Сельскохозяйственная биология, 2010, № 2, с. 100-102.

УДК 636.2:591.22:591.111.1

MOBILIZATION OF FREE RADICAL PROCESSES IN NEUTROPHILS OF BLOOD IN CALVES AT A DIFFERENT FORMS OF BRONCHOPNEUMONIA

M.I. Retskii, S.V. Shabunin, G.N. Bliznetsova, G.A. Vostroilova

In calves of the Red-and-White breed with acute and chronic form of bronchopneumonia the authors investigated the mobilization of oxidation processes in neutrophils that is accompanied by augmentation of formation by them of awake metabolites of oxygen. More expressed rising of a level of generation superoxide anion, activity of a superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) descend at calves with the acute form of disease and less expressed — at calves with a chronic bronchopneumonia owing to an attrition antioxidant potential of neutrophils.

Key words: bronchopneumonia, neutrophils, superoxide anion, superoxide dismutase, glutathione peroxidase.

Oxidative stress (activation of free radical processes due to excessive formation of awake metabolites of oxygen - AMO) is the pathogenesis symptom of many diseases (1). Induction of oxidative stress at inflammation initiates the development of antioxidant deficiency (2). Leukocytes are the important endogenous source of AMO; the most significant production of AMO is found in neutrophilous leukocytes.
In neutrophils (unlike monocytes, macrophages and lymphocytes), oxygen consumption and AMO production (so-called respiratory storm) doesn’t provide cell life support, but implement the exclusively effector functions (3) of defensive reaction of an organism in physiological conditions. At some pathological states with expressed inflammatory processes, respiratory storm may cause cell death of healthy tissues, may suppress their antioxidant defense system and facilitate the development of an acute form into the chronic disease (4).
Lung tissue with an intense oxygen exchange is one of the most sensitive to the damaging action of AMO, which initiates free radical oxidation of lipids and the expression of proinflammatory cytokines (5). Therefore, the phenomenon of oxidative stress is essential in pathogenesis of bronchopulmonary diseases (6).
The purpose of our study was to evaluate the intensity of free radical processes in neutrophils and their relation to the dynamics of respiratory pathology in calves.
Methods. The study was carried out in the cattle farm of "Voronezhpischeproduct" Ltd. (Novousmansky district, Voronezh region) in 2008. 20 calves the Red-and-White breed aged 1,5-2,0-months were separated in two groups depending on the severity of pathological process: group I included 8 animals with the acute bronchopneumonia, group II - 5 animals with the chronic form of disease (control - 7 clinically healthy calves).
The blood neutrophils were obtained by centrifugation in a double density gradient of fikoll with verografin (1,076 and 1,120) (suspension of 106 cells / ml). The resulting suspension was analyzed: the rate of generation of superoxide anion (O₂·) was determined as described (7), superoxide dismutase (SOD) activity - by the inhibition of adrenaline oxidation in alkaline medium (8), glutathione peroxidase (GPO) activity - with hydrogen peroxide as a substrate (9). Optical density was measured spectrometrically (Shimadzu UV-1700 spectrophotometer, «Shimadzu Corporation», Japan). The antioxidant potential of neutrophils was evaluated using so-called coefficient of reservation (CR), proposed by T.I. Gavrilenko et al. (10), which is defined as a ratio of MPO activity to the rate of generation of superoxide anion.
Statistical processing of data was performed using Statistica 5.5 application software.
Results. The authors established, that the dynamics of oxygen metabolism in neutrophils from calves with acute and chronic forms of bronhopnevmonia (groups I and II, respectively) was similar, but in animals of group I the more pronounced changes were observed:  an augmentation of О2· generation in cells (Table) by 1.35-fold (P <0,05) and 1.09-fold (P> 0,1) compared with control and group II.

This fact may flow from the increase in bactericidal activity of neutrophils due to the intense transfer of О₂·  into environment with its consequent conversion into hydrogen peroxide, as was evidenced by the higher activity of SOD and GPO in the neutrophils suspension.
The coefficient of reservation (CR) in calves with acute pneumonia was significantly higher (P <0,01). At the chronic form of disease, CR was insignificantly different from the value of clinically healthy animals (P> 0,1) owing to the attrition of antioxidant potential of neutrophils during the development of chronic pathological process. This is also proved by decrease in activity of SOD and GPO in neutrophils at the chronic form of bronchopneumonia.
The analysis of data obtained has revealed a direct correlation between the quantity of О₂· produced by neutrophils and GPO activity; in animals with acute bronchopneumonia, the coefficient of correlation was higher (r = +0,79, P <0,02) than at the chronic form of disease (r = +0,37, P> 0,05).
The progress of imbalance in the system “free radical oxidation - antioxidant protection” and the attrition of antioxidant potential of neutrophils are demonstrated by weakening the correlation between the level of О2· generation and the activity of selenium-containing GPO, which affects the reduction of inorganic peroxides, as was found in group II. This fact is connected to the attrition of chronic stress state at the development of chronic pathological process (11).
Thus, the development of pneumonia is accompanied with the mobilization of oxidation processes in neutrophils that is evidenced by augmentation of formation by them of awake metabolites of oxygen.
More expressed changes in the rate of О₂· generation, activity of superoxide dismutase (SOD) and glutathione peroxidase (GPO) descend in calves with the acute form of disease and less expressed — in calves with a chronic bronchopneumonia owing to an attrition of antioxidant potential of neutrophils.

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