The influence of pumpkin polysaccharides on oxidative stress indices in acute hypoxia with hypercapnia in rats

The aim. In the model of acute hypoxia with hypercapnia in an experiment on male rats, the protective effect of polysaccharides of pumpkin fruits in relation to the oxidative modification of protein molecules of skeletal muscle tissue is shown. Material and Methods. The experiment examined tissues of the cardiac and skeletal muscles taken from 18 sexually mature male rats of Wistar weighing 200-220 g, which were kept in the vivarium of Ryazan State Medical University. The animals were divided into three groups of 6 individuals. The first group of animals received a 10% solution of polysaccharides extracted from pumpkin fruits by intragastric feeding through a tube daily for 30 days at the dose of 0.1 g / kg body weight. The polysaccharides were extracted according to the following scheme: to remove extractives and colored molecules from the raw material, it was preliminarily treated with 4060% ethanol solutions. Then, a weighed portion of air-dried fruits was placed in a round bottom flask. After that it was poured with a 1% solution of ammonium oxalate and extracted in a boiling water bath for one and a half hours. After that the mixture was filtered, the filtrate was evaporated, and the obtained extract was precipitated with one and a half volume of 96% ethanol. The precipitate (PS) was separated, purified with ethanol, ether and acetone. The isolated PS was dried in vacuo for 12 hours over P2O5. On the last day of PS administration, the animals were exposed to acute normobaric hypoxia with hypercapnia once. For this, each animal was placed in a sealed chamber with a volume of 1000 ml for 30 minutes. The second group of animals received saline according to the introduction of PS. On the last day of the experiment, the animals were placed in a chamber with natural ventilation for 30 minutes. The third group of the animals remained intact until the end of the experiment and underwent hypoxia in the same way as the first group of the animals. After that tissues were taken from the anesthetized rats. Homogenates were obtained from skeletal and cardiac muscle tissues using a homogenizer, which were centrifuged for 10 minutes at 1000 g to precipitate nuclei and intact cells. The resulting supernatant was used for further studies. To assess the antioxidant ability of PS, the activity of the enzyme of the antioxidant system of cells, superoxide dismutase (SOD), was determined; to evaluate the protective effect of PS in acute hypoxia with hypercapnia, oxidative modification of proteins (OMB) was determined as markers of oxidative stress. For calculations in each sample, the concentration of total protein was determined by the Lowry method. Statistical data processing was performed using Microsoft Office Excel 2010. The level of differences was considered statistically significant with an error probability of p <0.05. Results. The results of evaluating the activity of SOD show that the state of hypoxia with hypercapnia, modeled in the experiment, causes an increase in enzyme activity by 2.74 times in the heart muscle and 2.34 times in the skeletal muscle. At the same time, the activity of SOD during hypoxia while taking polysaccharides is reduced by 1.52 times in the heart muscle and by 2.55 times in the skeletal muscle. Determination of the oxidative modification of proteins showed that polysaccharide intake did not influence the modification of the heart muscle proteins. However, the development of hypoxia in this model did not lead to the accumulation of oxidized modified derivative proteins. In skeletal muscle, the total area under the curve of the absorption spectrum of the oxidized modified protein during the development of hypoxia turned out to be 4.26 times greater, with the greatest increase in derivatives of ketone-dinitrophenolhydrozone (KDNPH). However, with PS administration, the accumulation of modified proteins was 2.05 times less than with hypoxia. PS administration reduced the accumulation of aldehyde-dinitrophenolhydrozone (ADNPH) of a basic and neutral nature by about 2.3 times, ketone-dinitrophenolhydrozone of a basic nature - by 1.77 times, and a neutral one - by 2.5 times. From the ratio of accumulated ADNPH and KDNPH, it follows that the intake of polysaccharides led to a decrease in the accumulation of ADNPH, which are the primary markers of oxidative stress, indicating the degree of fragmentation of protein molecules. A less effective decrease in the level of KDNPH of the main nature against the background of PS intake may indicate a possible mechanism of the protective effect of PS against neutral amino acids that make up protein molecules. These data indicate a greater resistance of the heart muscle to hypoxia and the effect of modulators such as polysaccharides compared with skeletal muscle tissue. Perhaps this is due to a more powerful activation of the antioxidant system, as indicated by a change in the activity of SOD. Moreover, the degree of damage to proteins was significantly reduced when prescribing PS due to a decrease in the accumulation of ADNPH derivatives and to a lesser extent due to a decrease in KDNPH derivatives, which indicates an increase in the resistance of tissue proteins to hypoxia. Conclusions. The effect of a single hypoxia with hypercapnia provoked an increase in the activity of superoxide dismutase in the tissues of the cardiac and skeletal muscles, while SOD of the heart showed itself more actively. The intake of polysaccharides restrained the increase in SOD activity during hypoxia, however, PS had a protective effect on the proteins of skeletal muscle tissue, which led to a decrease in their oxidative modification.

pumpkin fruit polysaccharides, hypoxia, oxidative modification of proteins