1. Introduction

In the multifaceted pathogenesis of hemorrhagic shock, along with disorders of cardiovascular system, microcirculation, acid-base status, water-electrolyte balance, hemocoagulation and blood rheology, a major role is played by hypoxia of complex genesis, which contributes to disruption of energy exchange, activation of lipid peroxidation and complex suppression of antioxidant defense system [1,3]. Recently, much attention has been paid to the development of effective antihypoxic agents of metabolic type of action for the treatment of extreme conditions [6,10,13]. One such remedy is a new antihypoxic remedy "Rheoambrasol", containing a complex of polysaccharide derived from local plant raw materials and metabolite of the Krebs cycle, capable of protecting cells from free radical oxidation and restore the disturbed energy metabolism in cells under hypoxic conditions [11].

2. Aim of the Study

The aim of the work is to study the effect of “Rheoambrasol” on the content of hypoxia-inducible factor HIF-1α, lipid peroxidation and antioxidant system in the liver in hemorrhagic shock.

2.1. Materials and Methods

The work was performed on a model of hemorrhagic shock on 40 male rabbits weighing 2.2 ± 0.2 kg.

Animals were divided into 4 groups:

Ι - intact (n=20),

ΙΙ - after hemorrhagic shock (n=16),

ΙΙΙ - after hemorrhagic shock and subsequent infusion of “Rheopoliglyukin” (n=7),

ΙV - after hemorrhagic shock and subsequent infusion of the new drug “Rheoambrasol” (n=8).

Hemorrhagic shock was modeled by fractional bloodletting from the femoral artery for 1 hour until the blood pressure dropped to 40 mm Hg. Art., followed by reinfusion of 1/3 of the released blood [11]. The total amount of blood released into the reservoir was 28.0±2.6 ml/kg. 1 hour after hemorrhagic shock, the animals received a single infusion of "Rheoambrasol" in the experimental group IV and the drug "Rheopoliglyukin" in group III (comparison) in a volume of 40 ml per 1 kg of body weight. 1a).

In dynamics, the content of hypoxia factor (HIF-1α) in the blood was studied, which was studied by the method of enzyme-linked immunosorbent assay (ELISA) using kits. The measurements were carried out on an MR 96 analyzer (Myndray, China).

The content of MDA, diene ketones, diene conjugates, glutathione reductase (GR), glutathione peroxidase (GPO), activity of superoxide dismutase (SOD) and catalase was determined in the liver homogenate.

MDA level was determined according to the method of L.I. Andreeva et al. using “TBK-AGAT” kits (AGAT). The products were calculated using the molar extinction coefficient and expressed in nmol/mg. Diene conjugates and diene ketones were determined according to the method of Titeeva G.R. and Korovina N.N. [3,7,15]

The activity of AOS enzymes was expressed in units (units) per gram of raw liver weight, and as specific activity [4]. The activity of catalase was determined by the method of M.A. Korolyuk et al. (1998), the principle of which is based on the ability of H²O² to form a stable coloured complex with molybdenum salts. Measurements were performed at a wavelength of 410 nm [8]. Catalase activity of the samples under study was determined by spectrophotometer and expressed in mmol/min/g protein.

SOD activity was determined according to the method of V.G. Mkhitryan et al. (1978), which was calculated by the percentage of inhibition (T%) of tetrazolium blue reduction in alkaline medium. SOD activity was expressed in units/min x mg of protein [5]. Superoxide dismutase (SOD) activity was expressed in mmol/min/mg protein [2,9]. Purified SOD preparation (ICN Biomedicals, USA) was used as a standard [14].

The activity of GPO was determined by the accumulation of oxidized glutathione (GSSG) as a result of lipoperoxide decomposition. The activity of the enzyme was expressed in units/min x mgHb per min. The activity of erythrocyte glutathione reductase (GR) was determined in the reaction medium of phosphate buffer at 340 nm and by NADPH*H decrease and expressed in μM NADPH2/min x g Hb (Vlasova S.N. et al., 1990) [12].

All measurements were made on a spectrophotometer "UNICO" (USA).

Statistical processing of the obtained data was performed using Student's test using "Microsoft Office Excel" and "Biostatistics 4.03" programs. The criterion of statistical reliability was p<0.05 m.

3. Results of the Study

The results of the study obtained in hemorrhagic shock showed that the condition of experimental rabbits, was extremely severe, as evidenced by a sharp, statistically significant increase in the content of the hypoxia factor (HIF-1α), by 6.8 times (p<0.001) (Fig. 1). Increased level of HIF-1α indicates the development of hypoxia and disorders of hepatocyte oxygenation in hemorrhagic shock.

Figure 1. Changes in the concentration of hypoxia-inducible factor (HIF-1α) in rabbits after hemorrhagic shock and infusion of infusion drugs

As is known, the accumulation of lipid peroxidation products in liver cells during hemorrhagic shock leads to inhibition of the enzymatic system of antioxidant defense. For this purpose, enzymes of antioxidant activity were determined in hepatocytes. The results of the study showed a decrease in catalase activity by 2.2 times (p<0.001 GPO - 1.5 times (p<0.001), and SOD - 1.7 times (p<0.001), and GR – 1.3 times (p<0.001).

Figure 2. Changes in MDA in the liver during hemorrhagic shock (HS) and after infusion of infusion drugs

It is known that accumulation of lipoperoxidation products in liver cells during hemorrhagic shock leads to inhibition of the enzymatic system of antioxidant protection. For this purpose, antioxidant activity enzymes in hepatocytes were determined. The results showed a 2.2-fold decrease in the activity of catalase (p<0.01) glutathione peroxidase - 1.5 times (p<0.01), and superoxide dismutase - 1.7 times (p<0.01), and glutathione reductase - 1.3 times (p<0.01).

Thus, in hemorrhagic shock there is a significant accumulation of highly toxic MDA, diene ketones and diene conjugates in the liver, and suppression of compensatory possibilities of antioxidant protection. To eliminate these changes we propose a new drug that has antihypoxant, antioxidant and membrane-protective effect and is able at the cellular level to restore the function and viability of hepatocytes cells, including liver cells.

After using “Rheoambrasol” in hemorrhagic shock we observed a 5.1-fold (p<0.01) decrease of high hypoxia-inducible factor (HIF-1α), and after using “Rheopolyglukin” it decreased 2.6-fold (p<0.01) (Fig. 1), indicating an antihypoxic effect of the new drug.

Experimental therapy with “Rheoambrasol” in GS in the liver reduced the intensity of LPO processes. Thus, the content of MDA decreased by 2.4 times (p<0.01), diene ketones - by 1.5 times (p<0.01), diene conjugates - by 1.8 times (p<0.01), relative to the group with hemorrhagic shock.

It should be noted that, compared with the result after “Rheopolyglukin” treatment, after “Rheoambrasol” administration the indicators of the recovery processes were more pronounced: the content of MDA was 75% lower (p<0.01), diene ketones - by 23.6% and diene conjugates - by 34.2% (p<0.01) (Table 1).

Table 1. Changes in the intensity of lipid peroxidation and the state of the antioxidant system of the liver in experimental hemorrhagic shock and after infusion of drugs (M±m)

Infusion therapy of hemorrhagic shock with the drug "Rheoambrasol" led to an increase in the activity of antioxidant system enzymes in the liver, relative to their values after hemorrhagic shock: catalase activity was restored 1.9-fold (p<0.01), superoxide dismutase 1.6-fold (p<0.01), glutathione reductase 1.4-fold (p<0.01) and glutathione peroxidase 1.3-fold (p<0.01) (Table 1). Compared with the values of these indicators obtained after infusion of “Rheopolyglukin”, 1 hour after administration of the new blood substitute "Rheoambrasol" in the liver catalase activity was higher by 36,5% (p<0,01), SOD - by 37,1% (p<0,01), GH - by 40,7% (p<0,01) and HPO - by 17.6% (p<0,01).

Survival of animals after “Rheoambrasol” infusion was 9 of 15 (66.6%) (p<0.05) and was 28.3% higher than in the comparative study with reopolyglucin infusion.

The conducted studies showed that hemorrhagic shock was accompanied by a sharp statistically significant increase in HIF-1α by 6.8 times (p<0.001), activation of LPO processes and suppression of the compensatory capacity of antioxidant protection in the liver in animals. Pharmacotherapy with “Rheoambrasol” leads to a decrease in the values of hypoxia inducible factor-HIF-1α by 5.1 times (p<0.001), inhibition of lipid peroxidation processes and a more pronounced restoration of AOS enzyme activity, compared with “Rheopolyglukin” therapy. This is due to the fact that the basis of the antihypoxic and antioxidant action of “Rheoambrasol” is the bioenergetic mechanisms of the succinate metabolite, which is oxidized along the succinate oxidase pathway of the respiratory chain into mitochondria. Due to this, during hypoxia, when the NADH-dependent oxidation pathway is limited, activity and the ability for oxidative phosphorylation at the second and third conjugation points are ensured, which helps to maintain a higher level of macroergs. Thus, the mechanism of the antihypoxic action of “Rheoambrasol” is associated with its specific effect on energy metabolism with an increase in the degree of cell energization [9].

The most powerful of all oxidation substrates is succinate, which is capable of restoring oxidized NAD+ of the initial part of the respiratory chain due to electron reverse transfer reactions, and the plant-derived polysaccharide facilitates the penetration of succinate into the cell and its subsequent oxidation in the respiratory chain, which enhances the antihypoxic and antioxidant properties of the drug "Rheoambrasol".

4. Conclusions

The new drug "Rheoambrasol" has an antihypoxant effect, reducing the level of hypoxia-inducible factor HIF-1α by 5.1 times (p1<0.05) and increases the survival rate of animals with hemorrhagic shock.

The use of a new drug "Rheoambrasol" in hemorrhagic shock contributes to the suppression of the intensity of lipid peroxidation processes and activates the activity of AOS enzymes in the liver.

"Rheoambrasol" in hemorrhagic shock turned out to be an effective means of protecting the liver from free radical oxidation, surpassing "Rheopolyglukin" in its effectiveness.