Anti-Apoptotic Effect of Synthetic Leu-Enkephalin Dalargin on Rat Leukocytes in Cold Stress Model in Vivo

In this research, a protective effect of synthetic analogue of leu-enkephalin dalargin on peripheral blood leukocytes of cold stress-exposed homeotherms has been investigated. The impact of this peptide and in vivo cold stress on cell composition of leukoconcentrate, leukocyte viability and DNA fragmentation degree in rat leukocytes, has been studied by using confocal microscopy. A decreased relative count of lymphocytes and an increased neutrophil one were established as significantly less pronounced in the animals injected with dalargin before cooling, than in non-handled ones. The dalargin administration was also shown to enhance the viability of peripheral blood leukocytes in rats exposed to cold stress. Preliminary administration of dalargin to animals significantly reduced both the degree of DNA fragmentation and a relative count of leukocytes with fragmented DNA in peripheral blood. Simultaneous introduction of opioid receptor antagonist naloxone to animals eliminated a protective effect of opioid receptor agonist dalargin. Our findings demonstrated the opioid receptor-mediated antiapoptotic effect of dalargin on peripheral blood leukocytes under in vivo cold stress.

in the ability to increase the cold resistance of homeothermic organisms. Obviously, this eff ect of dalargin may be explained by its impact on cell ability to maintain ionic homeostasis in energy defi ciency, resulted from a decreased ATP production. It is known, that under normothermy, a significant part of ATP is used during protein translation (25-30%) and to ensure the Na + -K + -ATPase function (19-28%) [7]. The ability of cells to reorient ATP metabolism for ensuring an active ion transport is crucial for successful resistance to cold stress exposure. A disorder in Na + and K + concentration gradients between intra-and extracellular media causes the membrane depolarization, thus inducing an uncontrolled Ca 2+ infl ux through potential-dependent Ca 2+ channels. As a result, an intracellular concentration of free cytosolic Ca 2+ increases, activating thereby the Ca 2+ -dependent phospholipases and proteases, which further accelerate the cell and mitochondrial membrane depolarization and, fi nally, cell apoptosis or necrosis occur. Cells in inactive state use 20-80% of all produced ATP to ensure the Na + -K + -ATPase function [43]. Dalargin as endogenous enkephalins and other opioids appears likely inhibit some energy-dependent processes in cells (protein synthesis, primarily), and thus preserves a defi cient ATP to maintain ionic homeostasis with active ion transport involvement. Such an eff ect can be realized both directly, by interacting with opioid receptors on the membrane of cell itself, and indirectly, i. e. through the neuroendocrine system and the factors it produces. Dalargin administration to rats before simulating the moderate hypothermia prevented the lipid peroxidation activation, a decrease in erythrocyte antioxidant protection and stabilized the structural and functional state of their membranes [34]. The study of the role of opioid receptors and their ligands in mechanism of counteracting the cold stress-induced apoptosis is among the fundamental tasks in cryobiology. The data [1,12,17,19,31] reported, that the receptors to enkephalins were present on surface of not only nerve cells but immunocompetent ones as well.
Proceeding from the mentioned above, the objective herein was to investigate a protective eff ect of synthetic analogue of leu-enkephalin (Dalargin drug) on peripheral blood leukocytes of cold stressexposed homeotherms.
The 6-month-old white male Wistar rats with average body weight of (183 ± 15) g were used in the work.
Rats were injected intraperitoneally with Dalargin drug (Peptidnyye Tekhnologii, Russia) as a 0.25% solution of pharmaceutical substance at a dose of 100 μg / kg body weight, and with Naloxone (Zdorovia Narodu, Ukraine) at a dose of 500 μg / kg.
To study an antiapoptotic eff ect of dalargin on leukocytes under in vivo cold stress, the rats before experiment were divided into 4 groups (n = 5 in each): the group 1 (intact) comprised the animals received neither drugs, nor cold stress; the group 2 (control) was the animals, injected with saline and exposed to cold stress; in the group 3 the rats were administered with dalargin and subjected to cold stress; those animals, injected intraperitoneally with opioid receptor antagonist naloxone together with dalargin, as well as exposed to cold stress, made the group 4.
To simulate the cold stress, the rats were placed in the closed tanks (8 cm height) with water and ice mixture. The water temperature in tanks was 0-2°С, the water level was 5 cm. Under such conditions, the animals were standing at the bottom of the tank, unable to rise far more above the water level throughout the experiment. The room temperature was 20°C. Every 10 min, the animals were removed from the water and a rectal temperature was measured with electronic thermometer at 2 cm depth. When a rectal temperature reached 20ºC, the animals were then removed from water, thoroughly wiped and left for self-warming at room temperature. In 24 hours, when a deep hypothermia (20ºC) was achieved, the animals were sacrifi ced by decapitation. The rectal temperature of rats of all the groups at the moment of decapitation was ≈38ºС (37.8-38.1ºС). After decapitation, the blood was collected for leukocyte isolation using citrate buff er (3% sodium citrate, 2% glucose, pH 7.4) as anticoagulant. Leukoconcentrate was obtained by centrifugation for 25 min at 300 g. The total count and population composition of peripheral blood leukocytes were determined in blood smear fi xed with May-Grunwald solution and stained by Romanowsky (both solutions produced by Mini-Med, Russia). The leukogram was counted in 200 cells. The obtained results were interpreted by the total проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 31, №/issue 1, 2021 ном Май-Грюнвальда і забарвленому за Романовським (обидва розчини виробництва «Міні-Мед», Росія). Лейкограму виводили з розрахунку на 200 клітин. Отримані результати інтерпретували за загальним вмістом лейкоцитів, відносним вмістом нейтрофілів і лімфоцитів, а також за їх співвідношенням.
Leukocyte viability was measured by using confocal microscopy with dual acridine orange (AO) / ethidium bromide (EB) staining at fi nal concentrations of 0.001% [22]. Photomicrography was performed with an inverted confocal microscope LSM 510 META (Carl Zeiss, Germany). The AO and EB fl uorochromes were excited using argon laser with 488 and 543 nm wavelength, respectively. The AO and EB emissions were recorded at 530 and 620 nm, accordingly. The image was recorded using multitrack technique. The resulting image was obtained via overlaying two images registered in diff erent (green and red) region using AimImage Examiner software (Carl Zeiss). The ratio of living, apoptotic and necrotic cell number was determined by the ratio of green, orange and red pixels in the image using the image-processing program ImageJ (Laboratory for Optical and Computational Instrumentation, USA) [32].
The DNA fragmentation in leukocytes was determined by the method of D.P. Evenson [8], modifi ed for cells containing signifi cant number of RNA, since the AO, when binding to RNA molecules exhibited fl uorescence within the same range as in case of binding to single-stranded DNA fragments. Leukoconcentrate (0.20 ml) was mixed with 0.40 ml of buff er solution, including 0.1% Triton X-100 (Sigma Aldrich, USA); 0.15 M NaCl and 0.08 M HCl, pH 1.4. After 30 s, the specimens were supplemented with 1.2 ml of the solution, prepared by mixing 370 ml of 0.1 M citrate buff er (pH 6.0); 630 ml of 0.2 M Na 2 HPO 4 ; 1 mM EDTA; 0.15 M NaCl and 100 μg / ml RNAase A (Sigma Aldrich, USA) and incubated at 37°C for 20 min. After incubation, a chromatographically purifi ed AO (Polysciences, Inc., USA) in an amount of 100 μg / ml was supplemented to specimens and 3 min later they were examined with confocal microscope. The AO excitation was performed using argon laser with 488 nm wavelength, and the emission was recorded in green (530 nm) and red (620 nm) regions. Cell nuclei without fragmented DNA were stained with AO and fl uoresced green, but those, having both native and fragmented DNA, were yellow-orange. The number of green and orange pixels of each image was counted using the ImageJ software package (USA) . The results obtained after processing the photomicrographs of each sample were summarized and the ratio of green and orange pixels, corresponded to that of native and fragmented DNA in all the cells, was determined. Also, the total number of cells and that with fragmented DNA were visually counted проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 31, №/issue 1, 2021 дилася й фрагментована ДНК, -жовто-помаранчевим кольором. Кількість зелених і помаранчевих пікселів кожного зображення підраховували, використовуючи пакет програм «ImageJ» (США). Отримані після обробки мікрофотознімків кожної проби результати підсумовували і знаходили співвідношення зелених і помаранчевих пікселів, що відповідало співвідношенню нативної і фрагментованої ДНК у всіх клітинах. Також на знімках візуально підраховували загальну кількість клітин і кількість клітин із фрагментованою ДНК для визначення процентного вмісту останніх.
In both techniques, 10 photomicrographs were taken from each sample of leukoconcentrate so that the total number of fl uorochrome-stained cells was at least 1000.
The results were statistically processed with STATA software package (StataCorp LLC, USA) using the Kruskal-Wallis H-test and the Mann-Whitney U-test.

Results and discussion
The prolonged stress is known to induce different immune responses [25]. The ratio of lymphocyte subpopulations in spleen, lymph nodes and thymus was established to change in animals under stress conditions [28], as a result the relative count of cytotoxic T-killers (CD8 + ), as well as natural killers increased. These changes can not but aff ect the population composition of peripheral blood. The reported data showed the body cooling within a limited time period to stimulate the immune system, but too long cold exposure inhibited its normal functioning [20]. In particular, it is known that in peripheral blood in case of stress exposure to a body, the lymphopenia and neutrophilia may occur [11]. Cold exposure was established to increase the leukocyte count in peripheral blood mainly due to a sharp rise of granulocyte count [13].
The Table demonstrates that in blood of animals, received neither drug nor cold exposure (group 1), there were 8,500 -11,200 leukocytes in 1 ml (in average (9,600 ± 1,100) / ml). Herewith, the relative count of lymphocytes was 59-71.5%, and for neutrophils it made 11.7-28.8%. This almost threefold predominance of lymphocytes over neutrophilic granulocytes in blood is specifi c for animals of this species [23]. In rats of groups exposed to cold stress, the total count of peripheral blood leukocytes was within the normal limits, but a signifi cant decrease in the relative count of lymphocytes and an increased count of neutrophils were revealed. In animals of group 3, these indices were signifi cantly diff erent from those in rats of group 1, herewith these changes were signifi cantly less pronounced than in groups 2 and 4 (p ≤ 0.01).
Preliminary administration of dalargin to rats in 24 hrs after reaching a rectal temperature of 20ºC neutralized a negative eff ect of in vivo cold stress on peripheral blood leukocytes (Fig. 1). In animals of all the groups exposed to cold stress, the relative count of viable nucleated cells in leukoconcentrate was lower than in group 1 (Fig. 1A).
Таким чином, введення щурам перед початком моделювання холодового стресу даларгіну The relative count of viable cells in leukoconcentrate of animals from group 3 was signifi cantly higher than in groups 2 and 4 (p ≤ 0.01), but lower as compared with group 1. The relative count of apoptotic cells in leukoconcentrate (Fig. 1B) in groups 2 and 4 was signifi cantly higher than in groups 1 and 3 (p ≤ 0.01). In cold stress-exposed animals (groups 2-4), the relative count of cells in leukoconcentrate, whose membranes were permeable for EB (necrotized) was signifi cantly higher than in group 1 (Fig. 1C).
Notably, that the 'viable' leukocytes included all the AO-stained cells which fl uoresced green, regardless of the nucleus size. It means that among such cells were leukocytes at pyknosis or nucleus fragmentation stage, but the cell membranes of which were not permeable for EB. Determination of a relative count of fragmented DNA and a percentage of leukocytes with fragmented DNA in peripheral blood leukocytes of rats enables estimating a number of cells, being at earlier stages of apoptosis. The Fig. 2 and 3 show the impact of cold stress, dalargin and naloxone on these indices to be similar to that detected by dual staining. In all the animals, a cold stress caused a signifi cant increase in both the degree of DNA fragmentation and the relative count of peripheral blood leukocytes with fragmented DNA. Preliminary administration of dalargin to animals signifi cantly reduced both indices, moreover the degree of DNA fragmentation in cells decreased to that in group 1. Dalargin and naloxone Примітки: відмінності статистично значущі порівняно з групою 1(*) та з групами 2 і 4 ( # ), p ≤ 0,01. Notes: diff erences are signifi cant as compared with group 1 (*) and groups 2 and 4 ( # ), p ≤ 0.01.
4. Введення експериментальним тваринам перед початком охолодження налоксону усуває антиапоптотичний ефект даларгіну на лейкоцити периферичної крові, що свідчить про участь в механізмі дії останнього опіатних рецепторів. thermore, the glucocorticoids, which concentration in blood plasma increases in response to stress impact, are known to enhance the neutrophil viability indices as well [15]. Therefore, in addition to direct eff ect on blood cells, dalargin likely aff ected the nervous system structures, thus stimulated a protective neuroendocrine response of body to cold stress. The probability of membranotropic eff ect of dalargin, stipulated by its surface-active properties, should also be taken into account. Presumably, this leu-enkephalin, in addition to affi nity to specifi c opioid receptors, has the ability to incorporate into lipid shelf of cell membranes, by altering their physicochemical properties and aff ecting the ion pump activity and passive permeability for various ions. Similar properties have been described for tyroliberin, enkephalins, oxytocin, and some other regulatory peptides [2,30].
Our fi ndings show the enkephalins to play an important role in adaptation of homeotherms to life under low temperatures. The mechanism of enkephalin action, in particular on immune competent blood cells, needs further research.

1.
In peripheral blood of experimental rats exposed to cold stress by cooling down to 20ºC, in 24 hrs after warming up, the count of lymphocytes was found to be signifi cantly reduced, and the neutrophil one was increased.
2. In dalargin-injected animals before cooling, the ratio of peripheral blood neutrophils to lymphocytes was signifi cantly lower versus the control.
3. The dalargin introduction to experimental rats prior to cooling signifi cantly reduced the apoptosis indices in nucleated blood cells.
4. The administration of naloxone to experimental animals before cooling eliminated an antiapoptotic eff ect of dalargin on peripheral blood leukocytes, thus indicating the involvement of the latter in the mechanism of action of opioid receptors.