CC BY
12
ISSN2079-8334. Ceim медицини та бюлогп. 2020. № 2 (72)
одночасного ураження токсикантами е статевонезрш щури.. Найвищий BMicT С-реактивного протешу зареестровано у сироватщ кровi щурiв старечого вку, у яких вiн тдвищився в 3,3 раза. Карболайн виявився ефективним в останнi термiни дослщження. Пiсля ураження спостерiгався зсув балансу мiж цитокiнами в сторону прозапальних процеЫв, на що вказувало тдвищення вмiсту прозапального та зниження вмюту протизапального iнтерлейкiну. У статевозрiлих щурiв ентеросорбент призвiв до вiрогiдного вщновлення дисбалансу цитокiнiв у сироватцi кровi пiсля ураження.
Ключовi слова: натрш нiтрит, тютюновий дим, метгемоглобiн, карбоксигемоглобш, цитокiни, С-реактивний протеш
Стаття надшшла 30.06.2019
одновременному поражению токсикантами были неполовозрелые крысы. В сыворотке крови пораженных старых крыс отмечено наиболее высокое содержание С-реактивного протеина (в 3,3 раза выше нормы). Карболайн оказал эффективное влияние на этот показатель в конце исследования. В сыворотке крови пораженных крыс всех возрастных групп наблюдался сдвиг цитокинового баланса в сторону провоспалительных процессов (повышение содержания интерлейкина-6 и снижение интерлейкина-4). Наиболее эффективное влияние оказал энтеросорбент на цитокиновый баланс у половозрелых крыс.
Ключевые слова: нитритно-табачная интоксикация, мет- и карбоксигемоглобин, цитокины, С-реактивный протеин
Рецензент Костенко В.О.
DOI 10.26724/2079-8334-2020-2-72-185-188 UDC 612.018.11-02:616.5-001
'I krainian Medical Stomatological Academy. Poltava
BIOCHEMICAL FEATURES OF POSTOPERATIVE SKIN WOUNDS HEALING AGAINST THE BACKGROUND OF DIABETES MELLITUS IN RATS WITH DIFFERENT
WAYS OF WOUND CLOSURE
e-mail: doctorloza@gmail.com
The study is devoted to identifying the features of free radical processes changes in skin homogenate of the rats with diabetes mellitus, after using different methods of surgical wounds closing. Analysis of the results suggests that the use of skin glue significantly reduces the intensity of the free radical oxidation course in the cells of scarred skin tissues in animals with simulated diabetes mellitus compared to the application of nodal sutures to the wound under similar conditions.
Key words: free radical oxidation, oxidative stress, abnormal scar.
The work is a fragment of the research project "Systemic and organ disorders due to emergency factors effect on the body, mechanisms of their development and pathogenetic correction ",state registration No. 016U003390.
Diabetes mellitus (DM) is considered to be one of the most important non-communicable diseases worldwide and is also one of the global health challenges of the 21st century [3]. It ranks fourth among the major causes of death in developed countries [15].
In 2017, 1,270,929 cases of diabetes were registered in Ukraine. Today, almost 50% of diabetes cases are undiagnosed. Development of late complications leads to a significant reduction in quality of life, reducing its duration by 10-30%, disability, increased mortality of patients by 2-3 times and significant budget expenditures for their treatment. The number of patients with diabetes mellitus is increasing by 57% annually. Such indices indicate a non-infectious epidemic of diabetes.
Wound healing is a coordinated process that goes through certain stages with different types of cells and their waste products participating, which regulate the healing process. It is proved that in patients with DM there is the wound healing process disorder, but not all features of this process in diabetes mellitus are completely understood.
At the same time, the quality and structure of the material, the chemical composition of the suture material effect the reaction of tissues to their introduction, as well as the final result of the operation. Free radical oxidation (FRO) is an important biochemical conversion process of lipids, proteins, nucleic acids and other compounds under the action of free radicals, and peroxide oxidation of lipids (LPO) and proteins is one of the further consequences [2, 13]. At all stages of the FRO, due to the interaction of free radicals and biological macromolecules, numerous intermediate products are formed. Such peroxidation compounds, in their excess, are characterized by pronounced cytotoxic activity. As a result, the processes of energy production in the cell are suppressed, the synthesis of proteins and nucleic acids is disrupted, which in its turn leads to the formation of pathological scar tissue.
The purpose of the study to identify the features of free radical processes changes in the skin homogenate of rats in different ways of surgical wounds closing, in the conditions of diabetes mellitus.
Materials and methods. An experiment was performed on 60 adult male rats weighing 240-280 g. The rats were simulated diabetes mellitus by intraperitoneal administration of streptozotocin
© Ye.O. Loza, M.I. Marushchak, 2020
("Sigma", USA) at the dose of 65 mg/kg body weight. 15 minutes before administration of streptozotocin the animals were intraperitoneally administered nicotinamide at the dose of 230 mg/kg body weight. All animals were obese, which was caused by keeping the animals on a high-fat diet for 4 weeks. By determining the concentration of glucose in the blood with ContourNext (USA) glucometer, the development of type 2 DM was confirmed. Under the thiopental anesthesia (40 mg/kg body weight), all experimental animals underwent straight incisions 2 cm long in the anterior-lateral region of the abdomen. In experimental group I (30 rats) surgical sutures "Vikril5 / 0" were used to close the postoperative wound. In experimental group II (30 rats) "Dermabond" skin glue (Ethicon, USA) was applied. Animals were sacrificed on the 3rd, 7th, 28th days after surgery by overdose of thiopental anesthesia (90 mg/kg body weight of rats).
The scarred skin pieces homogenate and the homogenate supernatant were under study.
To determine the degree of free radical oxidation processes activation, the content of reactive oxygen species (ROS) [5], lipid hydroperoxides (LHP) [5], diene conjugates (DC) and triene conjugates (TC) [1] and indices of blood plasma proteins oxidative modification were determined (OMP370 and OMP430) [10].
In order to study the antioxidant defense system, the activity of superoxide dismutase (SOD) in the supernatant was determined by the method of Chevari S. et al. [12]. Catalase activity was determined by the method of Korolyuk M.A. [6], sulfhydryl groups (SH groups) - according to Ellman G.L. [14].
Statistical processing of the obtained data was carried out by standard methods of variation statistics using a package of statistical software. The results are given as (M±m), where M is the mean value of the index, m is the standard error. The significance of discrepancies between the studied indices was determined using the Student's t-test.
Results of the study and their discussion. Our study showed that the activity of free-radical oxidation (FRO) increased during the wound process against the background of diabetes mellitus. However, in the skin homogenates of the animals treated with "Dermabond", all values were significantly lower than in the group of animals treated with interrupted sutures during all study periods. Thus, the number of ROS was greater in animals of group I, compared to those of group II, on the 3rd, 7th and 28th days of the experiment. They also showed a gradual decrease from the beginning of the study to the last time interval, the gradient of decrease being more significant in group II (table 1). The content of diene and triene conjugates in the skin homogenate of group I rats exceeded that of animals in group II on the 3rd day. In group I, the content of DC was (10.77±0.23) relative units (RU)/kg, TC -(9.72±0.41) RU/kg, in II - (7.66±0.37) RU/kg and (7.53±0.37) RU/kg, respectively. On days 7 and 28, a linear decrease in the content of primary LPO products was observed in both experimental groups I and II. Lipid hydroperoxides (LHP) content was also higher in group I rats, compared those in group II, on the 3rd day of the experiment and significantly decreased on the 7th and 28th days in the both groups of animals.
Table 1
Indices of free radical oxidation in the skin homogenate of rats with simulated diabetes mellitus using suture material and skin glue in the dynamics
Index ROS, % DC, RU/kg TC, RU/kg LHP, RU/h
Intact animals 19.20±0.37 4.05±0.08 4.09±0.18 5.05±0.06
Day 3 Group I 90.76±1.26 10.77±0.23 11.49±0.55 9.72±0.41*
Group II 74.67±1.06* 7.66±0.37* 9.71±0.19* 7.53±0.37*
Day 7 Group I 88.27±2.08 9.88±0.56 9.29±0.39 7.20±0.38*
Group II 43.93±1.31* 6.82±0.29* 7.84±0.20* 6.24±0.35*
Day 28 Group I 50.1±3.21 5.65±0.26 8.76±0.26 5.13±0.21
Group II 29.07±0.95* 5.46±0.16 6.51±0.26* 4.93±0.23*
Notes. Henceforward in the tables: * - the difference is significant between experimental groups I and II within one day
The content of OMP370 and OMP430 products in the skin homogenate of group I animals was by 2.3 and 1.5 times higher, respectively, similar to skin of those in group II on the 3rd day of the experiment. A slight decrease in these indices occurred in both experimental groups on day 7. On day 28, the content of OMP370 products in the skin homogenate of group I animals was by 3.6 times higher than the same index in the skin of group II animals (table 2).
Analysis of all indices permits to state that in the skin of animals treated with glue, the intensity of FRO processes is much lower compared to the imposition of interrupted sutures. Given the strengthening of the LPO processes, which cause the phenomena of primary and secondary alteration when using suture material to close wounds in rats with diabetes mellitus, the beginning of reparative mechanisms in them is delayed.
Table 2
Indices of proteins oxidative modification in skin homogenate against the background of diabetes mellitus using suture material of skin glue in the dynamics
Group/Index, % OMP370 OMP430
Intact animals 1.70± 0.06 0.33±0.46
Day 3 Group I 7.25±0.20 2.79±0.10
Group II 3.12±0.10* 1.84±0.09*
Day 7 Group I 6.78±0.17 2.10±0.11
Group II 2.67±0.11* 1.42±0.09*
Day 28 Group I 5.83±0.26 1.69±0.06
Group II 1.62±0.04* 1.02±0.06
At the early stages of scar formation (on the 3rd day), we observed an increase in the activity of the antioxidant defense system in the two groups presented. In the groups of animals to which interrupted sutures were applied, the activity of superoxide dismutase (SOD) and catalase was much higher than in the skin of the animals to which the glue was applied, and amounted to (199.81±7.59) RU and (175.02±8.31) kat/kg, respectively. Such data indicated the inevitable start of free radical oxidation due to the traumatic factor against the background of diabetes mellitus. A significant decrease in all parameters was determined in the skin of experimental animals of the both groups on the 7th day of the experiment. However, this difference in the change of SOD activity data was higher in group II animals than in the group treated with the skin glue,
where the index decreased by 1.4 times, while in group I the this index decreased by 1.15 times (table 3).
Table 3
Indices of the antioxidant defense system in the skin homogenate under the conditions of diabetes mellitus using suture material and skin glue
Index SOD, RU Catalase, kat/kg SH-groups, mmol/L
Intact animals 71.45±2.28 35.05±2.91 37.50±2.02
Day 3 Group I 199.81±7.59 175.02±8.31 90.44±2.00
Group II 118.57±3.51* 96.05±2.43* 68.50±1.42*
Day 7 Group I 173.19±7.05 146.72±7.19 71.50±2.77
Group II 84.72±1.81* 74.28±1.83* 62.90±1.27*
Day 28 Group I 140.51±5.93 111.12±5.13 64.18±2.25
Group II 83.18±2.29* 73.02±1.40* 62.27±1.33*
Similarly, the reduced glutathione indices were lower in rats of group II, compared to those in group I, on the 3rd and the 7th days from the beginning of the experiment.
Thus, in the animals in the wound process against the background of diabetes mellitus, the indices of FRO activity are much higher, compared to the control rats. The obtained data are confirmed by the results of other authors in the study of the oxidative-antioxidant system status of the liver in alloxandiabetic rats under the conditions of melatonin administration and in the study of the vanadium and chromium citrates effect on the antioxidant system in the blood of experimental rats [4, 9, 11]. The activity of superoxide dismutase and catalase, which are important components of the antioxidant system, in the skin homogenate of rats with diabetes mellitus is significantly reduced, compared to healthy animals. Similar activity of these indices was found in the study of the antioxidant defense system in rats under the action of carrageenan against the background of streptozotocin diabetes, as well as in the study of lipid peroxidation system and antioxidant defense enzymes in rats of different ages in experimental diabetes mellitus [7, 8, 11].
Conclusion
The use of skin glue significantly reduces the FRO intensity in the cells of scar tissue in animals with simulated diabetes mellitus compared to the imposition of interrupted sutures on the wound: in skin homogenates of animals, which were applied skin glue "Dermabond", all indices are significantly lower than in the group of animals which were imposed interrupted sutures during all terms of the study. In particular, in the long term of the study (the 28th day) the content of hydro lipid peroxides in experimental group I was 8.76±0.26 RU/kg, which is by 1.3 times more than in the animal group II - 6.51±0.26 RU/kg.
1. Vlizlo VV, Fedoruk RS, Ratych IB. Laboratorni metody doslidzhen u biolohii, tvarynnytstvi ta veterynarnoyi medytsyni.Dovidnyk. Lviv: 2012.761 s.[in Ukrainian]
2. Holovchak NP, Tarnovska AV, Kotsiumbas HI, Sanahurskyi DI. Protsesy perekysnoho okysnennia lipidiv u zhyvykh orhanizmakh: monohrafiia. Lviv: 2012.250 s.[in Ukrainian]
3. Dedova II, ShestakovaMV. Sakharnyy diabet: diagnostika, lecheniye, profilaktika. M.: 2011. 808 s.[in Russian]
4. Ivankiv YaI, Oleshchuk OM, Datsko TV, Fedoniuk LIa.Osoblyvosti pokaznykiv prooksydantno-antyoksydantnoho homeostazu, vuhlevodnoho obminu ta morfolohichni zminy pechinky za umov vvedennia melatoninu pry eksperymentalnomu diabeti 2 typu.Visnyk morfolohii. 2016; 2(22):253-8. [in Ukrainian]
5. Kamyshnikov VS. Spravochnik po kliniko-biokhimicheskim issledovaniyam i laboratornoy diagnostike. M.: 2004. 911 s. [in Russian]
ISSN2079-8334. Ceim медицини та бюлогп. 2020. № 2 (72)
6. Korolyuk MA, Ivanova LI, Mayorova IG. Metod opredeleniya aktivnosti katalazy. Lab. delo. 1988;1:16-8.[in Russian]
7. Levytskyi VA, Zhurakivska OIa, Miskiv VA. Aktyvnist perekysnoho okyslennia lipidiv ta fermentiv antyoksydantnoho zakhystu u shchuriv riznoho viku pry eksperymentalnomu tsukrovomu diabeti. Visnyk Ukrayinskoyi medychnoyi stomatolohichnoyi akademii: Aktualni problemy suchasnoi medytsyny. 2011; 4(36):103-6.[in Ukrainian]
8. Lisnianska NV. Osoblyvosti systemy antyoksydantnoho zakhystu shchuriv za umovy diyi karahinanu na tli streptozototsynovoho diabetu. Visnyk medychnykh i biolohichnykh doslidzhen. 2019; 2:19-23.[in Ukrainian]
9. Luhinich NM, Herush IV, Hryhorieva NP. Stan prooksydantno-antyoksydantnoi systemy pechinky aloksandiabetychnykh shchuriv za umov vvedennia melatoninu. Medychna ta klinichna khimiia. 2019; 21(2):30-4. [in Ukrainian]
10. Meshchyshen IF. Metod vyznachennia okysliuvalnoyi modyfikatsii bilkiv plazmy krovi. Bukovynskyi medychnyi visnyk. 1998; 2(1): 156-8.[in Ukrainian]
11. Sushko OO, Iskra RYa. Vplyv tsytrativ vanadiyu ta khromu na antyoksydantnu systemu v krovi shchuriv iz eksperymentalnym diabetom Visnyk ONU. Biolohiya. 2019; 24(1):106-15.[in Ukrainian]
12. Chevari S, ChabaI, Sekey Y. Rol superoksidreduktazy v okislitelnykh protsessakh kletki i metod opredeleniya yeye v biologicheskom materiale. Lab. delo. 1985; 11:678-681 .[in Russian]
13. Kurahashi T, Fujii J. Roles of antioxidative enzymes in woundhealing. J. Dev. Biol. 2015; 3:57-70.
14. Moffat JA, Armstrong PW, Marks GS. Investigations into the role of sulfhydryl groups in the mechanism of action of the nitrates. Canadian Journal of Physiology and Pharmacology. 1982; 60(10): 1261-6.
15. Whiting DR, Guariguata L, Weil C, Shaw J. International Diabetes Federation. Diabetes Atlas: Global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes research and clinical practice. 2011; 94:311-321.
Illlllllllii
БЮХ1М1ЧШ ОСОБЛИВОСТ1 ЗАГОСННЯ тСЛЯОПЕРАЦШНИХ РАН ШК1РИ НА ФОШ1 ЦУКРОВОГО Д1АБЕТУ У ЩУР1В ПРИ Р1ЗШИХ СПОСОБАХ ЗАКРИТТЯ РАН Лоза е.О.. Марущак М.1., Денефшь О.В..
Аветжов Д. С.. Кулянда О. О. Дослщження присвячено вивченню особливостей змш вшьнорадикальних процеЫв у гомогенат шюри щурiв при рiзних способах закриття операцшних ран, за умов цукрового дiабету. Аналiз отриманих результата дае змогу стверджувати, що застосування шюрного клею достовiрно знижуе штенсившсть переб^у ВРО у кйтинах рубцевозмшених тканин шюри тварин зi змодельованим цукровим дiабетом порiвняно iз накладанням на рану вузлових швiв за аналопчних умов.
Ключовi слова: вiльнорадикальне окиснення, оксидативний стрес, патолопчний рубець.
Стаття надшшла 13.06.2019 p.
DOI 10.26724/2079-8334-2020-2-72-188-192 UDC 616.36-0,02:611.018:599.323.4
БИОХИМИЧЕСКИЕ ОСОБЕННОСТИ ЗАЖИВЛЕНИЕ
ПОСЛЕОПЕРАЦИОННЫХ РАН КОЖИ НА ФОНЕ САХАРНОГО ДИАБЕТА У КРЫС ПРИ РАЗЛИЧНЫХ СПОСОБАХ ЗАКРЫТИЯ РАН Лоза Е.А., Марущак М.И., Денефиль О.В., Аветиков Д. С., Кулянда О. О.
Исследование посвящено изучению особенностей изменений свободнорадикальных процессов в гомогенате кожи крыс при различных способах закрытия операционных ран, при сахарном диабете. Анализ полученных результатов позволяет утверждать, что применение кожного клея достоверно снижает интенсивность течения свободно радикальных процессов в клетках рубцово-измененных тканей кожи животных с смоделированным сахарным диабетом по сравнению с наложением на рану узловых швов при аналогичныхусловиях.
Ключевые слова: свободно радикальное окисление, оксидативныйстресс, патологический рубец.
Рецензент Костенко В.О.
G.S. Maslova. R.I. Skrypnyk. O.F. Gopko. I.M. Skrypnyk I krainian Medical Stomatological Academy. Poltava
THE ROLE OF ARGININE/CITRULLINE CYCLE DISORDERS IN THE PATHOGENESIS OF DOXORUBICIN-INDUCED LIVER INJURY ASSOCIATED WITH NONALCOHOLIC
STEATOHEPATITIS IN RATS
e-mail: maslovaas1708@gmail.com
The article presents the results of an experimental study aimed to investigate the features of arginine / citrulline cycle disorders associated with doxorubicin-induced liver injury in rats, concomitant with NASH. The study involved adult fertile □utbrfl ClbCnnrQs (n=30; mUDrUts=15 (50%); faiUDrQs=15 (50%)) wDghtfl 160-220 g. The rats were assigned into 3 groups: Group I (n=10) involved rats with NASH, administered with CntrDQCCDnQ] 5 mg/kg dmorubonn fOr 3 dUES; GrQiDII (n=10) OnvOvQl rUts without NDSH, HflmDnHttJCfl with OntrQDJCCDnaH dDZQubCfflffl sCmCCD tDGrQiDQ-qgCma; GrQuDIII (n=10) - control group. It has been shown that administration of doxorubicin in rats with NASH leads to disorders of arginine/citrulline cycle, which are characterized by inhibition of arginase activity and activation of citrulline synthesis.
Key words: arginine, citrulline, arginase, doxorubicin, hepatotoxic reactions, nonalcoholic steatohepatitis.
The work is a fragment of the research project "Development of methods for prevention and treatment of the drug-induced internal organs damages ", state registration No. 0115U001087.
Doxorubicin, an anthracycline antibiotic, has been extensively used in oncology and oncohematology for over 30 years. Currently, it is considered one of the most effective antitumor drugs with pharmacological feature to accumulate in malignant cells. However, the use of doxorubicin is accompanied by a high risk of side effects due to its high toxicity [3, 8, 10, 13].
© G.S. Maslova, R.I. Skrypnyk, 2020
