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iSSn 2413-6077. iJmmR 2017 Vol. 3 issue 2

International Journal of Medicine and Medical Research 
2017, Volume 3, Issue 2, p. 59–63
copyright © 2017, TSMU, All Rights Reserved

dOI 10.11603/IJMMR.2413-6077.2017.2.8035

chRONIc ENTEROcOLITIs cOmbINEd WITh sTREPTOZOTOcIN-
INdUcEd dIAbETEs IN RATs: mEchANIsm Of OXIdATIVE sTREss 
dEVELOPmENT

1N. V. Lisnianska, 2М. І. Marushchak, 2І. V. Antonyshyn, 3О. P. Mialiuk
1CHERNIVTSI MEDICAL COLLEGE OF BUKOVINIAN STATE MEDICAL UNIVERSITY, CHERNIVTSI, UKRAINE;

2I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE;
3RIVNE STATE BASIC MEDICAL COLLEGE, RIVNE, UKRAINE

Background. Despite numerous studies on chronic inflammatory processes in colon, the complex problem 
of chronic enterocolitis (CEC) remains relevant.

Objective. The aim of the research is to evaluate the lipid peroxide oxidation and antioxidant defence sys-
tem in rats suffering from chronic enterocolitis development with underlying streptozocine-induced diabetes 
mellitus.

Methods. The study involved 106 white non-linear male rats. Diabetes mellitus (DM) was modeled by a 
single intraperitoneal administration of streptozotocin to animals (Sigma Aldrich, USA, at a dose of 60 mg/kg of 
body weight). CEC was induced by a free access of animals to 1.0% solution of carrageenan in drinking water for 
1 month.

Results. In the wall of small intestine of DM rats, lipid peroxide oxidation increases and the activity of enzyme 
link of antioxidant defence decreases reliably. The course of experimental CEC in rats is accompanied by the in-
crease in free radical oxidation in the wall of small intestine and simultaneous increase of enzyme activity of 
antioxidant defence system, compared to the control.

In cases of CEC with underlying DM in rats, the development of oxidative stress in the wall of small intestine 
is caused by the statistically significant increase in levels of diene conjugates and thiobarbituric acid reactive 
substances, (p<0.01) and the decrease in activity of SOD and catalase.

Conclusions. The activation of free radical reactions is an important non-specific mechanism of inflamma-
tion development in tissues of small and large intestine in cases of diabetes mellitus.

KEY WORDS: chronic enterocolitis; diabetes mellitus; lipid peroxidation; antioxidant defence 
system; experiment.

Corresponding author: Maria Marushchak, Department of 
Functional Diagnostics and Clinical Pathophysiology, I. Hor-
bachevsky Ternopil State Medical University, 1 Maidan Voli, 
Ternopil, Ukraine, 46001 
Phone number: +380979901202 
E-mail: marushchak@tdmu.edu.ua

Introduction
A difficult social and economic situation, 

deterioration of living conditions, aggressive-
ness of the environment and other negative 
stress factors contribute to an increase in inci-
dence of digestive diseases in the population. 
Digestive diseases are one of the most common 
among chronic diseases in developed coun-
tries. According to a number of studies, their 
share is 8–10% [1–5]. Chronic enterocolitis (CEC) 
is one of the diseases, which are based on the 
combination of elements of inflammation and 
dystrophy of mucous membrane with func-
tional disorders in small and large intestines. 
This disease takes account of about 10% of the 

total chronic pathology of digestive system 
organs, and its prevalence is 5–12 cases per 
1000 people [6]. Despite numerous studies on 
chronic inflammatory processes in colon, the 
complex problem of chronic enterocolitis re-
mains relevant [7, 8]. For a long time this term 
defined a variety of pathological conditions of 
intestine, which was due to the lack of suffi-
ciently clear notions about the nature of the 
disease. From the standpoint of modern con-
cepts, chronic enterocolitis should be conside-
red as a clinical and morphological phenome-
non characterized by pain and dyspeptic synd-
romes typical for intestinal diseases with 
mor phologically determined signs of epithe-
lium dystrophy, the decrease in crypts depth 
and development of various severity lympho-
plasmatic infiltration [9]. According to the offi­
cial data, the incidence of CEC in the world is 
50–230 cases per 100,000 people [10]. About 

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15–20% of the world’s population suffer from 
CEC. In the United States, over 20 million adults 
have symptoms of this disease. [11]. The an-
nual increase in number of enterocolitis pa-
tients in the world is 5–20 cases per 100,000 
people. Epidemiological calculations in the US 
proved that in white population, CEC is found 
in 3–5 times more often than in African Ameri-
cans, while Jews are threatened in 3.5 times 
more than non-Jewish people. The disease oc-
curs in all age groups, but the highest incidence 
is in 20–40-year-old individuals [12]. In the 
overall structure of gastroenterological patho-
logy in America, CEC ranks first and accounts 
for 28% of all cases of treatment by gastroen-
terologists. About 12% of the patients seek 
medical advice from a general practitioner with 
complaints specific to CEC. CEC is diagnosed 
the most commonly in young people: 13.5% of 
people are aged 15-34, 13% of them are 35–44 
years old and 9% are aged 45 and older. There-
fore, the disease causes great economic harm 
to the society both the cost of medical care and 
by indirect factors, which include a compensa-
tion for temporary disability. [13]. However, it 
should be noted that in modern literature there 
is controversial information on CEC as an inde-
pendent nosological form, and the presence of 
functional intestinal disease – an irritable 
bowel syndrome, is mentioned more often, 
while the changes in the intestinal mucosa are 
interpreted by the authors as morphofunc-
tional ones, which causes the complexity of 
clinical and laboratory diagnostics and different 
understanding of CEC [14, 15].

From the pathogenetic point of view, the 
diseases related to the class of free radical 
pathology are widespread, starting with birth 
(bronchopulmonary dysplasia, retinopathy in 
preterm infants, enterocolitis, etc.). [16, 17]. The 
weakening of antioxidant defence and uncon-
trolled enhancement of lipid peroxidation 
processes is one of the important links in the 
pathogenesis of autonomic dysfunction, at-
opic dermatitis, dental pathology, diabetes 
mellitus, as well as pathology of gastrointestinal 
tract, enterocolitis as well [18, 19]. In this case 
peroxide lipid oxidation products, including 
malonic dialdehyde, which destabilize cellular 
membranes, reach high concentrations in 
blood and tissues [20, 21]. To date, a large 
amount of data has been accumulated proving 
the participation of free radical processes in the 
pathogenesis of CEC [16]. 

Therefore, the aim of our study was to 
evaluate the lipid peroxide oxidation and anti-

oxidant defence system in rats suffering from 
chronic enterocolitis development with under-
lying streptozocine-induced diabetes mellitus.

Methods
The study involved 106 white non-linear 

male rats, which were kept on a standard viva-
rium diet of I. Horbachevsky Ternopil State 
Medical University. During the experiment the 
principles of the European Convention for the 
Protection of Animals used for Experimental 
and Other Scientific Purposes were followed. 
The experimental rats were divided into four 
groups: the 1st – control (intact animals), the 
2nd – animals with diabetes mellitus, the 3rd – 
animals with chronic enterocolitis, the 4th – ani-
mals with diabetes mellitus and chronic entero-
colitis. Diabetes mellitus (DM) was modeled by 
a single intraperitoneal administration of strep-
tozotocin to the animals aged 2 months, (Sigma 
Aldrich, USA, at a dose of 60 mg/kg of body 
weight) [22]. Right before its injection, strepto-
zotocin was dissolved in 0.1 molar citrate buffer 
(pH 4.5); the control group received the appro-
priate amount of citrate buffer. The study in-
volved the animals with the glucose rate at least 
10.8 mmol/L 2 weeks after streptozotocin ad-
ministration. Chronic enterocolitis was induced 
by a free access of animals to 1.0% solution of 
carrageenan in drinking water for 1 month [23, 
24]. Euthanasia of animals was performed by 
heart puncture under anaesthesia, in accor-
dance with the requirements of the Animal Care 
Committee [25].

The state of lipid peroxide oxidation (LPO) 
was evaluated by the concentration of diene 
conjugates (DC) and thiobarbituric acid reactive 
substances (TBARS). The content of DC was 
determined by direct spectrophotometry, the 
principle of which is to isolate native fatty acids 
by extraction with a mixture of equal volumes 
of heptane and isopropanol, followed by 
measuring the optical density of heptane phase 
of lipid extract. Absorption at a wavelength of 
232 nm evidences the content of DC [26]. To 
determine TBARS, we used the method of M. 
Mihara (1980), which consists in the formation 
of a coloured complex by the interaction of lipid 
peroxide oxidation products with thiobarbituric 
acid, by means of a standard set. The activity 
of antioxidant enzymes: catalase, was recorded 
simultaneously with the LPO processes [27], 
superoxide dismutase [28].

The obtained data were subjected to sta-
tistical processing [29, 30]. To verify the confor-
mity of the data samples with the normal dis-

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tribution law, the calculation by Shapiro-Wilk test 
was applied. Due to the lack of data matching 
to the normal distribution at the significance rate 
p<0.05, the median characteristics were esti-
mated: median (Me), first and third quartiles 
(Q25­Q75). The level of statistical significance of 
sample differences was evaluated using non-
parametric Mann-Whitney U test. Differences 
were considered statistically significant at the 
achieved rate of p<0.05.

Results
The increase of free radical oxidation pro-

cesses in small intestine wall in the presence of 
the studied pathologies has been established 
(p<0.05). For instance, in cases of streptozotocin-
induced diabetes, the content of DC increased 
statistically significantly by 66.93% and of TBARS 
by 71.22%, compared to the control values 
(Table 1). As the lipid peroxide oxidation 
increased, the activity of enzyme system of anti-
oxidant defence system decreased, in particular, 
the activity of SOD decreased by 38.02% and that 
of catalase by 58.82%.

Experimental CEC was accompanied by the 
increase of lipid peroxide oxidation in the wall 
of small intestine (DC by 35.16% and TBARS by 
27.65%, p<0.01) with simultaneous increase of 
enzyme activity of antioxidant defence system 
(SOD by 29.90% and catalase by 21.65%, p<0.01) 
compared to the control (Table 1).

The statistically significant activation of free 
radical oxidation processes in the rats with CEC 
combined with DM (the level of DC increased 
by 104.84% and of TBARS by 115.02% res-
pectively, p<0.01) was evidenced. A significant 
decrease in activity of SOD by 131.09% and of 

catalase by 21.65% (p<0.001), compared to the 
control, was found.

Discussion
Thus, in cases of experimental diabetes, the 

prooxidant-antioxidant disbalance in the wall of 
small intestine was characterized by the 
development of oxidative stress. According to 
the literature, the increase in blood glucose 
levels with its auto-oxidation is one of the causes 
of intensification of free radical oxidation [31].

In current studies there are no reliable data 
on the effect of systematic consumption of 
carrageenan on the body of an adult, child, 
foetus and when this supplement comprises 
the diet of pregnant women. Studying this 
problem in the clinic is very problematic, so 
there is an urgent need of studying the 
influence of ca rra geena n on meta bolic 
parameters in an experimental model.

The obtained data of experimental CEC 
prove that under the conditions of normal 
digestion due to acid hydrolysis, carrageenan 
splits into low and high molecular particles, 
which trigger free radical processes. Previous 
s t u d i e s  e v i d e n c e  t h e  i n v o l v e m e n t  o f 
macrophages in the absorption of carrageenan 
with the formation of heterolysosomes, which 
leads to the implementation of harmful effects 
of lysosomal enzymes. On the other hand, 
macrophages are established to be the source 
of free radicals [32]. In order to ensure anti-
oxidant equilibrium, an antioxidant defence 
system is activated simultaneously against the 
action of the external factor, proving the 
mobilization of protective and adaptive 
mechanisms associated with the excessive 

Table 1. Rates of free radical oxidation in the wall of small intestine of the rats with chronic 
enterocolitis combined with streptozotocin-induced diabetes, Me (Q25-Q75)

Rate Control DM(group 2)
CEC

(group 3)
DM+CEC
(group 4)

DC, st.un./g of tissue 2.89 
(2.50; 3.25)

4.83* 
(4.25; 5.29)

3.91* 
(3.71; 4.09)

5.92*
(5.76; 6.15)

р1,2<0.01
TBARS, mmol/mg 3.33 

(2.78; 3.80)
5.71* 

(5.53; 5.95)
4.26* 

(4.00; 4.57)
7.16*

(6.98; 7.31)*
р1,2<0.01

SOD activity, st. un/mg of 
proteins

17.17 
(16.28; 18.18)

10.64* 
(8.95; 12.02)

22.30* 
(21.43; 23.35)

7.43*
 (6.38; 8.08)
р1,2<0.001

Catalase activity,  
mmol·min/mg of proteins 

41.64 
(40.78; 42.55)

17.15* 
(16.16; 17.65)

50.66* 
(51.88; 54.55)

11.46* 
(10.85; 12.07)

р1,2<0.001

Notes: * – the difference between the control and experimental groups is statistically significant (р<0.05–0.001); р1 – the 
reliability value between the groups 2 and 4; р2 – the reliability value between the groups 3 and 4. 



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production of superoxide anion radical. Darren 
N. Seril et al. note that oxidative stress and free 
radical damage to mucous membranes of small 
and large intestines are one of the signs of 
peptic ulcer and, probably, one of the factors 
of carcinogenesis under these conditions [33].

The inflow of toxin (carrageenan) into the 
systemic circulation largely depends on the 
state of cytoplasmic membranes of organs and 
tissues that perform barrier functions, where 
the intestine is very important [34, 35]. One of 
the main pathogenetic factors regulating the 
permeability of membranes is the activation of 
free radical oxidation processes, which are 
crucial mechanism that provides the availability 
of lipid-protein complexes of membrane for 
phospholipase and proteases respectively [36]. 
This fact justifies the results obtained by us 
regarding the statistically significant activation 
of free radical oxidation processes in the rats 
with CEC combined with DM.

 Reducing SOD activity may be due to the 
damaging effects of free radicals on the me tallo-
protein complex of enzyme containing copper, 
zinc or manganese. Low activity of catalase may 
be associated with the increase in concentration 
of hydrogen ions, which leads to the develop-
ment of a proton form of an enzyme having an 
altered catalytic activity. It should be noted that 
in the experimental group 4 the rates of LPO 
were the highest and those of the antioxidant 

system were the lowest, compared to the other 
experimental groups. Therefore, the activation 
of free radical reac tions is an important non-
specific mechanism of inflammation develop­
ment in tissues of small and large intestine in 
cases of diabetes mellitus.

Conclusions
In the wall of small intestine of strepto-

zotocin-induced diabetic rats, lipid peroxide 
oxidation increases (DC content is higher than 
in the control by 66.93% and TBARS by 71.22%, 
respectively p<0.001) and the activity of enzyme 
link of antioxidant defence decreases reliably 
(SOD by 38.02% and catalase by 58.82%).

The course of experimental chronic entero-
colitis in rats is accompanied by the increase in 
free radical oxidation in the wall of small in-
testine (DC by 35.16% and TBARS by 27.65%, 
p<0.01) with simultaneous increase of enzyme 
activity of antioxidant defence system (SOD by 
29.90% and catalase by 21.65%, p<0.01), com-
pared to the control.

In cases of chronic enterocolitis with under-
lying diabetes mellitus in rats, the development 
of oxidative stress in the wall of small intestine 
is associated with the statistically significant 
increase in levels of DC (by 104.84%) and TBARS 
(by 115.02%), p<0.01 and the decrease in activity 
of SOD (by 131.09%) and catalase (by 21.65%).

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Received: 2017-08-29