animals housed under standard vivarium conditions (control group); group 2 (n = 20), animals with streptozotocin (STZ) injection-induced hyperglycemia; group 3 (n = 12), animals with STZ-induced glycemia that were administered with 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) solution intraperitoneally at a 1250 nmol/kg dose per day, starting from the second week; and group 4 (n = 8), animals housed under standard vivarium conditions that were administered with SkQ1 at a 1250 nmol/kg dose per day, during the second week of conducting the experiment. Group 2 also included rats (n = 8) that were administered intraperitoneally with appropriate aliquot quantity of citrate buffer equal to pH 4.4 once a week during 2 wk after 1-mo high-fat diet. Group 3 included animals (n = 8) that were administered intraperitoneally with appropriate amount of solution without SkQ1 (98% ethanol diluted eight times with normal saline solution). The experimental unit was a single animal. Rats were kept in laboratory, and they were divided through stratified randomization by their weight and age.
Hyperglycemia was induced by feeding rats a high-fat diet for 1 mo. STZ was administered intra-abdominally at a 30 mg/kg of animal weight dose with citrate buffer equal to pH 4.4. Hyperglycemia was verified by measuring the glucose level in the blood serum with a glucose oxidase test. SkQ1 solution was administered intraperitoneally at a 1250 nmol/kg dose per day. Appropriate aliquot quantity of citrate buffer equal to pH 4.4 was administered intraperitoneally.
To conduct the study, laboratory rats were selected of nursery rat males from Federal State-financed Organization of Health Service “Voronezh Hygiene and Epidemiology Center”. The rats used for the study weighed 200-250 g and aged 3-5 mo. Rats were kept at 12-h light day, room temperature, and access to water and food ad libitum for 2 wk before the study. Type of housing – plastic. Bedding material – sawdust. Number of cage companions - two rats per cage. The total number of animals used in experiment – 76 rats. The number of animals in each experimental group: Group 1 (n = 20 + 8); group 2 (n = 20 + 8); group 3 (n = 12); and group 4 (n = 8).
The number of animals was necessary for obtaining statistically significant results. The animals were divided through stratified randomization by their weight and age. The rats used for the study weighed 200-250 g and aged 3-5 mo.
The order in which the animals in the different experimental groups were treated and assessed: Group 2 compared with group 1; group 3 compared with group 2; and group 4 compared with group 1. Weight gain, increased water intake, and slowness in rats of the second group were seen. SkQ1 administration lowered glycemic level by 2.5 times, which was initially upregulated 2.7 times relative to the control. The glucose level of the animals of the fourth experimental group was not significantly different from the stated value. Moreover, glucose concentration was within the stated value in rats of the second experimental group, having been administered citrate buffer and of the third group, having been administered aliquot of 12% ethanol. The unit of analysis was group of animals.
Experiments were done at least in 8-20 biological and two analytical replicates. The results were compared with the control. The data were statistically analyzed using software package STATISTICA 6.0 with numerical variables – arithmetical mean (M), mean error (m), and statistical significance level (P). Normal distribution data were compared by applying Student's t-test for Bonferroni correction in independent samples. Significance level was set at aP ≤ 0.0167 and bP ≤ 0.0167.
It was found that influence of the mitochondrial-directed antioxidant elicits decrease of biochemiluminescence (BCL) parameters values that increase by pathology as well as the level of primary products of lipid peroxidation such as diene conjugates and carbonyl compounds, which indicate intensity of free radical oxidation. At the same time, the activity of aconitate hydratase (AH), considered as a crucial target of FR, which was decreasing during experimental hyperglycemia, increased. Apparently, increasing activity of AH influenced the speed of citrate utilization, whose concentration was decreasing after administering SkQ1 by pathology. Moreover, the previously applied anti-oxidant administration during hyperglycemia influenced the rate of antioxidant system mobilization. Thus, superoxide dismutase and catalase activity as well as the level of gene transcript under influence of SkQ1 at pathology were changing towards control groups values.
The results of this research suggest that the mitochondria targeted antioxidant SkQ1 might be a perspective substance for incorporation into the antioxidant therapy of type 2 diabetes mellitus (DM2). The experimentally revealed ability of this compound to lower the intensity of free-radical processes, acting as the key component of the pathogenesis of the DM2, may serve as the reason for this conclusion. Thus, after the introduction of SkQ1 to the animals with STZ induced hyperglycemia, the values of the BCL parameters reflecting the free radical oxidation intensity, the concentration of diene conjugates and carbonyl products of protein oxidation, the AH activity, and citrate content changed towards the control values. At the same time, the activity level of the antioxidant enzyme SOD and catalase changed to the direction of normal values.
The effects of SkQ1 on the stage of compensatory response occurrence in pathology may contribute to decrease of the degree of oxidative stress, normalization of antioxidant system functioning, and blocking of the development of decompensation, characterized by the inhibition of protective systems.
Thus, the mitochondria targeted antioxidant SkQ1 might be considered a perspective substance for incorporation into the antioxidant therapy of DM2.