49 b IO M E d Ic A L S c IE N c E S ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 Address for correspondence: Hanna Saturska, Department of Social Medicine, Health Care Management, Economy and Medical Statistics, I. Horbachevsky Ternopil State Medical University, 1 Maidan Voli, Ternopil, Ukraine, 46001 Tel. +380352527233 E-mail: saturska@tdmu.edu.ua DOI 10.11603/ijmmr.2413-6077.2016.1.6383 INfLUENcE Of TRImETAZIdINE mETABOLIc ThERAPY ON cONNEcTIVE TIssUE mETABOLIsm IN EXPERImENTAL dIffUsE IschEmIc NEcROTIc cARdIOscLEROsIs IN RATs WITh dIffERENT RATEs Of hYPOXIA REsIsTANcE H. S. Saturska, Yu. І. Bondarenko, U. V. Saturska I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE Background. The change in metabolism of the connective tissue elements of heart is the central chain in pathogenesis of diffuse ischemic necrotic cardiosclerosis (DINC), which occurs after repeated epinephrine injury of myocardial tissues. Objective. This study proves that trimetazidine (TM) metabolic therapy has a protective effect on the development of DINC in rats with different rates of hypoxia resistance. Methods. Male white rats were divided into three groups due to the different rates of hypoxia resistance by means of the method of hypobaric hypoxia: rats with low, middle and high rates of hypoxia resistance. Each group was divided into equal subgroups: a control group, a DINC group (injections of epinephrine hydrotartrate (0,5 mg/kg of body weight) and calcium gluconate (5 mg/kg of body weight) two times), a control group admin- istrated with trimetazidine dihydrochloride (10 mg/kg of body weight), a DINC group treated with TM every day (10 mg/kg of body weight) for all period of observation. Concentration of protein-bound oxyproline in blood serum was evaluated on the 7th, 14th and 30th days after the pathology simulation. Histological examination of Masson trichrome staining of myocardium was performed on the 30th days after the pathology simulation. Results. DINC increased the concentration of protein-bound oxyproline in blood serum on the 7th, 14th and 30th days after the pathology simulation, and followed by metabolic imbalances in diffuse connective tissue ele- ments, which are rich in collagens. DINC+TM increased the concentration of protein-bound oxyproline in blood serum less intensively. Conclusions. The intensity of metabolic imbalances in diffuse connective tissue elements is the highest in the low resistant animals to hypoxia. Those results are confirmed by histological examination of the myocardium of rats with different resistance to hypoxia. Fibrotic regions in myocardium are rich in collagens. It has been revealed that the most pronounced therapeutic effect of TM is observed in animals with low resistance to hypoxia, slight – in animals with medium resistance to hypoxia, and the lowest – in animals with high resistance to hypoxia. KEY WORDS: hypoxia, heart, diffuse cardiosclerosis, trimetazidine, oxyproline. Introduction The pathology of the cardiovascular system is the major medical and social problem, because it takes the main cause of morbidity and mortality [1-2]. The special attention is paid to the research on diagnostic markers of degra- dation and reparation of myocardial tissue [3-5], which would reflect the dynamic changes in myocardium and were predictors of diffuse cardiosclerosis [2-3]. The purpose of this inves- tigation was to determine the changes in the content of protein-bound oxyproline in blood as a diagnostic marker of metabolic activity of collagen at the experimental diffuse ischemic necrotic cardiosclerosis in rats with different rate of hypoxia resistance. Recently, we demonstrated that the use of trimetazidine as an endogenous cardioprotection inducer in the development of diffuse ischemic necrotic cardiosclerosis is manifested by de- crease in manifestations of oxidative and nitrooxidative stress, optimization of immune and cytokinе response, stabilization of humoral immune responsiveness [6]. The research was carried out to study the effects of TM on the improvement of connective tissue elements metabolism, indicating inhibition of cardio- sclerotic process. H. S. Saturska et al. International Journal of Medicine and Medical Research 2016, Volume 2, Issue 1, p. 49–53 copyright © 2016, TSMU, All Rights Reserved 50 b IO M E d Ic A L S c IE N c E S ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 Materials and Methods Animals and treatment Experiments were done on 192 male white rats (190-250 g) of the vivarium of Ternopil State Medical University, Ukraine. All animals re- ceived care in compliance with the “Guide for Care and Use of Laboratory Animals” (National Institute of Health Publication № 85-23, revised 1985). The studies were carried out according to the National Institute of Health Guide for Care and Use of Laboratory Animals and were approved by the local animal protection com- mittee. The experimental animals were divided into three groups according to different rates of hypoxia resistance by means of the method of hypobaric hypoxia [6] [Berezovskyi, 1975; Mar- kova, 1998]: rats with low, middle and high rates of hypoxia resistance. Each group was divided into equal subgroups: a control group, a diffuse ischemic necrotic cardiosclerosis group (injec- tions of epinephrine hydrotartrate (0,5 mg/kg body weight) and calcium gluconate (5 mg/kg of body weight) two times), a control group administered with trimetazidine dihydrochlo- ride (10 mg/kg of body weight) every day [6], diffuse ischemic necrotic cardiosclerosis group treated with trimetazidine dihydrochloride (10 mg/kg of body weight) every day for all period of observation (n=8 of each group). Evaluation of protein-bound oxyproline in blood serum Concentration of protein-bound oxyproline in blood serum was determined biochemically [7] on the 7th, 14th and 30th days after the pathol- ogy simulating. Histopathology study The tissue from myocardium ventricles was taken on the 30th day after pathology simula- tion, then put in 10 % neutral-buffered formalin solution for 5 days, embedded in paraffin, and sectioned. Histological examination of Masson trichrome staining of myocardium was per- formed [8]. Statistical analysis Statistical analysis was carried out by Orig- inPro Program. The results were presented as mean±standard deviation. Differences between experimental groups were analyzed with an unpaired two-tailed Student t-test [9]. Values were considered to be statistically significant at p<0.05. Results Before the DINC simulation the protein- bound oxyproline concentration in blood serum of rats with low hypoxia resistance was 17.8% (p<0.05) higher than in blood serum of rats with middle hypoxia resistance (Table 1), oxyproline concentration in blood serum of high hypoxia resistant animals was 21.9% lower (p<0.05) than in blood serum of rats with middle hypoxia resistance. After the DINC simulation protein- bound oxyproline concentration in blood serum of rats gradually increased at all groups. Under the influence of trimetazidine meta- bolic therapy the changes in protein-bound oxyproline concentrations in blood serum of the animals with low hypoxia resistance were less pronounced (Table 2). Concentration of this metabolite collagen in 7 days after pathology simulation with TM was by 11.0% (p<0.05) (Figure 1) lower than in the group of untreated animals at this stage of observation. On the 14th day after DINC simulation with TM, protein- bound oxyproline concentration in blood serum of the rats with low hypoxia resistance was by 25.3% lower (p<0.001) than in the untreated animals, and on the 30th day of observation it was by 33.9% (p<0.001) lower than in the Table 1. Protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (DInC) with innate hypoxia resistance in rats Hypoxia resistance rate in animals Control group (n=8) Stages of DINC observation 7 days (n=8) 14 days (n=8) 30 days (n=8) Low 49,55±0,59 р<0,05 57,45±1,78 р<0,01 р*<0,01 79,15±2,66 р<0,01 р*<0,01 104,84±3,42 р<0,01 р*<0,01 Middle 42,07±1,10 47,92±0,62 р*<0,01 58,24±1,00 р*<0,01 73,38±3,30 р*<0,01 High 34,52±0,92 р<0,05 38,53±0,55 р<0,01 р*<0,05 42,65±1,19 р<0,01 р*<0,01 56,43±2,84 р<0,01 р*<0,01 Notes: p<0.05 – significantly different from middle hypoxia resistant animals at all stages of observation; *р<0.05 – significantly different from the control group at all stages of observation. H. S. Saturska et al. 51 b IO M E d Ic A L S c IE N c E S ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 untreated animals with low hypoxia resistance at the similar stage of cardiosclerotic process development without any correction. In blood serum of the rats with middle hypoxia resistance, protein-bound oxyproline concentration on the 7th day after DINC simu- lation and trimetazidine correction was by 8.3% (p<0.05) (Fig. 1) lower than in the group of un- treated animals at this stage of observation. At the next stage of observation, on the 14th day of DINC simulation, protein-bound oxyproline concentration in blood serum of these animals was by 18.6% lower (p<0.001) than in the untreated ones, and on the 30th day of DINC observation – by 28.2% (p<0.001) lower than in the untreated rats. There was no significantly difference bet- ween the treated and untreated animals with high hypoxia resistance on the 7th day of DINC observation. On the 14th day after the pathology simulation, protein-bound oxyproline concen- tration was lower by 11.6% (p<0.05) than in the group of untreated animals at this stage of observation; and on the 30th day of DINC observation and correction with metabolic the- rapy, concentration of protein-bound oxyproline in blood serum was 28.4% (p<0.001) lower than in the untreated animals with high hypoxia resistance without any correction. Histological examination of the myocardium on the 30th day of DINC observation showed that at heart micropreparations of the healthy animals with different rates of hypoxia resis- tance, connective tissue was observed slightly in the form of thin collagen fibers (Figure 2), but in heart micropreparations of the animals with DINC (Figure 3) focal cardiosclerosis, perivascular sclerosis hyperelasticity of vessels Table 2. Influence of trimetazidine on protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (DInC) with innate hypoxia resistance in rats Hypoxia resistance rate in animals Control TM group (n=8) Stages of DINC+TM observation 7 days (n=8) 14 days (n=8) 30 days (n=8) Low 45,58±1,51 51,15±1,36 р*<0,05 р<0,001 59,09±1,85 р*<0,001 р<0,001 69,32±1,86 р*<0,001 р<0,001 Middle 40,58±1,83 43,97±1,34 40,58±1,83 р*<0,05 52,72±3,15 р*<0,05 High 34,90±0,96 р<0,05 36,33±0,91 р<0,001 37,72±1,52 р<0,001 34,90±0,96 р*<0,05 р<0,01 Notes: p<0.05 – significantly different from the middle hypoxia resistant of animals at all stages of observation; *р<0.05 – significantly different from the control group at all stages of observation. Fig. 1. Influence of trimetazidine on protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (DINC) with innate hypoxia resistance in rats. Notes: the indices of the control groups were presented in 100%; * – significantly different from the control group at all stages of observation, p<0.05; # – significantly different from the untreated rats at all stages of observation, p<0.05. 0 50 100 150 200 250 Control DINC 7 days DINC 14 days DINC 30 days LRH LRH+TM MRH MRH+TM HRH HRH+TM % * * * * * * * ** # # # # # # ### H. S. Saturska et al. 52 b IO M E d Ic A L S c IE N c E S ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 Discussion Evaluation of protein-bound oxyproline concentrations in blood serum in DINC simu- lation with and without trimetazidine correction proved that increased collagen production and products of its metabolism [4-5] can be used as a biological marker of the intensity of collagen synthesis in tissue infarction. So we can make the following conclusion: the intensity of metabolic imbalance of connective elements in cases of diffuse ischemic necrotic cardiosclerosis and trimetazidine correction depends on hy- poxia resistance of animals. In the low hypoxia resistance animals, maximum effect of trime- tazidine correction was manifested; however, more pronounced changes in oxyproline concentration were in DINC simulation without any correction. This effect was not enough for denoting differences between animals with different rates of hypoxia resistance. This matter is characteristic feature of animals with middle hypoxia resistance, but the changes were less pronounced. Animals with high hypoxia resistance were characterized by lower oxyproline concentration, which changed after DINC simulation, so the effect was manifested less, but in general, they are characterized by minimal metabolic disorders of connective tissue elements in the development of DINC and correction with trimetazidine [10-16]. The activity of connective tissue metabolism was studied in experimental diffuse ischemic ne cro- tic cardiosclerosis with different rates hy poxia resistance of a body. The investigations were based on the changes in concentration of pro- tein-bound oxyproline in blood serum that pro ved adequate metabolic changes in collagen [4–5]. Conclusions The development of the experimental diffuse ischemic necrotic cardiosclerosis at all stages of observation was accompanied by metabolic imbalance in connective tissue of heart, and was proved by the increase in oxyproline level in blood serum of animals with different rates of hypoxia resistance. The inten- sity of metabolic imbalances in diffuse connec- tive tissue elements was the highest in low hypoxia resistant animals. Those results were confirmed by histological examination of myo- cardium of rats with different rates of hypoxia resistance. Fibrotic regions in myocardium are rich in collagens. It has been revealed that the most pronounced therapeutic effect of TM is observed in animals with low hypoxia resistance, Fig. 2. Myocardium of the control rat. Masson trichrome staining of myocardium. х 400. Fig. 3. Myocardium of the low hypoxia resistant rat with DINC. Fibrotic regions in myocardium are rich in collagens and therefore appear in blue upon Masson trichrome staining. In addition, centralized nuclei as well as shape and size distribution of myofibers were detected that was the evidence of pronounced cardiomyocytes hypertrophy. 30-day DINC. Masson trichrome staining of myocardium. х400. inner membrane, cardiomyocyte hypertrophy, diffuse proliferation of connective tissue were presented. Fibrotic regions in myocardium are rich in collagens and therefore appear in blue upon Masson trichrome staining. In addition, centralized nuclei as well as shape and size distribution of myofibers were detected that was the evidence of pronounced cardiomyocytes hypertrophy. All the above-mentioned symptoms are the highest in the low hypoxia resistant animals, indicating the intense development of diffuse cardiosclerosis in animals with low hypoxia resistance and confirm the results obtained during evaluation of the concentration of protein-bound oxyproline serum of rats with different rates of hypoxia resistance. H. S. Saturska et al. 53 b IO M E d Ic A L S c IE N c E S ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 1 slightly less – in animals with medium hypoxia resistance, and the lowest – in animals with high hypoxia resistance. 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