THE DISTURBANCE OF OXIDANT-ANTIOXIDANT BALANCE IN RAT COLONIC MUCOSA AFTER ANTIBIOTIC THERAPY

Antibiotic treatment increases susceptibility to development of inflammatory bowel diseases (IBD) both in children and adults. Oxidative stress plays a prominent role in IBD pathogenesis. The aim of present study was to test an interrelationship between the morphological changes in rat colonic mucosa, the levels of antioxidant enzymes and redox sensitive transcription factors Egr-1 and Sp-1 after treatment with cephalosporin antibiotic ceftriaxone (Cf) with broad spectrum of action. Study was performed on male Wistar rats (180–230 g). Cf (50 mg/kg, i.m.) were injected daily for 5 days. The colonic levels of catalase and superoxide dismutase activity were measured by the colorimetric assays and zymography; levels of Egr-1 and Sp-1 – by Western-blot analysis; histological chan ges – by morphometric analysis. Body weight and diarrhea were recorded. Systemic administration of the ceftriaxone induced morphological and functional changes in rat colonic mucosa associated with initial stages of the acute inflammation. These changes were accompanied by a decrease of activity of superoxide dismutase and catalase. Levels of redox-sensitive transcription factors Egr-1 and Sp1 were significantly increased, which shows a disturbance of homeostasis of the intestinal barrier.


INTRODUCTION
The inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease are characterized by chronic non-specific inflammation and ulcers of the intestinal mucosa of unknown etiology. From 5 to 60 new cases per 100 thousand people are recorded annually and this rate is steadily increased [5,6].
In 2011, two independent groups reported about positive correlation between longterm use of antibiotics and the risk of IBD in children [2] and adults (average age -43.4 years) [12]. The mechanisms underlying this phenomenon are hypothetical and require empirical study.
A disruption of the intestinal barrier function is a crucial factor for the IBD pathogenesis [3]. We have found that a disruption of the intestinal barrier integrity at early stages of the IBD is associated with a development of hypoxia and oxidative stress in the epithelial cells with subsequent activation of the redox sensitive transcription factors [14].
Normal intestinal microbiota plays a crucial role in the formation and maintenance of the intestinal barrier homeostasis. Sterile environment (without bacteria) leads to a disruption of the intestinal barrier and immunity formation in germ-free animals. These animals have immature spleen and thymus; decreased number of lymphoid follicles and mature plasma cells which sensitize IgA; an imbalance at the level of anti-and proinflammatory cytokines; hypoplastic changes of Peyer patches [10]. Maintaining stability of microbiota composition is an essential factor for stimulating the expression of the antimicrobial peptides -defensins [1], the tight junction proteins [15] and mucins [11]. Considering a fact that antibiotics disturb the intestinal microbiota composition, we hypo thesized that antibiotic treatment may lead to a disruption of the intestinal barrier integrity via a shift in the redox status of the intestinal mucosa cells.
The aim of a present study was to test an interrelationship between the morphological changes in rat colonic mucosa, the levels of antioxidant enzymes and redox sensitive transcription factors Egr-1 and Sp-1 under treatment with cephalosporin antibiotic ceftriaxone.

MATERIALS AND METHODS
Male Wistar rats (180-230 g, n = 14) were bred and housed in a conventional animal facility of the ESC "Institute of Biology" Taras Shevchenko National University of Kyiv (Kyiv, Ukraine) under standard environmental conditions (12 h light/dark cycle at a constant temperature of 22 °C). All animals had unlimited access to animal chow and tap water throughout the study. To normalize gut microbiota, rats from all groups were kept in the same room and maintained by the same personal. Study was approved by the bioethical committee of ESC "Institute of Biology" Taras Shevchenko National University of Kyiv (Protocol No 8 issued by Nov, 2, 2015).
Animals were weighted before water or ceftriaxone treatment and next day after treatment stop. Diarrhea was estimated daily by a softness of feces. Animals were labeled as positive or negative for diarrhea sign.
Rats were euthanized by CO 2 inhalation followed by cervical dislocation next day after antibiotic withdrawal (6 th day of the experiment).
At autopsy, 2 cm of colonic tissue was embedded in 10% buffered formalin following paraffin for further histological analysis. Rest of colon was cut along the anti-mesenteric side and thoroughly rinsed in cold PBS. Colon was gently wiped with paper towel and flat by mucosa side up on ice. Mucosa was gently scraped by using metal spatula from the muscular layer and embedded in a liquid nitrogen for further biochemical assays.
Morphological signs of the colonic mucosa were estimated by the morphometric analysis on 3-5 µm histological sections stained with hematoxylin and eosin.
Catalase activity in colon was measured colorimetrically in a reaction with 0.03% H 2 O 2 solution. The reaction was stopped by the molybdate ammonium (Alfarus, Ukraine) and measurement was taken at a wavelength of 410 nm. The activity of superoxide dismutase (SOD) was determined by a zymography method, which was carried out in polyacrylamide gel with the addition of bromophenol blue to the samples. After the electrophoretic separation, the gel was incubated for 20 min in a solution of the nitroblue tetrazolium, TEMED and riboflavin (Sigma-Aldrich, Germany). Further it was kept under light until the manifestation of the reaction in the form of transparent spots on the lightblue background of the gel. Total concentration of proteins was measured by the Bradford method using a set of "Bio-Rad protein assay" (Bio-Rad, USA).
Separation and detection of proteins (150 µg proteins per sample) was run by the Western-blot analysis in 10% SDS polyacrylamide gel followed by transfer to the Hybond-ECL nitrocellulose membrane (Amersham Biosciences, USA) according to a standard protocol of Bio-Rad Company. Anti-Egr-1 (1:300) and Sp-1 (1:500) antibodies (Santa-Cruz, USA) were used to determine the level of the corresponding proteins in the colonic mucosa, followed by incubation with secondary HRP-conjugated antibodies (1:5,000, Santa-Cruz Biotech, USA). The loading controls were performed by using a mouse monoclonal antibody to β-actin (1:500) (Sigma-Aldrich, Germany).
Data are presented as M±SD. Statistical significance was determined by the Student's t-test. P-values less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
Аntibiotic treatment induced diarrhea in 30% rats. Despite of that, we did not detect a significant difference in body loss between control and antibiotic-treated rats. At the end of the experiment, the rats of control and experimental groups lost 5% and 6% of bodyweight, respectively.
The morphometric analysis of histological sections of rat colon revealed that 5 days of ceftriaxone administration increased the thickness of the mucous membrane (from 238.77±60.32 to 371.95±60.32 µm, P < 0.001, n = 5), the depth of intestinal crypts (from 224.0±32.0 to 310.4 ± 70.1 µm, P < 0.001, n = 5), and the area of the colonocytes nuclei (from 25.69±8.4 to 35.46±8.63 µm 2 , P < 0.001, n = 5). We observed a swelling and desquamation of the mucosa that indicated an increase of functional activity of colonocytes. It was accompanied by an increase in number of the goblet cells, but a decrease in their size (from 126.23±54.67 to 107.40±49.42 µm 2 , n = 5). These results indicate the pro-inflammatory changes and homeostasis disturbance of the intestinal barrier.
Lipid peroxidation processes (LPO) are major metabolic reactions, whose intensity is at certain level in the tissue. Disturbances of LPO after damaging effects are early and universal non-specific reactions in pathogenesis of many diseases. One of the pathogenic factors of LPO activation may be a deficiency of antioxidant activity in the colonic mucosa [8]. It is known that SOD catalyzes dismutation of the superoxide into oxygen and hydrogen peroxide. The hydrogen peroxide was formed during dismutation of the superoxide anion radical, is reduced to water, mainly with catalase and glutathione peroxidase.
An injection of ceftriaxone reduced the activity of SOD and catalase in rat colonic mucosa 1.1-and 4.5-folds, respectively (Fig. 1). A reduced activity of SOD and catalase may indicate a disturbance of physiological protection system from an excessive lipid peroxidation after administration of ceftriaxone. These data are consistent with the results of other study [13]. It was shown that the injection of different doses of cephalosporin antibiotics to rats for 15 days reduced the activity of catalase and SOD as early as at the 1 st -2 nd days after administration of the antibiotic. Despite a fact that the mechanisms of lipid peroxidation after antibiotic treatment have been reported earlier [8,13], changes at the level of redox sensitive transcription factors have not been studied. Egr-1 is an early response protein that refers to a group of redox-sensitive transcription factors. Its activity depends on intracellular balance between oxidants and antioxidants. DNA-binding domain of Egr-1 contains cysteine residues. Oxidation the thiol (SH)-groups of cysteine leads to a disruption of the inter/intramolecular disulfide bonds and, conformation changes of protein molecules that affect DNA binding activity of transcription factors. Egr-1 is an inducible factor that does not occur in normal colonic mucosa, but it is activated by an oxidative stress. Egr-1 can activate expression of many angiogenic factors (bFGF, PDGF-A, PDGF-B, VEGF, VEG-FR-1, angiopoentin-1, proteases), as well as pro-inflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1β, IL-2, monocyte chemotactic protein-1, tissue factor, GM-CSF) by interaction with the proximal promoter region of gene or by protein-protein interaction with other transcription factors [7,14].
The transcription factor Sp-1 plays an important role in the regulation of cellular processes such as metabolism, growth, differentiation, apoptosis and angiogenesis. It is involved in regulation of expression of the housekeeping genes, genes that direct development of erythroid, lymphoid and monocytic lineages. Sp-1 modulates gene transcription of tissue plasminogen activator which depends on the retinoic acid and cAMP. A protective role of Sp-1 was found during oxidative stress in brain neurons [9,14]. The regulation of gene expression by Egr-1 and Sp-1 transcription factors showed an interaction between them, since DNA-binding domains of these factors overlap [14,17]. We [14], and others [4,16], showed that Egr-1 and Sp-1 play an important role in pathogenesis of the inflammatory bowel diseases.
In present study, we showed an increase in the levels of Egr-1 and Sp-1 in rat colonic mucosa after administration of the ceftriaxone. The levels of Egr-1 were increased by 1.7-fold (Fig. 2, A), while Sp1 -1.6-fold (P < 0.05 vs. water-treated group) (Fig. 2, B). Considering a fact that an increase levels of Egr-1 and Sp-1 can be an early sign of the development of inflammatory bowel disease, our findings may indicate their involvement in the protective mechanisms in a response to side effects of the ceftriaxone.

CONCLUSIONS
Systemic administration of the cephalosporin antibiotic ceftriaxone leads to morphological changes in rat colonic mucosa whose nature indicates the initial stages of the acute inflammation. These changes are accompanied by a decrease in activity of the antioxidant enzymes in the colonic mucosa, and increase in the levels of redox-sensitive transcription factors Egr-1 and Sp1, that shows a disturbance in homeostasis of the intestinal barrier.