CHANGES OF ENERGY BALANCE OF RAT LIVER AND PANCREAS UPON GASTRIC HYPOACIDITY AND SEARCH OF APPROACHES FOR ITS RECOVERY

ИЗМЕНЕНИЯ ЭНЕРГЕТИЧЕСКОГО БАЛАНСА ПЕЧЕНИ И ПОДЖЕЛУДОЧНОЙ ЖЕЛЕЗЫ КРЫС ПРИ ГИПОАЦИДНОМ СОСТОЯНИИ ЖЕЛУДКА И ПОИСК ПОДХОДОВ К ЕГО ВОССТАНОВЛЕНИЮ Установлено, что в печени и поджелудочной железе крыс при длительной желудочной гипохлоргидрии нарушается энергетический баланс, о чем свидетельствует понижение содержания АТФ на фоне повышения уровня АДФ и АМФ. Мультипробиотик "Симбитер" способствует восстановлению равновесия адениловых нуклеотидов в исследуемых органах крыс с гипоацидным состоянием желудка. Ключевые слова: длительная желудочная гипохлоргидрия, поджелудочная железа, адениловые нуклеотиды, мультипробиотик.

Introduction.Cerebrovascular diseases are currently the actual problem of modern medicine since they are one of the leading cause of disability and the second most common cause of mortality and morbidity among the working population aged 40-60 years [5].A dominant role in the structure of all cerebrovascular diseases belongs to acute ischemic cerebrovascular accident -up to 70 % of all strokes are considered as ischemic (cerebral infarction).In accordance with the main mechanism of development there are atherothrombotic and cardioembolic stroke subtypes.In the first case, the acute cerebral blood flow failure is caused by blood clots formation and blockage of the lumen of blood vessels in the place of atherosclerotic plaque localization; occlusion of the arteries that nourish the brain by cardiac emboli takes place in the case of cardioembolic stroke [5].Clinical features that support the diagnosis of these pathologies include motor, language, visual, coordination and cognitive impairments.More complete laboratory screening of patients with ischemic cerebrovascular disease for currently identified prothrombotic states will probably increase the percentage of strokes attributed to disorders of hemostasis.One of the leading pathogenetic factors of cerebrovascular ischemia is the disruption of homeostasis that results in intravascular thrombosis, changes in blood rheology and microcirculation disturbances in ischemic tissues [12].Detailed definition of the molecular mechanisms underlying the development of these diseases is crucial not only in terms of the creation of new drugs directed action, guide the most effective care and therapy, but also will provide great perspectives in the field of clinical diagnostic of pathological manifestations of hemostasis.
Given that treatment of stroke is limited, the best way to reduce the negative effects of this pathological conditiontimely primary prevention, individual approach to treatment and rehabilitation.Study of the causes and mechanisms of stroke development and search of new biomarkers that can be used in diagnostics to identify individuals at high risk of disease development is very relevant for today.
Taking into account the involvement of blood coagulation system in the pathogenesis of ischemic strokes, the main goal of our work was to investigate the functional state of prothrombin -a precursor of key regulatory enzyme thrombin that converts soluble fibrinogen to fibrin monomers, further polymerization of that is the basis for the formation of a blood clots.
Materials and methods.Donor's plasma (n=66 for patients with atherothrombotic ischemic stroke and n=56 for patients with cardioembolic ischemic stroke) was obtained by cubital vein puncture using tubes with sodium citrate solution (38 g/l) in the final ratio of 9:1.Patients' plasma one day after the ischemic stroke was used in our experimental work.Determination of prothrombin time was supplied by kits and reagents of "Renam" company (Belarus).Prothrombin obtained by sorbing the vitamin K-dependent proteins on barium sulfate followed by extraction with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.2 M NaCl and 0.02 M EDTA.Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate was performed according to the method Laemmli using tris-glycine system.Immunoenzymatic assay and Western blot method were carried out in consist of standard protocols for soluble proteins [8,11].Prothrombin and its derivatives identification was done using rabbit polyclonal antibodies (Shijin International, Mongolia) to prothrombin.Mathematical and statistical processing of obtained results was performed using the computer program Origin 7.0.The difference between groups was analyzed by standard Student's t-test.P values less than 0.05 were considered statistically significant.Present data are typical for series of repeated experiments (at least five in each series).
Results and discussion.Hemostasis is one of the most complex physiological self-defense systems which ensure blood fluidity, the maintenance of the rheological properties in the physiological ranges and rapidly induces hemostatic plug formation in order to stop or limit bleeding [4,12].Haemostatic imbalance in one or another direction, due to the influence of both internal and external factors increases the risk of bleeding or thrombosis.The study of the functional state of prothrombin causes of considerable interest because of prothrombin involvement not only in the realization of a number of physiological reactions but also because of interfering in major physiopathological processes, in particular, the regulation of vascular tone, wounds healing, immune response implementation, development of inflammation, tumors, Alzheimer's disease, atherogenesis and others [11].The evolution of our knowledge of the physiology of hemostasis has numerous implications for therapy.It is known that changes in hemostatic system at ischemic stroke are accompanied by significant activation of blood coagulation system.Defects associated with the pathways of blood coagulation can be assessed with specific tests.In order to estimate the activity of factors of extrinsic pathway of blood coagulation, we determined prothrombin time (PT) in patients suffering from atherothrombotic and cardioembolic strokes.Determination of the prothrombin time was performed at different time points, in particular on the 1th day after ischemic stroke as well as on the 7th and 14th day after stroke development.According to obtained results (Table 1) prolongation of PT was observed in patients with both subtypes of stroke but only on the first day after stroke development.So, prolongation of PT by 1.12 and 1.19 times was established respectively for atherothrombotic and cardioembolic stroke.As data of anamnesis of patients allowed excluding hereditary coagulopathy, disorders of the functional state of the liver, caused by illnesses of different etiologies, presence of disseminated intravascular coagulation syndrome, PT prolongation is mainly due to the application at the period of blood sampling anticoagulation medications or can indicate the presence of nonphysiological inhibitors of coagulation cascade and phospholipid-dependent reactions in the bloodstream [7].So, one of the recently discovered and insufficiently studied risk factors for ischemic stroke development is a production of antibodies directed toward a phospholipidbinding protein, in particular to prothrombin.The ability of these antibodies reduce the functional activity of the target proteins promotes disturbance of pro-and anticoagulant balance.To clarify the characteristics of blood coagulation system, we measured the total pool of molecules of prothrombin origin.Using of polyclonal antibodies allowed us to estimate the content not only directly prothrombin, but also molecules containing epitopes of prothrombin origin, in particulr, intermediates of prothrombin activation, thrombin molecules and their complexes.As seen in the results presented in Figure 1, the increase of pool of prothrombin derivates occurs at both types of stroke.Analysis of Western blot results using specific polyclonal antibodies to prothrombin showed the presence of protein fractions in the range of molecular weights corresponding to both prothrombin and its degraded forms.Thus, the data presented in blotogram provide evidence that protein fractions with molecular weight about 72 and 37 kDa (track 2, 3) are formed, respectively, by prothrombin and thrombin molecules [4].It should be note that generation of thrombin can be confirm by accumulation in plasma N-terminal part of prothrombin moleculefragment F 1+2 [9], the molecular weight of that is about 40 kDa.Physiological activation of prothrombin, except formation of thrombin, can be accompanied by accumulation of a number of intermediates, some of that possess enzymatic activity (meizothrombin, meizothrombin 1) and other one belongs to the functionally inactive derivatives (prethrombin 1, 2, fragments 1, 2, 1+2).Protein fractions with molecular weight about 50 and 37 kDa (track 2, 3) may consist of prethrombin 1 and 2, which is agreed with literature, according to that their appearance in the bloodstream serves as an early marker of intravascular coagulation [4].Meizothrombin (mol.weight about 70 kDa) is one of the transient intermediates that are produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface.An undisputed and potentially important role for meizothrombin is as a mediator of vascular constriction.Meizothrombin has potent vasoconstrictive activity, approximately fivefold greater than α-thrombin.Meizothrombin participation in the mechanisms of thrombogenesis is related to its ability to influence on platelet hemostasis.Causing a secondary irreversible platelet aggregation meizothrombin helping to attract new platelets in formation of blood clots [3].The insensitivity of meizothrombin to the action of the main physiological thrombin inhibitors acquires special importance during anticoagulation therapy.An additional factor that result in increase of procoagulant potential of hemostasis under conditions of meizothrombin accumulation is the ability of meizothrombin to activate factors V, VIII, XIII of blood coagulation [2,3], and to catalyze the conversion of fibrinogen into insoluble fibrin, that promotes the formation and stabilization of fibrin clots.
Taking into account the fact that meizothrombin has both pro-and anticoagulant properties, that are depend on the degree of saturation of the molecule by sodium ions, it should be emphasized that hypersodiumia (characteristic feature of thrombotic states) causes realization mainly procoagulatic activity of meizothrombin [2,6].Presence in blotogram fractions with molecular weights about 100, 130, 250 kDa may be result of formation of prothrombin and its derivatives complexes with proteins at the term of cardioembolic stroke progression.So, for example, the appearance in the bloodstream of thrombin-antithrombin III complexes (molecular weight of 100 kDa) is regarded as one of the diagnostic criteria for thrombosis development [4].

Figure 1 .
Figure 1.Level of prothrombin molecules pool: 1 -control; 2 -patients with atherothrombotic stroke; 3 -patients with cardioembolic stroke; * -p ≤ 0,05Level of prothrombin pool at atherothrombotic stroke exceeded control value by 10%, while the development of cardioembolic stroke was accompanied by an increase in the content of prothrombin by 25% compared to the same reference group.As more expressed changes in prothrombin derivates content were observed in the case of