CC BY
8
ISSN 2520-2588 (Online)
Regulatory Mechanisms in JtSiosystems
¿m' v* ' Jfflfc Regulatory Mechanisms ISSN 2519-8521 (Print) ISSN 2520-2588 (Online)
Regul. Mech. Biosyst.,
in Biosystems 2019, 10(4), 438-444
doi: 10.15421/021965
Antioxidant protection enzyme activity in the blood serum and large intestinal mucosa of rats with prolonged gastric hypochlorhydria and given multiprobiotics
S. V. Pylypenko, A. A. Koval
Poltava V. G. Korolenko National Pedagogical University, Poltava, Ukraine
Article info
Received 0910.2019 Received in revised form
06.11.2019 Accepted 08.11.2019
Poltava V. G. Korolenko National Pedagogical University, Ostrohradskyi st., 2, Poltava, 36003, Ukraine. Tel.: +38-095-126-91-86. E-mail: pilipenko_s@ukr.net
Pylypenko, S. V., & Koval, A. A. (2019). Antioxidant protection enzyme activity in the blood serum and large intestinal mucosa of rats with prolonged gastric hypochlorhydria and given multiprobiotics. Regulatory Mechanisms in Biosystems, 10(4), 438-444. doi:10.15421/021965
The activity of antioxidant protection enzymes in the blood serum and colon mucosa in rats was studied under the conditions of 28-days administration of omeprazole on its own and omeprazole together with multiprobiotics "Symbiter" and "Apibact". Physiological and biochemical study methods were applied. It was found that after omeprazole administration, the activity of superoxide dismutase in the blood serum decreased, and the activity of catalase increased compared to the control. With the co-administration of omeprazole and multiprobiotics, the activity of superoxide dismutase increased compared to the group of rats that received omepra-zole only during the same time, but remained less compared to the control group. The content of reduced glutathione in the blood serum of rats after administration of omeprazole decreased, the activity of glutathione peroxidase and glutathione transferase increased, and the activity of glutathione reductase decreased compared to the control. With co-administration of omeprazole and multiprobiotics, the serum RG content was at the control level, the activity of glutathione reductase exceeded the control values. The activity of glutathione reductase decreased compared to the group receiving omeprazole only. The activity of glutathione reductase increased and did not differ from the control values. In the colon mucosa, superoxide dismutase and catalase activity decreased compared to control. With the combined administration of omeprazole and multiprobiotics, superoxide dismutase and catalase activity increased and even exceeded the control values. With the administration of omeprazole, the reduced glutathione content in the colon mucosa was lower than that in the control. The activity of glutathione peroxidase increased and glutathione transferase and activity of glutathione reductase decreased compared to the control. With co-administration of omeprazole and multiprobiotics to rats, the reduced glutathione content increased compared to the group of rats administered omeprazole only, and even exceeded that in the control.
Keywords: omeprazole; superoxide dismutase; catalase; glutathione; antioxidant protection enzymes.
Introduction
Today, an important problem for research remains the functioning of the digestive system in the conditions of prolonged gastric hypochlorhydria, which leads to hypergastrinemia, which in its turn is known to be a significant factor in the growth and development of tumours in the gastrointestinal tract (Burkitt et al., 2009). Most frequently carcinoid tumours of the stomach develop in patients in whom diseases are associated with hypergastrinemia (Stepanov et al., 2000). These are diseases such as chronic atrophic gastritis (Sipponen et al., 2002), Zolinger-Ellison syndrome with multiple endocrine neoplasia type 1 (Zhong et al., 2005), pernicious anemia (Kedika, 2009) and conditions after surgery for vagotomy (Waldum et al., 2005). Hypergastrinemia is also a consequence of long-term antisecretory drugs administration (Mokrasch & Teschke, 1984; Tazoe et al., 2008).
Widespread infection of Helicobacter pylori also contributes to the increase in the percentage of people with hypergastrinemia (LeBlanc & LeBlanc, 2014). Another, equally important, consequence of the prolonged decrease in the secretion of hydrochloric acid (HCl) in the stomach is the development of dysbacteriosis in various biotopes of the digestive tract. Gastric secretion acid denatures proteins, activates pepsinogen and enhances absorption of iron and calcium in the intestine. However, according to Howden and Hunt (Hütt et al., 2009), the main function of gastric juice is the inactivation of ingested microorganisms. It is known that decrease in gastric juice acidity for any reason can lead to excessive bacterial growth in the oral cavity, the stomach (Habig et al., 1974; Wat-
son et al., 2002; Willams & McColl, 2006, 2018; Pylypenko et al., 2018) and in the small and large intestine (Habig et al., 1974; Osefo et al., 2009).
Proton pump inhibitors (PPI) are widely used to treat disorders caused by the action of hydrochloric acid: gastroesophageal reflux disease (GERD), Barrett's esophagus, peptic ulcer, functional and unresearched dyspepsia, Zollinger-Ellison syndrome, and other less common acid-dependent diseases. Proton pump inhibitors are a compulsory component of eradication therapy for H. pylori infection and are used for the prevention and treatment of NSAID-gastropathy (Radchuk et al., 2010; Tsyry-uk et al., 2011). Acid-dependent diseases often require long-term drug treatment, and patients are constantly receiving PPI, which in its turn inhibits the protective effect of hydrochloric acid, leading to hyperga-strinemia and dysbiosis. Therefore, hypergastrinemia and dysbiosis can develop as a result of long-term antisecretory drug (proton pump inhibitors) administration (Osefo, 2009). Both prolonged hypergastrinemia and dysbacteriosis enhance the development of chronic inflammatory process, which plays a decisive role in the development of tumours. The purpose of our project was to study the activity of antioxidant defense enzymes in the blood serum and in the colon mucosa of rats under the condition of prolonged omeprazole alone and omeprazole combined with the Symbiter and Apibact multiprobiotics administration.
Materials and methods
The work was performed on sexually mature white adult rats, which were kept under standard vivarium ration. Throughout the experiment, the animals were not abused and inhumanely killed. The research meets the basic requirements of keeping and working with laboratory animals in compliance with the rules of the European Convention for the Protection of Vertebrate Animals Used in Experimental Research and Other Scientific Purposes (Strasbourg, 1986) and in accordance with ethical standards specified in Ukrainian law (Murzin, 2004).
The conclusion that the experimental studies complied with the generally accepted bioethical standards and the relevant international regulations was made at the meeting of the bioethical committee of the National Institute of Biology of the National Taras Shevchenko University of Ukraine (26.06.2013, No. 35). The studies were performed on 40 white nonlinear male rats weighing 160-180 g, which were kept in an accredited vivarium at the Institute of Biology of Taras Shevchenko National University of Kyiv in compliance with the "Standard Rules for Organizing, Equipping and Maintaining Experimental Biological Clinics (Vivarium)". All experiments were carried out in compliance with the Law of Ukraine No. 3447-IV "On the Protection of Animals from Cruelty".
All animals were divided into 4 experimental groups. Animals of group 1 served as control. They were administered intraperitoneally (i/p) 0.2 mL and orally (p/o) 0.5 mL of water by injection for 28 days once a day. Animals of group 2 were administered omeprazole and 0.5 mL water for injection once a day for 28 days. Animals of group 3 were co-administered the Symbiter® acidophilic concentrated multipro-biotic (Simbiter) with omeprazole once a day for 28 days. In group 4, omeprazole and "Apibact®" multiprobiotic were co-administered once a day for 28 days.
Omeprazole (manufactured by "Sigma-Aldrich", USA) was admi-mistered at a dose of 14 mg/kg dissolved in 0.2 mL of water for injection. Multiprobiotics Symbiter and Apibact (manufactured byNVK "OD Prolisok", Ukraine) were co-administered with omeprazole p/o at a dose of 140 mg/kg (1.4 ■ 1010 CFU/kg). Multiprobiotics were dissolved in 0.5 mL of water for injection.
Activity of superoxide dismutase (CF 1.15.1.1) in cells was determined by Cevari et al. (1985). The method is based on the ability of superoxide dismutase to compete with nitro blue tetrazolium (NBT) for superoxide anions formed as a result of the aerobic interaction of the nicotinamide adenine dinucleotide reduced form and phenazin metasulfate (FMS). As a result of this reaction, NBT is restored to form hydrazine tetrazolium. In the presence of superoxide dismutase, the percentage of NBT recovery decreases.
Serum aliquote, cell lysate, or mucous membrane homogenate (0.5 mg of protein) was added to a sample containing 0.15 M phosphate buffer and the total sample volume was 0.5 mL. The amount of 1 mL reagent I (57 pM HCT, 16 pM FMS in 0.15 M phosphate buffer with EDTA, pH = 7.8) was added to the sample. Absorbance of the samples was immediately measured at X = 540 nm with a spectrophotometer (SF-46, LOMO, Russia). Then, 0.035 mL of reagent II (98.5 pM NAD.H in Tris-EDTA buffer, pH = 8.0) was added to each sample, placed in the dark and the extinction was re-determined after 10 min under the same conditions. The samples were kept at 30 °C. The enzyme activity was expressed in units per minute per 1 mg of protein.
The catalase activity (EC 1.11.1.6) in cells was determined by (Ko-rolyuk et al., 1988). The principle of the method is that catalase destroys the H2O2 substrate, the non-destructed part of hydrogen peroxide, when interacting with molybdenum salts, forms a stable coloured complex. 2 mL of 0.03% hydrogen peroxide solution were added to the test tubes. The reaction was started by adding 0.1 mL of serum, cell ly sate, or mucous membrane homogenate (0.1 mg of protein). 0.1 mL of distilled water was added to the control sample instead of protein. The samples were kept at room temperature for 10 min, the reaction was stopped by adding 1 mL of 4% ammonium molybdate. The colour intensity was measured with a spectrophotometer (SF-46, LOMO, Russia) at X = 410 nm against a control sample, which was added to 2 mL of H2O instead of hydrogen peroxide.
Determination of the reduced glutathione content is based on mod-fications of the Koch and Lyle method (Howden & Hunt, 1987; Mikel-
saar & Zilmer, 2009). The method is based on the interaction of reduced glutathione with ortho-phthalic aldehyde (ORT) as a fluorescent reagent. This produces high-fluorescence products that are activated at 350 nm and have a well-pronounced peak at 420 nm. The ORT reagent was prepared before the experiment at a concentration of 1 mg/mL in methanol (Mikelsaar & Zilmer, 2009). It is shown that the reaction depends on the acid-alkaline characteristics of the medium, and takes place at pH 8.0, since a change in pH below the specified level is accompanied by a decrease in the fluorescence intensity, and conversely, the change of pH towards the alkaline medium causes the conversion of the reduced glutathione form into the oxidized one. The fluorescence intensity for ORT-GSH reaction directly and linearly depends on the concentration of GSH within the range of 10 ng to 4 pg GSH.
The samples were thawed and homogenized on ice in a small Teflon Potter-Elvelgem homogenizer with addition of 1.5 mL of 0.01 M HCOH to precipitate proteins (Binder, 2010). Next, it was centrifuged in cold (+4 °C) at 20,000 g for 15 min to obtain a supernatant, in which the content of reduced glutathione was determined.
Sampling for GSH concentration was carried out according to the following scheme. To 0.1 mL of supernatant 100 pL of 0.1 M phosphate buffer was added with (1:4 (V/V) 37% formalin (Tkachenko, 2014): 0.1 M Na2HPO4- 12H2O) (the samples were kept for 5 min at room temperature); 2 mL of 0.1 M phosphate buffer containing 1 mM Na2HPO412H2O, 1mm NaH2PO42H2O, pH 8; 100 pL of orthophtha-lic aldehyde. After the 45-minute incubation at room temperature, the fluorescence intensity at 420 nm was measured with 350 nm activation with a spectrofluorophotometer (RF -1501, Shimadzu, Japan). A calibration curve was constructed to determine the GSH concentration. GSH content was expressed in nmol per 1 mg of protein.
To determine the activity of glutathione-dependent enzymes, the serum, cell suspension, mucous membrane homogenate were centri-fuged in cold (+4 °C) at 20,000 g for 15 minutes to obtain a supernatant, which was used to determine the enzymes activity.
The glutathione peroxidase activity (CE 1.11.1.9), as well as other glutathione-dependent enzymes, was determined by Vlasova (Vlasova & Perslegina, 1990). This enzyme activity was determined by accumulation of GSSG. The reaction mixture consisted of: 1 mL of 0.3 M phosphate buffer containing 12 mm sodium azide; 6 mm EDTA, pH 7.4; 0.5 mL of 2.5 mM GSSG; 0.2 mL of supernatant; 0.5 mL of 1.8 mM H2O2. The reaction was started by adding 0.5 mL of 1.8 mM hydrogen peroxide, stopped after 2 min by adding 0.5 mL of 10%o trich-loroacetic acid. After centrifugation at 3000 rpm for 15 min, the extinction of GSSG was determined at a wavelength of 260 nm. The enzyme activity was expressed in GSSG U-mol (mcM) per 1 mg of protein per min by the formula: A = a - b / c x t, where: a - the amount of oxidized glutathione in the control sample, taking into account non-enzymatic oxidation (nmol/mL); b - amount of oxidized glutathione in the test sample, taking into account non-enzymatic oxidation (nmol/mL); c -amount of protein in the test sample (mg); t - incubation time (min).
Glutathione transferase activity (CE 2.5.1.18) in cytosol was determined by the rate of the conjugate formation with 1-chloro-2.4-dinitro-benzene (CDNB), characterized by a maximum absorption at 346 nm (Hissin & Hilf, 1976; Vlasova & Perslegina, 1990).
To determine the glutathione transferase activity, a mixture was prepared: 1.5 mL of 0.1 phosphate buffer (pH 6.5), 0.2 mL of 10 mM GSH, 0.1 mL of the test sample supernatant. The reaction was started by adding 0.02 mL of 0.1 M CDNB. The increase in optical density was recorded for 3-5 min at 340 nm with a spectrophotometer (SF-6305, Lomo, Russia) and expressed in nanomoles (nM) of glutathione conjugate (GSR) per 1 mg of protein per min.
Glutathione reductase activity (CE 1.8.1.7) was determined by Vlasova et al. (1990), with modification for microcuvette measurement, with a total reaction mixture volume of 535 pL. Determination of the enzyme activity was performed by reducing the NADPH content. The reaction mixture consisted of 350 pL of phosphate buffer (0.05 M pH 8.0), 35 pL of 1 mM EDTA, 50 pL of 7.5 mM GSSG; 50 pL of parietal cells cytosolic fraction; 50 pL of 1.2 mM NADPH. The enzyme activity was determined by reducing the amount of NADPH at 37 °C for 8 min, at a wavelength of 340 nm with a spectrophotometer (SF-
6305, Lomo, Russia). The activity was expressed in NADPH nano-moles (nM) per 1 mg of protein per min.
Statistical data processing was performed in the Statistica 8.0 (StatSoft Inc., USA, 2009) software package. To test the samples for the type of distribution of the studied index the Shapiro-Wilk W-test was used. If the distribution ofthese samples did not correspond to the Gaussian distribution, then a nonparametric method was used to compare the samples -the Mann-Whitney U-test was applied for the comparison of two independent samples. In this case, the data obtained were presented as Median. The differences between the values in the control and experimental groups were determined using the ANOVA, where the differences were considered significant at P < 0.05 (with Bonferroni correction). The results were defined as means ± standard error (x ± SE).
Results
After 28 days of omeprazole administration, the activity of superoxide dismutase (SOD) in the blood serum, which eliminates the superoxide anion radical due to its dismutation into hydrogen peroxide, decreased by 54.5% (P < 0.05) compared to Hie control (Fig. la). Hie activity of catalase increased by 211.5% (P < 0.001 ) (Fig. lb).
0.14
¡1
й -S S p
0.12
0.10
0.08
0.06
0.04
0.02
0.00
7#
With co-administration of omeprazole and the multiprobiotic, Symbiter SOD activity in the blood serum increased by 60% (P < 0.05) compared to the group of rats that received only omeprazole during the same time (Fig. 1a). At the same time, SOD activity remained 27.3% (P < 0.05) lower than in the control group. With co-administration of omeprazole and the multiprobiotic "Apibact", the blood serum SOD activity increased by 80% (P < 0.05) compared to the group of rats that received only omeprazole for 28 days and was statistically significantly different from the control group (Fig. 1a). In the experiments with co-administration of ome-prazole and the multiprobiotic Symbiter and omeprazole and the multi-probiotic "Apibact", catalase activity in the rat blood serum decreased by 31.1% (P < 0.05) and 10.7% (P > 0.05), respectively, compared to the group of rats that received only omeprazole during the same time (Fig. 1b). However, this index remained significantly higher than in the control group (by 114.5% (P < 0.05) and 178.2% (P < 0.01), respectively). Activity of SOD and catalase in the mucous membrane of the rat colon after 28 days of omeprazole administration decreased by 35.9% (P < 0.001) and 45.6% (P < 0.05), respectively, compared to the control (Fig. 1a, b).
9 t
7 --
б --
5 --
< I
1 i 4 --
< СЙ
3 --
2 --
1 --
\\\ W V4V -.w-www
VWyVV-
www
VWyVV-
www
VWyVV-
www VVVVV-Vi
VVVVV-Vi www VVVVV-Vi
V44V44V
VWyVV-
VVVVV-Vi www
*/#
С
О
О+S
O+A
b
С
О
О+S
O+A
Fig. 1. Activity of superoxide dismutase (a) and catalase (b) in the blood serum of rats (x ± SE): C - the control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and "Symbiter" multiprobiotic
co-administration (n = 10); O + A - group of rats after 28 days of omeprazole and "Apibact" multiprobiotic co-administration (n = 10); * -P < 0.05, ** - P < 0.01, *** - P < 0.001 compared to the control;# - p < 0.05 compared to Hie group of animals administered omeprazole
S I
;> s
0.8 0.7
0.6
0.5 --
'g-i 0.4 --
0.3 --
0.2 --
0.1 --
0.0
С
Tsfe
.v-
XWWv лч-л-л
.www
О
7##
7#
7 T
5 -
4 -
ÈvS
3 f
< с
О+S
O+A
b
2 -h 1 0
С
тттрт
\\YWV\ \WWYi ЧЧЧ.ЧЧЧ.Ч
www
\VV\4VV
www
\VVWVV
О
7##
О+S O+A
Fig. 2. Activity of superoxide dismutase (a) and catalase (b) in the mucous membrane of the rat colon (x ± SE): C - control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and multiprobiotic "Symbiter" co-administration (n = 10); O + A - group of rats after 28 days of of omeprazole and multiprobiotic "Apibact" co-administration (n = 10); * - P < 0.05 compared to the control; # - P < 0.05, ## - P < 0.01 compared to the group of animals administered omeprazole
a
a
In the mucosa of rats which were co-administered omeprazole and the multiprobiotic "Symbiter" and omeprazole with the multiprobiotic "Apibact" for 28 days, SOD activity increased by 96.0% (P < 0.05) and 168.0%, respectively (P < 0.01 compared to the group of rats administered omeprazole only, Fig. 1a). However, the activity of SOD in groups of rats, which for 28 days were coadministered omeprazole and multiprobiotics together, exceeded even the control values. SOD activity under the conditions of omeprazole and "Symbiter" multiprobiotic co-administration was higher by 23.6% (P < 0.05), and under the conditions of omeprazole and "Apibact" multiprobiotic co-administration higher by 71.8% (P < 0.01) compared to the control.
The activity of catalase in the colon mucous membrane after prolonged co-administration of omeprazole and multiprobiotics also increased: after the omeprazole and multiprobiotic "Symbiter" co-administration it increased by 64.7% (P < 0.05) compared to the group of rats which were administered omeprazole only and statistically did not reliably differ from the controls; after omeprazole and multiprobiotic "Apibact" co-administration, the catalase activity increased by 179.4% (P < 0.05) compared to the rats treated with omeprazole only, which by 51.9% (P < 0.05) exceeded this index in the control (Fig. lb). An important role in the implementation of antiradical and antiperoxide protection of cells is played by 0.30 -r
0.25
0.20
0.15 --
0.10
0.05
0.00
TTTTTT" ■■■■■
V4V.VV V4V.VV V4V.VV V4V.VV V4V.VV V4V.VV V4V.VV V4V.VV
the glutathione system, which consists of reduced glutathione and a set of enzymes - glutathione peroxidase, glutathione transferase and glutathione reductase. The components of the glutathione link in the antioxidant protection inhibit most of the free radical reactions, ensure the non-radical reduction of lipohydroperoxides, inactivate various toxic substances and contribute to maintenance of antioxidant protection (Freitas et al., 2009). In this respect, our further experiments were aimed at studying the gluta-thione antioxidant defense system functioning in the blood serum and colon mucosa of rats with prolonged gastric hypochlorhydria and under the conditions of omeprazole and multiprobiotics co-administration.
A study of the reduced glutathione (VG) content in the blood serum of rats after 28 days of omeprazole administration showed its decrease by 15.8% (P < 0.05) compared to the control (Fig. 3a). This may be due to both increased consumption of glutathione by glutathione-dependent enzymes (glutathione peroxidase, glutathione transferase) and glutaredoxin, and by the direct oxidation or reduction with participation of glutathione by SH groups ofproteins (Oktyabrsky & Smirnova, 2007).
After 28 days of gastric juice hypoacidity in the blood serum, glutathione peroxidase activity increased by 40.0% (P < 0.05, Fig. 3b), glutathione transferase activity increased by 31.6% (P < 0.05, Fig. 4a) and gluMiione reductase activity decreased by 14.3% (P < 0.05, Fig. 4b). SO t
70
è-o
■= ¡л
а о <
60 --
50 --
40 --
30 --
20 --
10 --
w w\ twKw ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\ ■.www лч\ч\\
О
О+S
O+A
b
О
О+S O+A
Fig. 3. The content (x ± SE) of reduced glutathione (a) and activity of rat serum glutathione peroxidase (b): C - the control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and "Symbiter" multiprobiotic co-administration (n = 10); O + A - group of rats after 28 days of omeprazole and "Apibact" multiprobiotic co-administration (n = 10); * - P < 0.05 compared to the control;# - P < 0.05 compared to the group of animals administered omeprazole
250 -r
•I 200
t ou
■ S S
-Э о
Oil й с
О с
¿■ei
■г СЛ
•è О
О
С
150 -
100 --
50 --
W, sw WWW WWW WWW
www www www
\V-l44V
www www www
WWW
www www www www www www www
*/#
Ï
V OÛ
S £
~ s
'S о
ш S
■4- С
о
1.2
1.0 --
0.8
0.6 --
0.4
0.2
0.0
WjSW W4W4 WV-W WWW
www
W4W4 WV-W WWW WV-W WWW
www
W4W4 WV-W WWW 4WW\ WWW
www
С
О
О+S
O+A
b
С
О
О+S
O+A
Fig. 4. Activity (x ± SE) of glutathione transferase (a) and glutathione reductase (b) in the blood serum of rats: C - the control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and "Symbiter" multiprobiotic co-administration (n = 10); O+A - group of rats after 28 days of omeprazole and "Apibact" multiprobiotic co-administration (n = 10); * - P < 0.05 compared to the control; # - P < 0.05 compared to the group of animals administered omeprazole
Under conditions of 28-day co-administration of omeprazole and "Symbiter" or "Apibact" multiprobiotics, the serum RG content remained at the control level (Fig. 3a). Activity of glutathione peroxidase increased and exceeded the control values in the group of rats co-administered ome-
prazole and "Symbiter" multiprobiotic by 57.8% (P < 0.05), and in the group of rats co-administered omeprazole and Apibact multiprobiotic - by 36.8% (P < 0.05, Fig. 3b). Activity of glutathione transferase in groups of rats co-administered omeprazole and "Symbiter" multiprobiotic and ome-
С
С
a
a
prazole and "Apibact" multiprobiotic for 28 days decreased by 52.7% (P < 0.05) and 25.9% (P < 0.05), respectively, compared to the group of rats, which were administered omeprazole only (Fig. 4a).
In this case, the glutathione transferase activity in the blood serum in the group of rats administered omeprazole and "Apibact" multiprobiotic, reached the control values (Fig. 4a). The activity of glutathione reductase in the blood serum of rats co-administered omeprazole and multiprobio-tics increased and was not statistically reliably different from the similar values in the control group (Fig. 4b).
After 28 days of omeprazole administration, the reduced glutathione (RG) content in the colon mucosa decreased and was by 19.6%o (P < 0.05) lower than in the control group (Fig. 5a). In this group of rats, glutathione peroxidase activity increased by 24.4% (P < 0.05, Fig. 5b), and gluta-thione transferase and glutathione reductase activity decreased by 26.3% (P < 0.05, Fig. 5a) and 23.6% (P < 0.05, Fig. 5b), respectively, compared to the control.
a:
О
§ .s
II
3
ob gl -о j= 8 с ■§!
50 j 45 -40 -35 30 25 20 15 10 5
*/#
7#
44.4k.44V SVSSV VV4VVV-W.WV VV4VVV-W.WV VV4VVV-W.WV VV4VVV-W.WV VV4VVV-W.WV 4W4W
о 00 о
С
О
О+S
O+A
В s
Ml "г
b
7 --
б --
5 --
4 --
§ з --
2 --
1 --
_îfc
.\Vikw .44444V .444444"1 .444444"1 .444444"1 .444444"1 .444444"1 .444444"1 .444444"1
7#
С
О
О+S O+A
Fig. 5. The content of reduced glutathione (a) and the activity of glutathione peroxidase (b) in the mucous membrane of the rat colon (x ± SE): C - the control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and "Symbiter' multiprobiotic co-administration (n = 10); O + A - group of rats after 28 days of omeprazole and "Apibact" multiprobiotic co-administration (n = 10); * - P < 0.05 compared to the control;# - P < 0.05 compared to the group of animals administered omeprazole
Fig. 6. The activity of glutathione transferase (a) and the activity of glutathione reductase (b) in the mucous membrane of the rat colon (x ± SE): C - the control group (n = 10); O - group of rats after 28 days of omeprazole administration (n = 10); O + S - group of rats after 28 days of omeprazole and "Symbiter" multiprobiotic co-administration (n = 10); O+A - group of rats after 28 days of omeprazole and "Apibact" multiprobiotic co-administration (n = 10); * - P < 0.05 compared to the control
After the 28-day co-administration of omeprazole with "Symbiter" multiprobiotic and omeprazole with "Apibact" multipibiotic to rats, the content of reduced glutathione in the colon mucosa increased by 58.3% (P < 0.05) and 61.8% (P < 0.05), respectively, in comparison with the group of rats which were administered omeprazole only; by 27.2% (P < 0.05) and by 30.1% (P < 0.05) exceeding this index compared to the control (Fig. 4a). The activity of glutathione peroxidase (Fig. 4b), glutathione transferase (Fig. 5a) and glutathione reductase (Fig. 5b) in the mucous membrane of the rat colon after 28 days of omeprazole and multiprobiotics co-administration did not undergo significant changes compared to the similar indices in the group ofrats which were administered omeprazole only.
Discussion
Therefore, we can assert that under the conditions of prolonged gastric hypoacidity, depletion of SOD in the serum occurs as a result of excessive synthesis of reactive oxygen forms by phagocytic cells in development of the digestive tract inflammation, induced by dysbiosis and hypergastrinemia. SOD activity can also be reduced due to its inactiva-tion by hydrogen peroxide, which is accumulated in the blood serum. The evidence of hydrogen peroxide accumulation is growth of catalase activity in the blood serum (Fig. 1b). There are reports that omeprazole inactivates one of the SOD isoforms, which may also cause the reduction in SOD activity (Elchuri & Oberley, 2005). Finally, there may be one more reason for the decrease in SOD enzymatic activity in these conditions, namely: the oxidative modification of histidine residues that bind copper and zinc in the active center of the SOD molecule (Hoso-kawa et al., 1985).
a
a
Under the action of free radicals, including hydrogen peroxide, the residue of His-118 is oxidized to 2-oxyhistidine. Other histidine residues under the influence of free radicals are converted into aspartate or aspa-ragine (Liochev & Fridowich, 2002). With regard to the increase in the content of H2O2 in the blood serum, H2O2 can be formed, first, in the spontaneous dismutation of the superoxide radical, the content of which is increased as a result of the SOD activity reduction. Secondly, hydrogen peroxide is formed during the reaction of the superoxide radical with the peroxide radical. Therefore, the increase in catalase activity may be a consequence of the activated bypass pathways in hydrogen peroxide formation (Ozbakis et al., 2007). The literature analysis indicates that the obtained data on the antioxidant properties of multiprobiotics are expected. After all, the phenomenon of the LPO process inhibition by different strains of lactic acid bacteria is described in a number of works (Hütt et al., 2009). In addition, it has been shown that the Apibact multiprobiotic reduces the content of LPO products in the pancreas and liver of rats under prolonged gastric hypoacidity induced by omeprazole (Dvorshchenko & Ostapchenko, et al., 2010). Significant changes in the activity of antioxidant enzymes in the rat mucosa were observed after prolonged administration of omeprazole. Increase in the content of TBA-reactive substances in the mucous membrane of the colon indicates an impairment of the prooxidant-antioxidant balance, and testifies to the development of inflammation in this organ (Pellicano et al., 2006; Pylypenko & Koval, 2018; Pylypenko et al., 2018).
In the colon, oxidative / nitrosative stress, which was caused by hypergastrinemia and dysbacteriosis and was accompanied by an increase in the TBA-reactive substances and NO production, is also the result of the SOD enzymes and antioxidant protection system's catalase depletion, which may be due to accumulation of free-radical compounds in the mucous membranes, affecting the intestinal mucosa and causing the inflammatory process in the intestine. The results suggest that after prolonged inhibition of HCl secretion in the stomach, the glutathione antioxidant defense link is depleted in the colon mucosa, as a result of the over-activation of free radical reactions and LPO, and the cells cannot resist the oxidative stress against the background of inflammatory process induced by hypergastrinemia and dysbiosis. Oxidative stress is important for removal of pathogenic microorganisms, although overproduction of reactive oxygen forms or impaired endogenous antioxidant protection can lead to disastrous effects on the cells and tissues of the host organism (Friis-Hansen, 2006; Boyko et al., 2016). The cause of glutathione depletion in the antioxidant system may also be due to the direct action of omeprazole, which is metabolized with the participation of glutathione (Zavros & Merchant, 2005). We believe that the work that revealed the antioxidant properties of omeprazole (Burdan et al., 2001; Biswas et al., 2003; Kaur et al., 2009) cannot be compared with our results, since the authors studied the effects of short-term use of proton pump inhibitors in the treatment of gastroduo-denal ulcer.
Therefore, intensification of LPO reactions and NO generation in the balance disorders in the functioning of the antioxidant protection enzymes and depletion of the glutathione link of the antioxidant system indicate a decrease in the antioxidant potential of the colon mucosa against the background of prolonged gastric hypochondria. Prolonged co-administration of omeprazole and multiprobiotics led to a significant normalization of the antioxidant system's enzyme activity, including its glutathione link. Reasons for intensification of LPO reactions in the large intestinal mucosa under the conditions of the prolonged HCl secretion inhibition in the stomach may be, on the one hand, insufficient activity of antioxidant enzymes, on the other, increased generation of reactive oxygen forms and NO. Indeed, it has been proved that the inducer of free radical lipid oxidation in dysbacteriosis is an excess of reactive oxygen forms and nitrogen generated by phagocytes.
Suppression of hydrochloric acid secretion in the stomach leads to dysbacteriosis throughout the digestive tract, which only intensifies over time. In the colon dysbiosis, the balance between obligate, optional and transient microflora is significantly impaired. In the development of dysbiosis, the concentration of obligate representatives of anaerobic suc-ralitic flora (bifidobacteria, lactobacilli, etc.) decreases first of all, and against this background there is an increase in the level of opportunistic
microorganism populations (Escherichia, enterococci, clostridia) and an increase in their aggressive potential (Makarchuk et al., 2019). This is accompanied by a decrease in the physiological activity of the microbi-ome, which are expressed in the development of various bowel dysfunctions and in the emergence of local and systemic pathological processes. Accumulation of the opportunistic microflora to the "critical level", accompanied by the natural activation of its virulent properties, can lead to the development of serious pathological disorders. Downturn of dysbiosis is associated with disorders of metabolic, morphoki-netic, functional, and sometimes structural changes in the colon, with the activation of lipid peroxidation, antioxidant defense suppression, decrease of reticuloendothelial system activity, and immune processes, decrease of the liver detoxication function, synthesis of vitamins, enzymes, mediators, short-chain fatty acids, accumulation of toxic compounds. Simultaneously, the processes of fermentation and nutrient uptake, in which the biofilm indigenous microbiota takes an active part are decreasing, physiological regeneration of colonocytes is suppressed, intercellular membranes are damaged, the tissue antigens are formed, permeability of the gastrointestinal tract is increased with development of alimentary allergy (Yankovskyy et al., 2017).
Almost all acute and chronic diseases of the digestive system, as well as other organs and systems, are accompanied by intestinal dysbi-osis of varying severity (Kharchenko & Anokhina, 2005). Therefore, to ensure complete clinical cure, a compulsory component of complex therapy for patients with any gastrointestinal disease, as well as a number of other diseases that depend on the condition of the digestive tract, should include the correction of microecological disorders. To do this, probiotics are used created on the basis of living cells of lactobacilli and bifidobacteria (Sanders, 2011; Koch et al., 2012).
The modulating effect of multiprobiotics on the processes of free radical oxidation in the colon mucosa under the conditions of inflammatory process development is obviously the result of the elimination of oxygen metabolism disorders in phagocytes and protection of mucous membranes from aggressive action of reactive oxygen and nitrogen forms. The literature analysis shows that the obtained data on the anti-oxidant properties of multiprobiotics are expected, and the phenomenon of the LPO process inhibition by different strains of lactic acid bacteria is described (Forman et al., 2009). In addition, the "Apibact" multiprobiotic has been shown to reduce the content of LPO products in the pancreas and liver of rats under the conditions of long-term gastric juice hypoacidity caused by omeprazole (Dvorshchenko & Ostapchenko, 2010).
Consequently, multiprobiotics activate the antioxidant protection enzymes in the mucous membrane of the colon by reducing the intensity of free radical lipid oxidation, normalize the quantitative and qualitative composition of microflora, reduce gastrin levels in the blood serum and directly neutralize reactive oxygen forms and H2O2 and activate other links of the antioxidative defense (Metz, 2012).
Conclusions
Prolonged gastric juice hypochlorhydria led to changes in the functioning and depletion of antioxidant protection enzymes: compared to the control, the blood serum SOD activity decreased by 54.5% (P < 0.05) and the catalase activity increased by 211.5% (P < 0.05). Prolonged gastric juice hypochlorhydria led to changes in the functioning and depletion of antioxidant defense enzymes: compared to the controls in the colon mucosa, SOD and catalase activity decreased by 35.9% (Р < 0.001) and 45.6% (P < 0.05). Under the conditions of prolonged gastric hypochlorhydria, the functioning of the glutathione antioxidant protection system was impaired in the colon mucosa. Under the conditions of prolonged gastric hypochlorhydria, functioning of the glutathione antioxidant defense system was impaired in the colon mucosa. Prolonged administration of multiprobiotic drugs against the background of gastric hypochlorhydria significantly reduced the inflammatory process manifestations in the mucous membrane of the large intestine, which was expressed in the normalization of the antioxidant system enzymes activity, including its glutathione link.
References
Binder, H. J. (2010). Role of colonic short-chain fatty acid transport in diarrhea. Annual Review of Physiology, 72, 297-313.
Biswas, K. Bandyopadhyay, U., Chattopadhyay, I., Varadaraj, A., Ali, E., & Banerjee, R. K. (2003). A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical. Journal of Biological Chemistry, 278, 10993-11001.
Boyko, O. O., Zazharska, N. M., & Brygadyrenko, V. V. (2016). The influence of the extent of infestation by helminths upon changes in body weight of sheep in Ukraine. Visnyk of Dnipropetrovsk University. Biology, Ecology, 24(1), 3-7.
Burdan, F., Burak, B., & Sek, A. (2001). Level of malondialdehyde after short-time omeprazole administration. Medical Science Monitor, 7, 89-92.
Burkitt, M. D. Varr, O. A., & Pritchard, D. M. (2009). Importance of gastrin in pathogenesis and treatment of gastric tumors. World Journal of Gastroenterology, 15(1), 1-16.
Dvorshchenko, K. O., Berehova, T. V., & Ostapchenko, L. I. (2010). Vplyv mul-typrobiotyku "apibakt®" na perekysne okysnennya lipidiv u pidshlunkoviy zalozi shchuriv za umov tryvaloyi hipoatsydnosti [Apibact® multiprobiotics influence on lipid peroxidation in rat pancreas gland in prolonged hypoacidity]. World of Medicine and Biology, 2, 55-57 (in Ukrainian).
Elchuri, S., Oberley, T., Qi, W., Eisenstein, R., Roberts, L., Van Remmen, H., Epstein, C., & Huang, T. (2005). CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. On-cogene, 24, 367-380.
Fabian, E., & Elmadfa, I. (2007). The effect of daily consumption of probiotic and conventional yoghurt on oxidant and antioxidant parameters in plasma of young healthy women. International Journal for Vitamin and Nutrition Research, 77(2), 79-88.
Forman, H. J., Zhang, H., & Rinna, A. (2009). Glutathione: Overview of its protective roles, measurement, and biosynthesis. Molecular Aspects of Medicine, 30(1-2), 1-12.
Freitas, M., Lima, J. L., & Fernandes, E. (2009). Optical probes for detection and quantification of neutrophils' oxidative burst. A review. Analytica Chimica Acta, 649, 8-23.
Friis-Hansen, L. (2006). Achlorhydria is associated with gastric microbial overgrowth and development of cancer: Lessons learned from the gastrin knockout mouse. Scandinavian Journal of Clinical and Laboratory Investigation, 66, 607-622.
Habig, W. H., Parst, M. J., & Jakobv, W. B. (1974). Glutathione-S-transferases. The first enzymatic step in mercapturie acid formation. Journal of Biological Chemistry, 249(22), 7130-7139.
Hissin, P. J., & Hilf, R. (1976). A fluorometric method for determination of oxidized and reduced glutathione in tissues. Analytical Biochemistry, 74, 214-226.
Hosokawa, S., Nishitani, H., & Umemba, K. (1985). Relationship between hae-modialysis anaemia and cooper and zince. International Urology and Nephrology, 17, 365-371.
Howden, C. W., & Hunt, R. H. (1987). Relationship between gastric secretion and infection. Gut, 28, 96-107.
Hutt, P., Andreson, H., Kullisaar, T., Vihalemm, T., Unt, E., Kals, J., Kampus, P., Zilmer, M., & Mikelsaar, M. (2009). Effects of a synbiotic product on blood antioxidative activity in subjects colonized with Helicobacter pylori. Letters in Applied Microbiology, 48(6), 797-800.
Kaur, I. P., Kuhad, A., Garg, A., & Chopra, K. (2009). Probiotics: Delineation of prophylactic and therapeutic benefits. Journal of Medicinal Food, 12(2), 219-235.
Kedika, R., Souza, R., & Spechler, S. (2009). Potential anti-inflammatory effects of proton pump inhibitors: A review and discussion of the clinical implications. Digestive Diseases and Sciences, 54(11), 2312-2317.
Koch, H., Cisarovsky, G., & Schmid-Hempel, P. (2012). Ecological effects on gut bacterial communities in wild bumblebee colonies. Journal of Animal Ecology, 81(6), 1202-1210.
Korolyuk, M. A., Korolyuk, M. A., Ivanova, L. I., Majorova, I. G., & Tokarev, V. E. (1988). Metod opredeleniya aktivnosti katalazy [Method for determination of catalase activity]. Laboratornoye Delo, 1, 16-18 (in Russian).
LeBlanc, A. M., & LeBlanc, J. G. (2014). Effect of probiotic administration on the intestinal microbiota, current knowledge and potential applications. World Journal of Gastroenterology, 20(44), 16518-16528.
Liochev, S., & Fridowich, I., (2002). Zinc superoxide dismutase and H2O2. The Journal of Biological Chemistry, 277(38), 34674-3468.
Makarchuk, V. V., Pylypenko, S. V., & Koval, A. A. (2019). Vplyv tryvaloho za-stosuvannya blokatoriv protonnoyi pompy na mikrofloru shlunka ta tovstoyi kyshky u shchuriv [Effect of prolonged use of proton pump blockers on gastric and colon microflora in rats]. Ukrainian Journal of Medicine, Biology and Sport, 18, 278-283 (in Ukrainian).
Metz, D. C. (2012). Diagnosis of the Zollinger - Ellison syndrome. Clinical Gast-roenterology and Hepatology, 10, 126-130.
Mikelsaar, M., & Zilmer, M. (2009). Lactobacillus fermentum ME-3 - An antimicrobial and antioxidative probiotic. Microbial Ecology in Health and Disease, 21(1), 1-27.
Mokrasch, L. C., & Teschke, E. J. (1984). Glutathione content of cultured cells and rodent brain regions: A spesific fluorometric assay. Analytical Biochemistry, 140, 506-509.
Oktyabrsky, O., & Smirnova, G. (2007). Redoks-regulyatsiya kletochnykh funktsiy [Redox regulation of cellular functions]. Biokhimiya, 72(2), 158-174.
Osefo, N., Ito, T., & Jensen, R. T. (2009). Gastric acid hypersecretory states: Recent insights and advances. Current Gastroenterology Reports, 11(6), 443-441.
Ozbakis, G., Odabasoglu, F., & HaliciEmail, Z., Suleyman, H., Cadirci, E., Bayir, Y. (2007). Gastroprotective and antioxidant effects of amiodarone on indo-methacin-induced gastric ulcers in rats. Archives of Pharmacal Research, 30(11), 1426-1434.
Pellicano, R., De Angelis, C., Resegotti, A., & Rizzetto, M. (2006). Zollinger -Ellison syndrome in 2006: Concepts from a clinical point of view. Panminerva Medica, 48(1), 33-40.
Pylypenko, S. V., & Koval, A. A. (2018). Vplyv multyprobiotykiv na vmist nitryt-ioniv ta aktyvnist syntazy oksydu azotu u syrovattsi krovi y u slyzovykh obo-lonkakh shlunku ta tovstoyi kyshky shchuriv za umov tryvaloyi hipoatsydnosti shlunkovoho soku [Influence of multiprobiotics on nitrite-ion content and nitric oxide synthase activity in serum and in gastric and colon mucous membranes of rats under the conditions of prolonged hypoacidity of gastric juice]. Ukrainian Journal of Medicine, Biology and Sport, 14, 300-305 (in Ukrainian).
Pylypenko, S. V., Koval, A. A., & Bazhan, A. H. (2018). Funktsionuvannya systemy NO v syrovakttsi krovi i slyzoviy obolontsi shlunka shchuriv za umov tryvaloyi hipoatsydnosti shlunkovoho soku ta zastosuvannya multyprobiotykiv [Functioning of the NO system in the blood serum and the gastric mucosa of rats under the conditions of prolonged gastric juice hypoacidity and the use of multi-probiotics]. Biolohiya ta Ekolohiya, 4(1), 86-93 (in Ukrainian).
Pylypenko, S. V., Koval, A. A., & Makarchuk, V. V. (2018). Impact of multipro-biotics on the content of TBA-reactive substances in the blood serum and mucous membranes of the stomach and colon in rats with long-term gastric hypochlorhydria World of Medicine and Biology, 65, 218-222.
Radchuk, O. M., Berehova, T. V., Yashchenko, A. M., & Rybalchenko, V. K. (2010). Vplyv multyprobiotykiv "Symbiter atsydofilnyi" kontsentrovanyi ta "Apibakt" na tsytotopohrafiyu lektynovykh retseptoriv za nayavnosti tryvaloyi hiperhastrynemiyi [Effect of "Symbiter acidophilic" concentrated and "Apibact" multiprobiotics on the cytotopography of lectin receptors in the presence of prolonged hypergastrinemia]. Problemy Ekolohichnoyi ta Me-dychnoyi Henetyky i Klinichnoyi Imunolohiyi, 98, 46-58 (in Ukrainian).
Sanders, M. E. (2011). Impact of probiotics on colonizing microbiota of the gut. Journal of Clinical Gastroenterology, 45, 115-119.
Sipponen, P., Harkonen, M., & Alanko, A. (2002). Diagnosis of atrophic gastritis from a serum sample. Clinical Laboratory, 48, 505-515.
Tazoe, H., Otomo, Y., Kaji, I., Tanaka, R., Karaki, S., & Kuwahara, A. (2008). Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions. Journal of Physiology and Pharmacology, 59, 251-262.
Tkachenko, Y. I. (2014). Pitaniye, endoekologiya cheloveka, bolezni. Sovremen-nyy vzglyad na problemu ikh vzaimosvyazey [Nutrition, human endoeco-logy, diseases. A modern view of their correlations problem]. Terapeutic Ar-hive, 2, 67-71 (in Russian).
Tsyryuk, O. I., Yashchenko, A. M., Kharchuk, Y. V., Kharchenko, M. M., & Berehova, T. V. (2011). Vplyv tryvaloho vvedennya omeprazolu na slyzovu obolonku shlunka [Effect of prolonged omeprazole administration on the gastric mucosa]. World of Medicine and Biology, 2, 71-75.
Vlasova, S. N., Shabunina, Y. I., & Perslegina, I. A. (1990). Aktivnost' glutationzavi-simykh fermentov eritrotsitov pri khronicheskikh zabolevaniyakh pecheni u de-tey [Activity of glutathione-dependent erythrocyte enzymes in children with chronic liver diseases]. Laboratornoye Delo, 8, 19-22 (in Russian).
Waldum, H. L., Gustafsson, B., & Fossmark, R (2005). Antiulcer drugs and gastric cancer. Digestive Diseases and Sciences, 50, 39-44.
Watson, S. A., Morris, T. M., & McWilliams, D. F. (2002). Potential role of endocrine gastrin in the colonic adenoma carcinoma sequence. British Journal of Cancer, 87(5), 567-573.
Willams, C., & McColl, K. E. L. (2006). Review article: Proton pump inhibitors and bacterial overgrowth. Alimentary Pharmacology and Therapeutics. 23(1), 3-10.
Zavros, Y., & Merchant, J. L. (2005). Modulating the cytokine response to treat Helicobacter gastritis. Biochemical Pharmacology, 69(3), 365-371.
Zhong, D., Xie, Z., & Chen, X. (2005). Metabolism of pantoprazole involving conjugation with glutathione in rats. Journal of Pharmacy and Pharmacology, 57(3), 341-349.
