State of the antioxidant protection system of rat liver in ischemia and reperfusion

Purpose: determination of the state of the antioxidant protection system of the cytosolic fraction and suspension of rat liver mitochondria after experimental ischemia and reperfusion. Materials and methods: the study was conducted using white mature rats, divided into 3 groups: the control group (n = 15); The 2nd group of animals (n = 15), from which the liver was taken after 15 minutes of liver ischemia; the 3rd group of rats (n = 15), from which the liver was taken after a 15-minute reperfusion period, followed by a 15-minute ischemic period. Mitochondrial suspension and cytosolic fraction were isolated from liver tissue. Results: the obtained research results showed the presence of certain pathobiochemical changes in the suspension of mitochondria and the cytosolic fraction after ischemia or reperfusion. In the mitochondrial suspension during the reperfusion period it was found an adaptive increase in the activity of glutathione peroxidase by 39% and glutathione reductase by 61%. In the cytosolic fraction, it was the most remarkable increase of the total antioxidant capacity by 38% already during ischemia and a progressive decrease in the level of reduced glutathione form by 26% in ischemic and 55% in reperfusion period. The change in the state of the antioxidant system occurred against the background of an increase in the number of products of oxidative modifications of biomolecules by 40% during ischemia and 2.2 times after reperfusion. Conclusion: The results indicate the need to develop not only a mitochondria-oriented correction of oxidative disorders, but also active support for the components of the cytosol, which provide the main accumulation of free radical damage products and their subsequent removal from the cell, which is essential for survival.


EXPERIMENTAL PHYSIOLOGY
Disorders of oxidative homeostasis, including hypoenergetic states and oxidative stress, are the leading pathobiochemical mechanisms of the development and progression of ischemic and reperfusion injuries of various organs. During the ischemic period, a sharp decrease in the oxygen tension in the tissue naturally leads to a transition to less effective anaerobic energy processes. At the same time, the oxygen residues against the background of dysregulation of the respiratory chain form their active forms to a greater extent, which already in the period of turning off the blood flow stimulate the development of free-radical damage. The resumption of blood supply and the transition to the reperfusion period are accompanied by an even more powerful intensification of oxidative stress against the background of relative hyperoxia. Currently, it is known that the damage of the reperfusion period makes the leading contribution to the total damage of the organ and the development of its subsequent dysfunction [1][2].
Understanding the sequence of events occurring in the dynamics of ischemic-reperfusion syndrome injuries at different levels can contribute to the development of new pathogenetically justified methods of correction of such pathological processes [3][4][5]. The relevance of correction of such disorders is due to the fact that hypoxia and reoxygenation are universal pathological processes and are the basis of many disorders of cardiovascular activity, found in surgical practice, including organ transplantation.
The so-called Pringle technique is often found in surgical Hepatology. It is a compression of the hepaticduodenal ligament in order to prevent blood loss during surgical interventions on the liver parenchyma. The use of this technique is limited by the risks of postoperative liver failure due to ischemic reperfusion injuries. The similar complex of pathobiochemical processes develops during the liver transplantation, which is often the only way to treat hepatitis and cirrhosis [6][7][8].
The purpose of this study was to determine the state of the antioxidant protection system of the cytosol fraction and suspension of the rat liver mitochondria after experimental ischemia and reperfusion.

Material and methods
White non-linear male rats weighing 220-250 grams (n=50) were used for the study. All laboratory animals were kept in the conditions of the vivarium of FSBEI HE KubGMU of the Ministry of Health of Russia in appropriate conditions. All experimental work with the participation of animals was performed in a specialized operating vivarium, while all painful manipulations were carried out after preliminary general anesthesia using Zoletil 100 (Virbac, France) 10 mg / kg intramuscularly. The study was carried out in accordance with the requirements of the "European Convention for the Protection of Vertebrate Animals" (Strasbourg, 1986) and after approval by an independent ethical committee (protocol No. 51 of 05.23.2017).
Experimental animals were divided into 3 groups: control group is the group of rats that underwent a medians laparotomy without further modeling of the pathological process (pseudo-operated animals, group 1, n=20); group 2 is group of rats (n=15) that underwent vascular liver exclusion by clamping an analog of the hepatoduodenal ligament for 15 minutes after which the liver was taken without restoring blood flow; group 3 is a group of rats (n=15) whose liver was taken after a 15-minute reperfusion period following a 15-minute ischemic period. The liver was immediately placed in a cold medium consisting of at least one-third of the frozen medium to isolate the mitochondrial suspension [9]. In the laboratory of the Department of fundamental and clinical biochemistry the liver immediately after the experiment was crushed and homogenized in the same sucrose medium (0.25 M sucrose containing EGTA and Mg ions in 0.02 M tris-HCl buffer with pH = 7.4). After homogenization they were centrifuged at 1000 g for 5 minutes to remove large fragments whole undisturbed cells, red blood cells, ect. Further after centrifugation for 10 minutes at 12,000 g the supernatant was used as a cytosolic (postmitochondrial) fraction and the pellet was washed again under the same conditions and prepared from it a working mitochondrial suspension. Protein concentration was determined in the cytosolic fraction and mitochondrial suspension using the Bradford method [10]. The calculation of the content of metabolites or enzyme activity in the subsequent was ЭКСПЕРИМЕНТАЛЬНАЯ ФИЗИОЛОГИЯ based on the protein content in the sample.
The state of the antioxidant defense system was evaluated by determining the total antioxidant capacity of ferrum-reducing method FRAP [11], catalase activity, glutathione peroxidase activity, glutathione reductase, reducible glutathione count [12][13]. The level of intensity of oxidative processes revealed the accumulation of diene conjugates in the studied biofluids.
Statistical data handling was performed using Stat plus LE and Excel for Windows. The nature of the data division was evaluated using the Shapiro-Wilk statistic, the data were presented as a median and the 1st and 3rd quartiles (Q1 and Q3). The nonparametric Mann-Whitney U-test was determined to assess the significance of differences between the indicators of the studied groups of animals. The differences were considered statistically significant at p<0.05.

Results and discussion
Determination of indicators of antioxidant defense system and mitochondrial fractions of rat liver cytosol after ischemia or ischemia / reperfusion showed the presence of characteristic differences at different times and in different subcellular compartments. In the mitochondrial suspension (table. 1) after 15 minutes of ischemia and without restoring of blood flow a slight increase in total antioxidant capacity was noted by 15% relative to the target. In the same period an increase 7% activity of glutathione reductase was observed. These changes are associated more likely with rapid adaptive changes aimed at maintaining adequate oxidative homeostasis. At the same time the catalase activity was reduced by 27%. This is due to the relatively low rate of formation of hydrogen peroxide during this period. The activity of glutathione peroxidase did not undergo significant changes although this enzyme is primarily responsible for the utilization of peroxides in mitochondria. The strengthening of free radical processes already in the ischemic period is confirmed by an increase in the accumulation in the mitochondrial suspension of intermediate products of lipid peroxidation, i. e. diene conjugates the content of which in the mitochondria of the liver of rats of 2nd group increased by 12.5%. Given the short duration of the pathological process of only 15 minutes of vascular excretion the recorded changes should not seem insignificant. GSH, nmol/mg of protein 10,1 (9,6/10,3) 9,6 (9,5/10,2) 9,5 (9,4/10,1) Diene conjugates, conv. units 0,08 (0,07/0,09) 0,10* (0,09/0,10) 0,08 (0,07/0,09) Note:* -statistically significant differences (р<0,05) from the same index of the 1st group; ^ -statistically significant differences (р<0,05) from index of the 2nd group.
The more significant changes in state of the system of antioxidant protection of mitochondria were determined after 15 minutes of reperfusion in livers of rats from the 3rd (Table 1). In this group, the total antioxidant capacity kept increasing up to 30% in comparison with the control values. The increase in activity of all studied enzymes was also revealed. The activity of glutathione peroxidase increased by 39%, while the activity of glutathione reductase increased by 61% in comparison with indices of the control group. The catalase activity also increased by 27% in comparison with the values of the 2nd group, but the value of this index remained a bit lower than the control values. The concentration of reduced glutathione in mitochondrial suspension of liver in rats of the 3rd group remained at the level of the control group which certainly proved high capacity of antioxidant system of mitochondria. The level of diene conjugates determined within the mitochondrial suspension of liver in the 3rd group did not differ from the level of control group which proved the rapid recovery of mitochondrial structure and function after relatively long period of ischemic and reperfusion damage.

Table 2
Indices of the system of antioxidant protection of cytoplasmic fractions received from rat liver after ischemic and reperfusion damage (Ме(р0,25/р0,75))

Studied indices
Test groups
In cytoplasmic (post-mitochondrial) fraction, the changes in enzymic activity were considered to be less reactive, but the more significant changes of non-enzymic components and the accumulation of diene conjugates were also detected ( Table 2). So, at the ischemic period, the activity values of glutathione peroxidise increased by 40%, but returned to their initial level at the reperfusion period. The activity of glutathione reductase changed in a similar way. In test animals of the 2nd group, the activity of this enzyme in the cytoplasmic fraction of liver increased by 8%, while in animals of the 3rd group it did not differ from the control values. The catalase activity significantly increased after 15-minute reperfusion, in particular by 42% in comparison with the control values. The revealed changes may result from the primary catalase localization within the cytosol and peroxisomes as well as from the more important role of this enzyme in protection of cytosol and its structures in comparison with mitochondria. The total antioxidant capacity changed within the cytosol fraction in the same way that in mitochondria, but more significantly. In test animals of the 2nd -3rd groups, the studied index increased by 37-38% in comparison with the values of the control group. The significant decrease in level of reduced glutathione in cytoplasmic fraction of rat liver was also revealed -by 26% after the ischemic period and by 55% after the reperfusion period respectively. The content of diene conjugates gradually increased in liver of test animals in all the studied groups. In cytoplasmic fraction of rat liver of the 2nd group, the level of diene conjugates increased by 40% while in rats of the 3rd group -by 2,2 times. We believe that the received results are conditioned by the adaptative character of changes in system of antioxidant protection with the increase in enzymic activity and in total antioxidant capacity. Furthermore, the main adaptative processes within the cytolysis take place at the ischemic period, while in the mitochondrial suspension they take place during reperfusion. In such conditions the cytosol plays the role of the buffer receiving the greater part of products of oxidative modifications of biomolecules and providing the main amount of reduced equivalents of glutathione for the further functioning of antioxidant system. Another highly reliable thesis is that during the process of excretion of mitochondrial suspension the damaged and destroyed organelles remain in the cytoplasmic fraction as well as their components; thus, the artificial image of "wellness" of metabolic systems of mitochondria is represented against the background of extensive pathobiochemical disorders within the cytoplasmic fraction. In our opinion, the chosen experimental model of pathobiochemical process is relatively mild and against its background the mitochondrial damage should not be expected; that's why the first thesis is considered to be more reliable. The whole image of pathological process is probably more complex and is composed of the buffer influence of cytosol and depletion of damaged mitochondrial components into it. One way or another, it is clear that for the objective assessment of the state of antioxidant system during the development of ischemic and reperfusion damages the indices of both mitochondrial suspension and cytoplasmic fraction should be evaluated. Furthermore, by the analysis of mitochondrial suspension the attention should be paid to the changes in enzymic activity of metabolic glutathione, while in the cytoplasmic fraction the attention should be paid to the changes in catalase activity, content of reduced glutathione, and products of oxidative modifications of biomolecules.

Conclusion
The results of the study proved the peculiarities of pathobiochemical changes in suspension of mitochondria and cytoplasmic fraction after 15 minutes of ischemia and the same period of reperfusion. In mitochondrial suspension, the adaptative increase in activity of glutathione peroxidase and glutathione reductase was detected, especially at the reperfusion period. In cytoplasmic fraction, the increase of total antioxidant capacity was the most significant at the ischemia period with the progressing decrease in the level of reduced glutathione against the background of growing number of products of oxidative modifications of biomolecules. The received results prove that it's necessary not only to develop the mitochondrial-oriented correction of oxidative disorders, but also to actively support the cytoplasmic components which provide the storage of products of free-radical damages and their further removal from the cells which is extremely important for surviving.