DOI 10.1007/s10517-018-3981-5

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Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  164,  No.  3,  January,  2018 PHARMACOLOGY AND TOXICOLOGY

Signal Mechanism of the Protective Effect of Combined Preconditioning by Amtizole and Moderate Hypoxia O. S. Levchenkova, V. E. Novikov, E. S. Abramova, and Zh. A. Feoktistova

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 164, No. 9, pp. 298-301, September, 2017 Original article submitted March 21, 2017 The content of regulatory proteins involved in adaptation to hypoxia and ischemia was studied in brain rat homogenate under conditions of normoxia and after bilateral ligation of the common carotid arteries. Preconditioning with amtizole in combination with moderate hypoxia increased the levels of HIF-1α, erythropoietin, vascular endothelial growth factor under conditions of normoxia. During experimental ischemia, combined preconditioning led to stabilization of the content of these regulatory proteins at the level of intact control and to a decrease in glycogen synthase-3β kinase activity. This pattern of changes in regulatory proteins was noted during the early and late periods of preconditioning. Key Words: preconditioning; HIF-1α; erythropoietin; vascular endothelial growth factor; glycogen synthase-3β kinase The possibility of pharmacological preconditioning (PC), i.e. induction of the mechanism of metabolic adaptation to hypoxia and ischemia by pharmacological agents, including those with marked antihypoxic effect, is actively discussed in the scientific literature [2,6,11]. Substances with antihypoxic activity can themselves induce metabolic adaptation or potentiate the action of hypoxic PC [4,9]. In our previous studies, the effect of pharmacological (administration of amtizole) and hypoxic (moderate hypobaric hypoxia) PC on the life expectancy of animals in different periods of adaptation to hypoxia and ischemia was studied on models of acute hypoxia and global incomplete cerebral ischemia. A higher efficacy of the combined PC (pharmacological and hypoxic) in comparison with PC with moderate hypoxia or course administration of amtizole was received [5,8]. Here we studied signaling stage in the mechanism of realization of combined PC with amtizole and hypobaric hypoxia. Department of Pharmacology, Smolensk State Medical University, Smolensk, Russia. Address for correspondence: os.levchenkova@ gmail.com. O. S. Levchenkova

MATERIALS AND METHODS The study was performed on white Wistar rats (n=36) weighing 200-220 g obtained from Stolbovaya Breeding Center, Russian Academy of Sciences. All studies were conducted in accordance with Rules of Laboratory Practice (Order No. 708n of the Ministry of Health and Social Development of the Russian Federation, August 23, 2010). At the preliminary stage, the animals were divided to highly and low-resistant to hypoxia [2]. One month after testing, the rats demonstrating low resistance to hypoxia were taken into the main experiment, because low-resistant specimens develop urgent and long-term adaptation to hypoxia, which, in turn, is associated with expression of HIF-1α [7,13]. Combined PC was performed during 6 days with alternation of antihypoxant amtizole and hypobaric hypoxia (HBH). On days 1, 3, and 5 of the experiment, the animals received intraperitoneal injections of amtizole in a dose of 25 mg/kg (2.5% solution was prepared ex tempore; the substance was synthesized at the Department of Pharmacology, S. M. Kirov Military Medical Academy). On days 2, 4, and 6, moderate HBH was modeled; the animals were placed under a glass bell and the air was evacuated with a Komov­ 0007­-4888/18/16430320 © 2018 Springer Science+Business Media, LLC

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skii pump to rarefaction of 410 mm Hg, which corresponded to an altitude of 5,000 meters above see level (HBH-5000), the exposure was 60 min. The levels of HIF-1α, erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glycogen synthase-3β kinase (GSK-3β) in the brain homogenate were measured by ELISA. Plates were used to quantify measurement these proteins in homogenates of rat tissue (SEA028Ra, SEA143Ra, SEA798Ra, SED317Ra 96 Tests ELISA for EPO, VEGF, HIF-1α, and GSK-3β, Cloud-Clone Corp.). The procedure was conducted in accordance with the protocol provided by the manufacturer. Two experimental series with 3 groups in each (control group and two experimental ones) were performed. In series I, the level of regulatory proteins was assessed under conditions of normoxia: 1 h after termination of combined PC (early adaptation period), 48 h later (late adaptation period), and in intact animals (control). In series II, the level of regulatory proteins was evaluated 1 day after ischemia modeling. Acute brain ischemia was simulated in rats anesthetized with chloral hydrate (8% solution; 400 mg/kg intraperitoneally) by simultaneous bilateral ligation of the common carotid arteries (CCA) in 1 h (early period; group 1) and 48 h (late period; group 2) after termination of combined PC, animals with ischemia served as the control. Statistical processing of the results was performed using BioStat 2009 software. Nonparametric Mann— Whitney U test was used to compare the data. The differences were significant at p≤0.05.

RESULTS Despite the fact that HIF-1α undergoes proteasomal degradation under conditions of normoxia, its content in the brain of intact rats was relatively high, which fully agrees with published data [7]. Combined PC with amtizole for 6 days increased the level of HIF-1α in brain homogenate in comparison with that in the intact control group both in early (p=0.022) and late (p=0.037) period of PC (Table 1). In the group of animals with ischemia, the level of HIF-1α one day after CCA ligation was higher than in the intact group (p=0.092), which agrees with a previous report [14], where a significant increase in HIF-1α protein expression immediately after simulation of cerebral ischemia with a peak in 12 h and further gradual decline was demonstrated. Combined PC prior to simulation of ischemia was followed by a decrease in the level of HIF-1α in the brain homogenate 1 day after CCA ligation. In the group of animals in whom ischemia was modeled 1 h after the last session of PC, the HIF-1α level did not differ from intact control. In animals with ischemia modeled during the late period after PC (48 h), HIF-1α level was lower than in the ischemic group and in the corresponding group of PC without ischemia and did not differ from intact control. In series I (normoxia), EPO content in the brain homogenate was higher in both experimental groups. It is natural that the expression of EPO increases against the background of HIF-1α accumulation. In

TABLE 1. Effect of Amtizole and Moderate Hypoxia on the Level of Regulatory Proteins in the Brain before and after Bilateral CCA Ligation in Rats (n=6; Me (25%; 75%) Group

HIF-1α, ng/ml

EPO, pg/ml

VEGF, pg/ml

GSK-3β, ng/ml

1.7

431.65

86.9

1.8

(1.52; 1.87)

(344; 447.5)

(75.6; 98.2)

(1.52; 2)

Amtizole+HBH-5000 1 h after PC

2.1* (2.1; 2.5)

606.6* (590.9; 611)

107* (94.9; 108)

2 (1.9; 2.1)

Amtizole+HBH-5000 48 h after PC

2.85* (2.07; 3.17)

686* (658.4; 707.7)

99.75 (96.1; 100.2)

1.7 (1.6; 1.9)

Series I Intact control

Series II Ischemia

2.0&

584.4*

82.8

2.25

(1.82; 2.17)

(484.4; 649.7)

(77.5; 91.4)

(2.1; 2.3)

Amtizole+HBH-5000 (ischemia after 1 h)

1.6 (1.35; 2.15)

485.5+ (390.4; 520.2)

78.45+ (71.2; 81.2)

1.16*о (0.7; 1.6)

Amtizole+HBH-5000 (ischemia after 48 h)

1.55ох (1.32; 1.77)

516.9* (500.1; 627.8)

92.3# (89.8; 106.4)

1.25*о (1.3; 2.2)

Note. p<0.05 in comparison with *control, oischemia group, +the corresponding parameter 1 h after PC, x48 h after PC, #ischemia after 1 h; &p<0.1 in comparison with the intact control group.

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the control group with ischemia, EPO content was also higher than in the intact control (p=0.025). In series II, the level of EPO in experimental groups decreased in comparison with the corresponding groups in series I. Thus, the level of EPO in the early period of PC did not differ from the intact control and was significantly lower than in the corresponding group of PC without ischemia (p=0.0039). During the late period of PC, the level of EPO was higher than in the intact control (p=0.016) and did not differ significantly from the group with ischemia. Analysis of the combined use of amtizole and hypoxia on the level of VEGF in rats under conditions of normoxia, we found that PC increased level of VEGF 1 h after the last presentation of PC (p=0.05), and no significant differences were found 48 h later. Analysis of VEGF content in brain homogenate of rats with cerebral ischemia reflecting induction of HIF-1α-dependent angiogenesis revealed no difference from the intact control group. Combined PC before ischemia modeling in the early adaptation period (ischemia after 1 h) resulted in a decrease of VEGF in comparison with the corresponding group without ischemia (p=0.01); no differences from intact control and control with ischemia were observed. In the late adaptation period (ischemia after 48 h), the parameter differed from that in the group of early PC with ischemia; no differences from other groups were found. The role of angiogenic factor VEGF in the development of the cytoprotective effect under the action of PC is ambiguously interpreted in scientific literature. On the one hand, VEGF belongs to HIF-1α-dependent genes and is a marker of long-term adaptation to hypoxia [5]. At the same time, the neuroprotective effect of ischemic PC assessed 24 h after cerebral ischemia that was modeled 48 h after ischemic PC (late period) was associated with significant drop of VEGF in the brain [12]. Analysis of GSK-3β activity showed that combined PC does not affect the level of the enzyme under normoxic conditions. In control rats with cerebral ischemia, the level of GSK-3β did not differ significantly from the intact control on the next day after surgery. After preliminary combined PC, the content of GSK-3β was significantly lower in both early and late period of PC in comparison with intact control and the control group with ischemia. This fact indicates that the mechanism of them neuroprotective action of combination of amtizole with hypoxia includes modulation of GSK-3β activity. Amtizole and hypoxia are not direct inhibitors of GSK-3β under normoxic conditions, but can reduce activity of this enzyme of the protective kinase cascade under conditions of experimental ischemia. It is known that inhibitors of GSK-3β

suppress opening of mitochondrial pore, which in turn can prevent apoptosis of cells [1]. Thus, regulatory proteins HIF-1α, EPO, and VEGF play a signal role in the induction of adaptation processes in the mechanism of the protective effect of the combined PC with amtizole under conditions of hypoxia and ischemia. Under the influence of combined PC, their content in the brain of experimental animals increased in normoxia and was stabilized at the level of control in ischemia. In addition, this mode of PC affects GSK-3β activity; inhibition of this enzyme in ischemia can result in potentiation of the protective effect due to inhibition of induction of mitochondrial pore. This pattern of changes in regulatory proteins was noted in the early and late period of PC. The detected mechanisms do not exclude participation of other signaling molecules in the realization of PC effect [10]. ELISA used for analysis of regulatory proteins involved in signal transduction during PC is more convenient and easy in comparison with traditional Western blotting [7,11]. The immunoenzyme method is more often used for analysis of HIF-1α and other proteins in the blood serum [3], in this work it demonstrated good reproducibility on brain tissue homogenate.

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