Wpływ flawonoidu kwercetyny na wskaźniki układu tlenku azotu u szczurów z przewlekłym zapaleniem jelit i cukrzycą indukowaną streptozotocyną

Іnna Krynytska1, Mariia Kushynska2, Iryna Pavlenko3, Mariya Marushchak1

1Department of Functional and Laboratory Diagnostics, I. Horbachevsky Ternopil State Medical University, Ternopil, Ukraine

2Department of Medical Biology, Parasitology and Genetics, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine

3Department of Anesthesiology and Intensive Care, Danylo Halytsky, Lviv National Medical University, Lviv, Ukraine

Abstract

Introduction: Chronic hyperglycemia is accompanied by significant physiological, biochemical, and histological changes, e.g. development of oxidative and nitrosative stress that affects the motor activity of the intestine.

The aim: The present study was designed to evaluate the indices of nitric oxide (NO) system in blood serum and a colon tissue supernatant of rats with chronic enterocolitis combined with streptozotocin-induced diabetes.

Materials and methods: The total NOS activity was performed by monitoring the rate of conversion of L-arginine into citrulline. The total contents of NO metabolites was assessed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (NO2) and also nitrate ions reducted to nitrites (NO3).

Results and conclusions: Thus, in rats with modeled chronic enterocolitis activation of nitroxydergic process in blood serum and colon tissue has been established. Herewith more pronounced intensification of nitroxydergic processes was observed in rats with chronic enterocolitis combined with streptozotocin-induced diabetes. The liposomal form of quercetin – Lipoflavon significantly reduces nitrosative stress in rats with chronic enterocolitis combined with streptozotocin-induced diabetes.

Wiad Lek 2018, 71, 8, -1508

Introduction

Diabetes Mellitus (DM) is an endocrinological disorder and not a single disorder which is a group of metabolic or heterogeneous affliction resulting from an irregularity in insulin secretions and insulin actions or both [1, 2]. DM represents a massive public health issue, being one of the major causes of morbidity and mortality in the modern societies [3, 4]. Recently, it was recorded that only in 2012 at least 1.5 million deaths induced from diabetes [3]. The number of patients in the developed countries of the world is 2-6% of the total population. The latest International Diabetes Federation estimates indicate that 415 million (1 in 11 persons) have diabetes, and this will increase to 642 million or almost 10% of the general population by 2040 [5].

Prevalence of diabetes is seen to vary in different populations with different ethnic and racial backgrounds, geographic location, e.g. from 10 % in Japan to 40 % in Pima Indians [6]. It has been established that prevalence of diabetes is increasing among the population of the world depending on the region, level of economic development of the country, gender and age. It should be noted that about 75% of these patients live in countries with low and middle income [7].

As is known, chronic hyperglycemia is accompanied by significant physiological, biochemical, and histological changes in patients suffering from DM [8]. In physiological conditions in the mucous and muscular shell of the digestive organs are expressed constitutive forms of NO synthases (cNOS) – an endothelial (eNOS) and neuronal (nNOS) that are calcium-dependent and provide biosynthesis of insignificant quantities of nitric oxide, which regulates secretion, motility, absorption, blood flow, supports structure and function of the mucosal barrier, the process of intercellular integration [9]. DM increases the activity of inducible NO-synthase (iNOS), which is localized in the endothelial, epithelial, immune cells and smooth myocytes of the intestine [10], whereas the expression of nNOS in NO-ergic neurons decreases [11].

Hyperglycemia induces the development of oxidative stress and changes in activity enzymes of antioxidant system protection that affects the motor activity of the intestine [12]. It is known that antioxidants, in particular flavonoid quercetin, positively influence on the main links of the pathogenesis of diabetes mellitus, including limiting the frequency of damages caused by oxidative stress [13].

The aim

The aim of the research was to study the indices of nitric oxide system in blood serum and a colon tissue supernatant of rats with chronic enterocolitis combined with streptozotocin-induced diabetes and the specific pharmacological activity of flavonoid quercetin.

Materials and methods

The experiments were performed on 48 white nonlinear mature male rats, which were kept on a standard diet at the vivarium of I. Ya. Horbachevsky Ternopil State Medical University. The animals were kept and experiments were conducted in accordance with the European Convention for the Protection of vertebrate animals used for experimental and other scientific purposes [14].

The animals were divided into 4 groups: the 1st – control group (n=12), the 2nd – animals with diabetes mellitus (n=12) , the 3rd – animals with chronic enterocolitis (n=12), the 4th – animals with diabetes mellitus and chronic enterocolitis (n=12).

Diabetes mellitus (DM) was induced by a single intraperitoneal administration of streptozotocin (Sigma Aldrich, USA, at a dose of 60 mg/kg body weight) [15]. Immediately prior to the administration, streptozotocin was dissolved in 0.1 M citrate buffer (pH 4.5). The control group received a corresponding amount of citrate buffer. The experimental group included animals with glucose level of at least 10.8 mmol/L in 2 weeks after streptozotocin administration.

Chronic enterocolitis (CEC) was modeled by free access of animals to 1.0% solution of carrageenan in drinking water during 1 month [16].

Liposomal form of flavonoid quercetin – “Lipoflavon” («Pharmstandart-Biolik», Ukraine) was injected intraperitoneally at the dose of 10 mg/kg body weight (counting on quercetin) for 2 weeks.

Blood serum and a colon homogenate were used for our investigation. Glucose levels in the blood were measured using a Precision Xtra Plus glucometer (MediSense UK Ltd., Great Britain).

NOS activity assay in the supernantant of colon homogenate was performed by monitoring the rate of conversion of L-arginine into citrulline [17]. Total protein was measured by Lowry assay [18]. Briefly, the samples aliquots that contained 300 mg protein were used to determine the total NOS activity. They were incubated for 60 min at 37°C in a total volume of 1 mL substrate mixture (pH 7.0) of the following composition (µmol/mL): KH2PO4 – 50, MgCl2 – 1, CaCl2 – 2, NADPH («Sigma», USA) – 1, L-arginine – 2. The reaction was stopped by adding 0.3 mL of HClO4 (CN=2 mol/L). As a control samples that contained the full substrate mixture previously denatured by HClO4 (CN=2 mol/L) were used. The mixture was centrifuged at 3500 g for 10 min and the non-protein supernatant mixtures were used to test L-citrulline by highly specific method for color reaction with antipyrine. Its sensitivity is 0.2 mg of L-citrulline in 1 mL, so it can be used to study the NOS activity. Protein-free aliquot samples were mixed with 2 mL of reagent (1 mL of 59 mmol/L diacetyl monoxime («Sigma», USA) + 1 mL of 32 mmol/L antipyrine («Sigma», USA) + 55 µmol/L Ferrous (II) sulphate in H2SO4 (CN=6 mol/L)) and boiled for 15 min in a water bath. After cooling the value of extinction was determined at 456 nm. The citrulline content was determined using a calibration graph. Total NOS activity was expressed as pmol of L-citrulline/min per 1 mg of protein.

Quantitative assessment of total concentration of NO2–+NO3– was performed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (NO2–) and also nitrate ions reducted to nitrites (NO3–) [19]. Reduction was performed using zinc dust in acidic environment. Nitrites with sulphanilic acid underwent a reaction of diazotisation. Obtained diazotisation compound with N-1-naphthtylethylendiamin formed azo dye. Optical density of the obtained colour solution was evaluated by spectrophotometry at absorption maximum and wavelength 536 nm. The total concentration of NO2–+NO3– in the studied sample was estimated by the equation: Х = (Y – А) / B, where X – concentration of NO metabolites in µmol/L; Y – optical density of the studied sample; B – regression coefficient; A – intercept.

All of the data were processed using the software package Statistica 6.1 for Windows. Intergroup comparisons were performed using Mann–Whitney–Wilcoxon U test. The median (Me) and interquartile range (IQR [Q25-Q75]) were used. Differences with p-value < 0.05 were considered as significant.

Results and discussion

Total NOS enzymatic activity in the colon tissue supernatant of rats was significantly increased in all experimental groups as compared to control animals (54.8, 30.6 and 79.2 % respectively), but was greater in the rats with chronic enterocolitis combined with streptozotocin-induced diabetes (table I). Lipoflavon significantly reduces total NOS activity by 16,5 % compared to group with chronic enterocolitis combined with streptozotocin-induced diabetes.

The total concentration of NO2+ NO3in blood serum of rats with streptozotocin-induced diabetes was significantly increased by 2.1 times, with chronic enterocolitis – by 43.2 % and with chronic enterocolitis combined with streptozotocin-induced diabetes – by 2.6 times vs control group. Comparison of total concentration of NO2+ NO3in blood serum and colon tissue supernatant was great important. It was determined that NO production disorders took place unidirectionally towards the oxidative burst. Thus, the total concentration of NO2+ NO3in colon tissue supernatant of rats with streptozotocin-induced diabetes was significantly increased by 2.8 times, with chronic enterocolitis – by 1.9 times and with chronic enterocolitis combined with streptozotocin-induced diabetes – by 3.4 times vs control group. Lipoflavon significantly reduces total contents of NO metabolites by 17,8 % in blood serum and by 29,8 % in colon tissue supernatant compared to group with chronic enterocolitis combined with streptozotocin-induced diabetes.

Nitric oxide (NO) is a messenger molecule functioning in variety of physiological activities in different cell types and tissues, formed by the enzymatic oxidation of L–arginine under the influence of cytochrome P450–like hemoproteins, NO–synthases (NOS) [17]. There are three isoforms of this enzyme: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS) or macrophagal [20]. Pathophysiology studies have shown that NO could be a messenger of an inhibitory neurotransmitter in human intestine [21]. NO plays not only a significant role in providing for the processes of nervous impulses modulation, regulation of motility and secretion, but also cytoprotection of the large intestine [22].

In the mucous membrane of the large intestine, eNOS is identified in the endothelial and epithelial cells, smooth muscles, thrombocytes and T-cells, while nNOS is mainly localized in the central and peripheral nerves, but also is detected in myocytes, epithelial cells, mast cells, and neutrophils [22].

NO can regulate the functional state of smooth muscles of the intestine with the participation of several mechanisms. As a lipophilic molecule, NO easily diffuses through cell membranes into the neighbouring cells (e.g. from endothelial to myocytes) where cyclic guanosine monophosphate decreases concentration of free calcium and activates myosin light chain kinase [23]. NO signaling is carried out in three main mechanisms: (1) activation of soluble guanylate cyclase (sGC) through binding to its heme group (thus forming a Fe2+-nitrosyl complex), which leads to the formation of cGMP, which in turn stimulates protein kinase G; (2) S-nitrosylation: active forms of nitrogen reversibly nitrosilate thiol groups of target protein cysteins; (3) the formation of peroxynitrite (ONOO-) with subsequent nitration of the tyrosine and tryptophan residues in proteins, leading to the engagement of mitogen-activated protein kinases, protein kinase C isoforms, transcription factor NF-κB, etc., in the process of signaling [24, 25].

Normally, endogenous NO inhibits the motility of digestive organs [26]. Increasing the activity of iNOS leads to a significant reduction in motor activity [27], whereas inhibition of NO-ergic blockage of smooth muscle of the large intestine causes its intensification [10].

The destructive changes in the intestinal mucosa in case of chronic enterocolitis are associated with increased number of reactive oxygen species, enhanced synthesis of NO, expression of iNOS by epithelial cells, macrophages, and neutrophil infiltraton into the damaged mucous membrane.

S.V. Hridneva notes that in case of chronic enterocolitis endothelial functions are impared, which manifest in the activation of free radical oxidation processes andor a decrease in the activity of the antioxidant system, which explains the excessive production of ROS [28].

Our previous studies also have shown that the number of ROS evaluated by flow cytometry in rats with DM, increased by 3,0 times, in rats with CEC – by 2,0 times, and in rats with combined pathology – by 3,4 times vs control values [2].

In case of streptozotocin-induced hyperglycemia, the total activity of NOS and total concentration of NO2+ NO3 in our research increased, which may cause a decrease in the tone of smooth muscles and disrupt the motor and evacuation function of the large intestine.

Hyperglycemia is accompanied by the muscle relaxation, with rising of iNOS activity in the muscular membrane, which increases the contents of NO. It should be noted that the part of the nitrogen oxide synthesized by iNOS interacts with the superoxide radical, which leads to the formation of ONOO-, which causes endothelial dysfunction, nitrosylation of cytoplasmic proteins, activates the processes of lipoperoxidation. Increase activity of iNOS during muscles relaxation is also associated with enlarged formation of Н2О2 and the influence of proinflammatory cytokines that activate expression of mRNA iNOS [12]. Therefore, TNF-α is very important in the development of both DM and CEC. The mechanisms by which TNF-α initiates and enhances inflammation in the intestine are very complex and have so far been poorly understood. A direct negative effect of TNF-α is associated with damage to enterocytes, which leads to increased of the epithelium permeability. In addition to direct effects on the integrity of the intestinal epithelial barrier, TNF is a powerful inducer of matrix metalloproteinases in stromal cells of the intestine; it induces the production of keratinocyte growth factor, leading to hyperplasia of crypts and causing increased expression of the major histocompatibility, complex class II antigens [29].

In case of diabetes mellitus the production of ROS and peroxynitrite, which cause the development of oxidative and nitrosative stress, is increasing. This may be due to auto-oxidation of glucose, enhanced glycosylation of cellular proteins, activation of the polyol pathway, and increased formation of superoxide radical in the respiratory chain of mitochondria [12]. Moreover in diabetes mellitus, advanced glycation end products have a damaging effect on the DNA of cells and tissues, accumulating in ganglia, villi and membranes of enterocytes of the intestine [30].

The positive effect of quercetin on the level of glycemia in case of diabetes is realized through inhibition of glucose absorption in the intestine and increase in its absorption by peripheral tissues [31]. Quercetin also accelerates the use of glucose in hepatocytes and skeletal muscle cells by activating of key enzymes for the glycosylation of hexokinase and pyruvate kinase, decreases the activity of glycogen phosphorylase and stimulates the formation of glycogen in hepatocytes and skeletal muscles [32]. In vitro, quercetin has established itself as an inhibitor of α-glucosidase activity [33]. The anti-inflammatory activity of quercetin is confirmed by data on the blockade of key enzymes of the cascade of arachidonic acid – 5-lipoxygenase and cyclooxygenase. This property helps to stabilize the processes of lipid peroxidation of biomembranes and to decrease oxidative and nitrosative stress. The presence of an unsaturated (double) bond in the C2-C3 position, the character of hydroxylation / methoxylation of the rings A and B, the presence of the 3 OH-ring C promote its lipoxygenase / cyclooxygenase inhibitory activity [13].

Conclusions

Thus, in rats with modeled chronic enterocolitis activation of nitroxydergic process by significant increase in total concentration of NO2+ NO3 and total NO synthases activity in blood serum and colon homogenate supernatant has been established. Herewith more pronounced intensification of nitroxydergic processes was observed in rats with chronic enterocolitis combined with streptozotocin-induced diabetes. The liposomal form of quercetin – Lipoflavon significantly reduces nitrosative stress in rats with chronic enterocolitis combined with streptozotocin-induced diabetes.

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Study limitation:

This research was conducted only on a small size of rats’ population. It is therefore essential to validate our findings with greater sample sizes to determine the features of nitroxydergic processes.

Authors’ contributions:

According to the order of the Authorship.

Conflict of interest:

The Authors declare no conflict of interest.

CORRESPONDING AUTHOR

Mariya Marushchak

Department of functional and laboratory diagnostics,

I. Horbachevsky Ternopil State Medical University

Majdan Voli 1, 46001, Ternopil, Ukraine

tel: +380979981202

e-mail: marushchak@tdmu.edu.ua

Received: 08.07.2018

Accepted: 15.10.2018

Table I. The effect of flavonoid quercetine on the indices of nitric oxide system in blood serum and colon homogenate supernatant of rats with chronic enterocolitis combined with streptozotocin-induced diabetes, Me [Q25-Q75]

Group of animals

Control

(n=12)

DM

(group 2)

(n=12)

CEC

(group 3)

(n=12)

DM+CEC

(group 4)

(n=12)

DM+CEC

+

Lipoflavon

(group 5)

(n=12)

Blood serum

NO2+NO3, µmol/L

38.2

[30.8; 48.1]

81.7*

[76.1;84.4]

54.7*

[50.8; 58.2]

101.2*

[93.3;104.1]

p1<0,05

p2<0,002

83.2*

[78.8;88.4]

p3<0,05

Supernatant of colon homogenate

NO2+NO3, µmol/L

15.9

[12.3; 17.8]

44.5*

[40.3;48.9]

30.0*

[25.8; 36.4]

54.7*

[50.5; 57.3]

p1<0,05

p2<0,01

38,4

[33.7; 42.4]

p3<0,01

Total NOS, pmol of L-citrulline/min per 1 mg of protein

47,1

[39; 52]

72,9*

[65,7; 76,7]

61,5*

[53,1; 68,3]

84,4*

[75,7; 89,2]

p1<0,05

p2<0,01

70.5*

[67.6;74.1]

p3<0,05

Notes:

* – The difference between the control and the experimental group is statistically significant (р<0.05-0.001);

p1 – The reliability value between groups 2 and 4;

p2 – The reliability value between groups 3 and 4;

p3 – The reliability value between groups 4 and 5.