Effect of cold helium plasma on the catalytic activity of certain erythrocyte dehydrogenases of rat blood Влияние гелиевой холодной плазмы на каталитическую активность некоторых дегидрогеназ эритроцитов крови крыс

: The work is aimed at clarifying the effect of cold helium plasma on the catalytic properties of lactate-and aldehyde dehydrogenase in rat blood erythrocytes. The effect was studied in 20 white Wistar rats. Upon completion of the full course of exposures (1 exposure per day for 5 days), blood samples were taken from all animals with subsequent erythrocyte isolation performed by standard differential centrifugation for assessing the activity of lactate dehydrogenase (LDH) and aldehyde dehydrogenase (AlDH). When assessing LDH activity, both direct and reverse reactions were considered. Gas flow microwave ionisation was applied to the synthesis of cold plasma using a special device developed at the Institute of Applied Physics, Russian Academy of Sciences. The plasma treatment was established to provide stimulation of LDH activity in both direct and reverse reactions. In the direct reaction, erythrocytic LDH activity in rats with plasma-treated skin almost doubled (94 %) against 48 % of activity growth in the reverse reaction. Rat blood erythrocyte AlDH tends to moderate inactivation, with catalytic properties observed to decrease by 13 %. Thus, treating the skin of healthy rats with cold helium plasma was demonstrated to stimulate the energy metabolism of blood cells, with moderate activity inhibition for AlDH presenting one of the detoxification enzymes. The nature of the observed shifts indicates their adaptability. In general, according to the obtained data, the modulation of free radical processes was confirmed to play a key role in the molecular-cellular mechanisms of the cold helium plasma action on the biological system.


INTRODUCTION
In recent decades, the use of cold plasma in various therapeutic treatments has kindled the interest of biomedical specialists 1 [1][2][3][4]. The effectiveness of this treatment approach is confirmed by in vitro (on colonies of various microorganisms [1,5,6]) and in vivo (on infected skin abrasion models [5][6][7]) studies on the antibacterial activity of cold plasma-based therapies. The relevance of the antibacterial cold plasma effect is predetermined by the urgent need to find an alternative to antibiotics in the context of rapidly forming resistance in existing strains of pathogenic microorganisms [3,[5][6][7], though this has tended to eclipse research into other aspects of its effect on biological systems [1,8,9]. It should be noted here that both direct (at the place of treatment [4,6,9,10]) and indirect (including systemic [1,5]) realisation of these effects is possible. These particular aspects of the issue under discussion are discussed in less detail in the literature.
In previous in vitro experiments conducted by the authors, cold plasma was demonstrated to affect the oxidative and energy metabolism of biological systems along with their physical and chemical properties, as well as leading to an increase in antibacterial activity [8,[11][12][13]. Moreover, shifts were recorded in a number of metabolic parameters for blood [12] and the state of systemic hae-modynamics and microcirculation in animals with dermal administration of the studied treatment [14,15]. Although one possible cause of these transformations involves the effect on the catalytic activity of blood enzymes, no such confirmation of this hypothesis is provided in the experimental literature.
Against this background, the present study was aimed at clarifying the effect of cold helium plasma on the catalytic properties of lactate dehydrogenase (LDH) and aldehyde dehydrogenase (AlDH) in rat blood erythrocytes.

EXPERIMENTAL PART
The study was performed on 20 healthy sexually-mature male Wistar rats. The animals were divided into two groups equal in number. The first group (n = 10) presented a control group with no manipulations performed other than a single blood sampling. Rats of the second group (n = 10) were treated daily over the course of 5 days with cold helium plasma applied to a pre-epilated dorsal surface. A single treatment time comprised 1 min. Cold plasma was synthesised using a special device developed at the Institute of Applied Physics, Russian Academy of Sciences and based on the phenomenon of gas flow microwave ionisation [8,[11][12][13][14][15]. Grade A bottled helium was used as a source gas for obtaining the cold plasma.

ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY
Upon completion of the full course of exposure to cold helium plasma, blood samples were taken from all animals. Subsequent isolation of an erythrocyte suspension was carried out by differential centrifugation according to the standard method for evaluating the activity of lactate dehydrogenases (LDH) and aldehyde dehydrogenase (AlDH). The LDH activity was determined in the erythrocyte hemolysate admixed with distilled water (1:40 vol.) according to the method of G.A. Kochetov 2 . Both direct and reverse reactions were considered for evaluation of LDH activity.
AlDH activity was determined spectrophotometrically according to the method described by B.M. Kershengolts and E.V. Serkina (1981). The pro-tein content was specified using the modified Lowry method.
Statistical data processing was carried out using Microsoft Excel 2007 and the Primer of Biostatistics 4.03 software program.

RESULTS AND DISCUSSION
Cold helium plasma was shown to contribute to significant changes in the catalytic properties of erythrocyte enzymes. The studied treatment was observed to stimulate LDH activity in both direct and reverse reactions (see Fig. 1). However, the extent of these shifts is not the same: at the end of the course of exposure to cold plasma, the catalytic enzyme activity in both reactions is practically equalised, while, in the intact animals of the first group, a moderate predominance of the reverse reaction occurs. In this regard, in rats treated with cold plasma, the activity of erythrocyte LDH in the direct reaction almost doubled (94 %, p < 0.05 as compared to the intact animal level). Conversely, in the reverse reaction an increase of 48 % (p < 0.05) was observed. Indirectly, this can be seen as indicating a stimulating effect of the considered treatment on the intermediate element of energy metabolism with an increase in the production of pyruvate, the primary substrate of the Krebs cycle [16,17].
A different aspect of the modification was revealed with respect to AlDH (see Fig. 2). The specified enzyme related to the enzyme deto-xification system tends to a moderate inactivation with catalytic properties decreased by 13 % (p < 0.05 relative to the level detected for intact rats of the first group). This may be due to increased production of free radicals induced by external exposure to cold plasma. Although these compounds are intensively utilised by the antioxidant system of blood and tissues, their secondary (malon-dialdehyde) and tertiary (Schiff's bases) products require the involvement of appropriateenzymes in the detoxification process [18].

Рис. 2. Активность альдегиддегидрогеназы эритроцитов крови крыс (в нмоль НАДН/мин*мг белка; * -статистическая значимость различий по отношению к уровню, характерному для интактных животных)
This is confirmed by the increase in the concentration of malondialdehyde in rat blood erythrocytes exposed to the cold helium plasma demonstrated by the authors in previous studies [12,13]. Moreover, no stimulating effect of cold plasma on the intensity of free radical reactions in the membranes of these blood cells was registered. For this reason, together with the results obtained in the framework of this study, the plasma effect can be characterised as training and pro-adaptive.
It is also important to emphasise that the presented data indirectly indicate the consistency of the working hypothesis on the induction of free radical processes as one of the main systemic effects caused by cold helium plasma to the body, since all detected metabolic transformations resulted from the studied treatment are directly associated with a short-term increase in the intensity of free radical oxidation in the blood and tissues.

CONCLUSION
Thus, according to the dynamics of the catalytic properties of erythrocyte lactate dehydrogenase in direct and reverse reactions, treating the skin of healthy rats with cold helium plasma was established to stimulate the energy exchange of blood cells with simultaneous moderate inhibiting AlDH activity -one of the detoxification enzymes. The nature of the observed shifts indicates their adaptability. In general, according to the obtained data, the modulation of free radical processes was confirmed to play a key role in the molecular-cellular mechanisms of the cold helium plasma action on the biological system.