Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration
Aim. Determination of the Cckbr, Gast and Reg1a genes expression in rat duodenal epithelial cells upon long- term hypoacidity and with the administration of the multiprobiotic Symbiter. Methods. The experiments were carried out on white non-strain male rats. The hypoacidic state was induced through...
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Інститут молекулярної біології і генетики НАН України
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Dranitsina, A.S. Savko, U.V. Dvorshchenko, K.O. Ostapchenko, L.I. 2019-06-15T16:28:00Z 2019-06-15T16:28:00Z 2014 Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration / A.S. Dranitsina, U.V. Savko, K.O. Dvorshchenko, L.I. Ostapchenko // Вiopolymers and Cell. — 2014. — Т. 30, № 5. — С. 365-371. — Бібліогр.: 23 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008B3 https://nasplib.isofts.kiev.ua/handle/123456789/154538 577.21:612.34:616.33-008.821.14 Aim. Determination of the Cckbr, Gast and Reg1a genes expression in rat duodenal epithelial cells upon long- term hypoacidity and with the administration of the multiprobiotic Symbiter. Methods. The experiments were carried out on white non-strain male rats. The hypoacidic state was induced through intraperitoneal injection of omeprazole for 28 days. The level of genes expression was determined by semi-quantitative analysis with RT-PCR Results. The elevation of mRNA levels of the Cckbr and Gast genes in rat duodenal villus and crypt epitheliocytes, the increased expression of the Reg1A gene in crypt epithelial cells were shown as well as the appearance of the Reg1a gene expression in villus epitheliocytes upon hypoacidic conditions were shown. The content of mRNAs of the above mentioned genes decreased or remained at the control level upon the treatment of hypoacidic rats with the multiprobiotic Symbiter. Conclusions. Long-term gastric hypoacidity is accompanied by the changes in expression of the Cckbr, Gast and Reg1a genes in rat duodenum, whereas upon administration of the multiprobiotic Symbiter the pattern of studied gene expression did not changed in the most cases. Мета. Дослідити експресію генів Cckbr, Gast і Reg1a у дванадцятипалій кишці щурів за умов тривалої гіпоацидності та за введен- ня мультипробіотика Симбітер. Методи. Досліди виконано на білих нелінійних щурах-самцях. Гіпоацидний стан моделювали інтраперитонеальним введенням омепразолу протягом 28 діб. Рівень експресії генів визначали напівкількісним аналізом результатів ЗТ-ПЛР. Результати. Показано зростання рівня мРНК генів Cckbr і Gast в епітеліоцитах ворсинок і крипт дванадцятипалої кишки щурів; підвищення рівня експресії гена Reg1a в епітеліоцитах крипт, а також появу експресії цього гена в епітеліальних клітинах ворсинок за гіпоацидного стану. При введенні мультипробіотика Симбітер за тих же умов вміст мРНК досліджуваних генів зменшувався або був на рівні контрольних значень. Висновки. Стан тривалої шлункової гіпоацидності супроводжується зміною експресії генів Cckbr, Gast і Reg1a у дванадцятипалій кишці щурів, тоді як за введення мультипробіотика Симбітер патерн експресії цих генів у більшості випадків подібний до контролю. Цель. Исследовать экспрессию генов Cckbr, Gast и Reg1a в эпителиоцитах двенадцатиперстной кишки крыс в условиях длительной гипоацидности, а также при введении мультипробиотика Симбитер. Методы. Исследования проведены на белых нелинейных крысах-самцах. Состояние гипоацидности желудка моделировали интраперитонеальным введением омепразола в течение 28 дней. Уровень экспрессии генов определяли полуколичественным анализом результатов ОТ-ПЦР. Результаты. Показано повышение уровня мРНК генов Cckbr и Gast в эпителиоцитах ворсинок и крипт двенадцатиперстной кишки крыс; увеличение уровня экспрессии гена Reg1a в эпителиоцитах крипт, а также появление экспрессии этого гена в эпителиальных клетках ворсинок при гипоацидности. При введении мультипробиотика Симбитер в тех же условиях содержание мРНК проанализированных генов уменьшалось или было на уровне контрольных значений. Выводы. Состояние длительной желудочной гипоацидности сопровождается изменением экспрессии генов Cckbr, Gast и Reg1a в двенадцатиперстной кишке, тогда как при введении мультипробиотика Симбитер паттерн экспрессии исследованных генов в большинстве случаев близок к контролю. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Genomics, Transcriptomics and Proteomics Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration Експресія генів Gast, Cckbr, Reg1a в епітеліоцитах дванадцятипалої кишки щурів за умов тривалої шлункової гіпоацидності та за введення мультипробіотика Экспрессия генов Gast, Cckbr, Reg1a в эпителиоцитах двенадцатиперстной кишки крыс в условиях длительной желудочной гипоацидности и при введении мультипробиотика Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| spellingShingle |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration Dranitsina, A.S. Savko, U.V. Dvorshchenko, K.O. Ostapchenko, L.I. Genomics, Transcriptomics and Proteomics |
| title_short |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| title_full |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| title_fullStr |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| title_full_unstemmed |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| title_sort |
expression of gast, cckbr, reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration |
| author |
Dranitsina, A.S. Savko, U.V. Dvorshchenko, K.O. Ostapchenko, L.I. |
| author_facet |
Dranitsina, A.S. Savko, U.V. Dvorshchenko, K.O. Ostapchenko, L.I. |
| topic |
Genomics, Transcriptomics and Proteomics |
| topic_facet |
Genomics, Transcriptomics and Proteomics |
| publishDate |
2014 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Експресія генів Gast, Cckbr, Reg1a в епітеліоцитах дванадцятипалої кишки щурів за умов тривалої шлункової гіпоацидності та за введення мультипробіотика Экспрессия генов Gast, Cckbr, Reg1a в эпителиоцитах двенадцатиперстной кишки крыс в условиях длительной желудочной гипоацидности и при введении мультипробиотика |
| description |
Aim. Determination of the Cckbr, Gast and Reg1a genes expression in rat duodenal epithelial cells upon long- term hypoacidity and with the administration of the multiprobiotic Symbiter. Methods. The experiments were carried out on white non-strain male rats. The hypoacidic state was induced through intraperitoneal injection of omeprazole for 28 days. The level of genes expression was determined by semi-quantitative analysis with RT-PCR Results. The elevation of mRNA levels of the Cckbr and Gast genes in rat duodenal villus and crypt epitheliocytes, the increased expression of the Reg1A gene in crypt epithelial cells were shown as well as the appearance of the Reg1a gene expression in villus epitheliocytes upon hypoacidic conditions were shown. The content of mRNAs of the above mentioned genes decreased or remained at the control level upon the treatment of hypoacidic rats with the multiprobiotic Symbiter. Conclusions. Long-term gastric hypoacidity is accompanied by the changes in expression of the Cckbr, Gast and Reg1a genes in rat duodenum, whereas upon administration of the multiprobiotic Symbiter the pattern of studied gene expression did not changed in the most cases.
Мета. Дослідити експресію генів Cckbr, Gast і Reg1a у дванадцятипалій кишці щурів за умов тривалої гіпоацидності та за введен- ня мультипробіотика Симбітер. Методи. Досліди виконано на білих нелінійних щурах-самцях. Гіпоацидний стан моделювали інтраперитонеальним введенням омепразолу протягом 28 діб. Рівень експресії генів визначали напівкількісним аналізом результатів ЗТ-ПЛР. Результати. Показано зростання рівня мРНК генів Cckbr і Gast в епітеліоцитах ворсинок і крипт дванадцятипалої кишки щурів; підвищення рівня експресії гена Reg1a в епітеліоцитах крипт, а також появу експресії цього гена в епітеліальних клітинах ворсинок за гіпоацидного стану. При введенні мультипробіотика Симбітер за тих же умов вміст мРНК досліджуваних генів зменшувався або був на рівні контрольних значень. Висновки. Стан тривалої шлункової гіпоацидності супроводжується зміною експресії генів Cckbr, Gast і Reg1a у дванадцятипалій кишці щурів, тоді як за введення мультипробіотика Симбітер патерн експресії цих генів у більшості випадків подібний до контролю.
Цель. Исследовать экспрессию генов Cckbr, Gast и Reg1a в эпителиоцитах двенадцатиперстной кишки крыс в условиях длительной гипоацидности, а также при введении мультипробиотика Симбитер. Методы. Исследования проведены на белых нелинейных крысах-самцах. Состояние гипоацидности желудка моделировали интраперитонеальным введением омепразола в течение 28 дней. Уровень экспрессии генов определяли полуколичественным анализом результатов ОТ-ПЦР. Результаты. Показано повышение уровня мРНК генов Cckbr и Gast в эпителиоцитах ворсинок и крипт двенадцатиперстной кишки крыс; увеличение уровня экспрессии гена Reg1a в эпителиоцитах крипт, а также появление экспрессии этого гена в эпителиальных клетках ворсинок при гипоацидности. При введении мультипробиотика Симбитер в тех же условиях содержание мРНК проанализированных генов уменьшалось или было на уровне контрольных значений. Выводы. Состояние длительной желудочной гипоацидности сопровождается изменением экспрессии генов Cckbr, Gast и Reg1a в двенадцатиперстной кишке, тогда как при введении мультипробиотика Симбитер паттерн экспрессии исследованных генов в большинстве случаев близок к контролю.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154538 |
| citation_txt |
Expression of Gast, Cckbr, Reg1a genes in rat duodenal epithelial cells upon long-term gastric hypoacidity and after a multiprobiotic administration / A.S. Dranitsina, U.V. Savko, K.O. Dvorshchenko, L.I. Ostapchenko // Вiopolymers and Cell. — 2014. — Т. 30, № 5. — С. 365-371. — Бібліогр.: 23 назв. — англ. |
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UDC 577.21:612.34:616.33-008.821.14
Expression of Gast, Cckbr, Reg1a genes in rat
duodenal epithelial cells upon long-term gastric hypoacidity
and after a multiprobiotic administration
A. S. Dranitsina, U. V. Savko, K. O. Dvorshchenko, L. I. Ostapchenko
Educational and Scientific Center «Institute of Biology»,
Taras Shevchenko National University of Kyiv
64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
alevtina.dranitsina@gmail.com
Aim. Determination of the Cckbr, Gast and Reg1a genes expression in rat duodenal epithelial cells upon long-
term hypoacidity and with the administration of the multiprobiotic Symbiter. Methods. The experiments were car-
ried out on white non-strain male rats. The hypoacidic state was induced through intraperitoneal injection of
omeprazole for 28 days. The level of genes expression was determined by semi-quantitative analysis with RT-PCR
Results. The elevation of mRNA levels of the Cckbr and Gast genes in rat duodenal villus and crypt epitheliocytes,
the increased expression of the Reg1a gene in crypt epithelial cells were shown as well as the appearance of the
Reg1a gene expression in villus epitheliocytes upon hypoacidic conditions were shown. The content of mRNAs of
the above mentioned genes decreased or remained at the control level upon the treatment of hypoacidic rats with
the multiprobiotic Symbiter. Conclusions. Long-term gastric hypoacidity is accompanied by the changes in ex-
pression of the Cckbr, Gast and Reg1a genes in rat duodenum, whereas upon administration of the multiprobio-
tic Symbiter the pattern of studied gene expression did not changed in the most cases.
Keywords: gastric hypoacidity, duodenal, expression of Cckbr, Gast, Reg1a genes, multiprobiotics.
Introduction. In recent decades, proton-pump inhibi-
tors (PPI) of gastric parietal cells, such as omeprazole,
appear to be the most effective and probably safe thera-
peutic agents for the acid-related disorders treatment
[1–3]. The development of dysbiosis is one of the key
consequences of long-term hypoacidity [3–5].
Colonization of gastrointestinal tract (GIT) by op-
portunistic microbiota forms stable sources of endoge-
nous infection and in addition to the effect of hypergast-
rinemia promotes gastric carcinogenesis and tumorige-
nesis in other parts of GIT as well as in associated or-
gans [3–7].
It is well known, that the Gast gene encodes the GIT
hormone gastrin, which is expressed exclusively in both
enteroendocrine G-cells of gastric antrum mucosa and
proximal part of duodenum, and exerts a trophic impact
on some GIT cells [4, 8, 9]. According to the literature
data, the overexpression and «adventive» expression of
gastrin may be associated with the development of gas-
trinoma under most pathological conditions [8].
The Cckbr gene (encodes gastrin/cholecystokinin
receptor type II or B – CCKBR) mediates certain phy-
siological functions of gastrin [10]. To date, there is suf-
ficient evidence for the presence of CCKBR protein in
small intestine, but the majority of reports suggest that
the normal duodenal cells are characterised by low levels
of the Cckbr expression. On the other hand, a high ex-
pression level of the CCKBR protein is mostly found in
clinical disorders as well as in certain GIT tumors [9].
The Reg1a gene gives rise to a regenerative protein,
specifically – regenerating islet-derived protein 1a [11–
13]. This protein is localized in the small intestine
Paneth cells, as well as in the non-mature columnar cells
of human intestinal crypts, but has not been yet found
365
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 5. P. 365–371 doi: http://dx.doi.org/10.7124/bc.0008B3
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
in functional villous epithelium [12]. Therefore, such
topographical distribution appears to be associated with
the growth and differentiation processes of actively
proliferating intestinal epithelium cells [13]. It has been
also established that the Reg1a gene expression in high-
ly differentiated villi may be connected to tumorigene-
sis [11–13].
In clinical trials it was proved that probiotics in ge-
neral are able to cure dysbiotic states, as well as to in-
stantly reduce GIT lesions ratio [14, 15]. The multipro-
biotics of the «Symbiter® acidophilic» concentrated
group, also referred to as Symbiter, are characterized
by action complexity, wide bioactivity array, high level
of biological safety and close to that of natural human
and animal microbial population composition [14, 15].
The analysis of recent scientific literature revealed
lack of data on the pattern of above mentioned genes ex-
pression in duodenum under experimental or natural hy-
poacidic conditions. The data concerning possible pro-
biotic effects on the gene expression in duodenal cells
upon hypoacidity are also nearly absent.
Consequently, the aim of current investigation was
to determine the expression of Cckbr, Gast and Reg1a
genes in rat duodenal epithelial cells upon long-term
injection of omeprazole and with «Symbiter» admini-
stration.
Materials and methods. The International recom-
mendations on performance of medical and biological
investigations with the use of animals according to Eu-
ropean Convention for the Protection of Vertebrate Ani-
mals Used for Experimental and other Scientific Pur-
poses were followed. Experiments were conducted on
white non-strain male rats with initial weight around
180–200 g.
All experimental animals were divided into four
groups. The first one (control) consisted of rats injected
with 0.2 ml of physiological solution abdominally and
0.5 ml of water orally. The second group was treated
with «Symbiter» (manufactured by LLC «O. D. Proli-
sok») orally (0.14 ml/kg) for 28 days. In the third animal
group hypoacidity was modelled by daily intraperito-
neal injection of omeprazole (14 mg/kg) for 28 days
[15]. Finally, the rats of the fourth experimental group
were given the same dose of «Symbiter» simultaneously
with omeprazole. Every experimental group consisted
of 6 animals. The crypts and the villi of duodenal epithe-
lial cells were extracted by a rapid low-temperature (4
oC) method, which included intestine washing under the
constant stirring in different buffers with the consequen-
tial gradual collection of villus or crypt fractions [16].
RNA was isolated following Chomczynski and
Sacchi [17]. cDNA was synthesized in 20 �l of reaction
mix, containing 2 �g of RNA, 1 mM dNTP, 50 U of re-
verse transcriptase «MultiScribeTM Reverse Transcrip-
tase», the corresponding buffer, 20 U of ribonuclease in-
hibitor «RNase Inhibitor» («Applied Biosystems», USA),
and 20 pmol (1.0 µM) of reverse primer. The synthesis
was performed upon 37 oC for 2 h. Polymerase chain re-
action was conducted in 30 �l of the reaction mix con-
taining 10 �l of cDNA, the PCR buffer, 200 �M of each
dNTP, 30 pmol (1.0 �M) of each primer, 2.5 mM of
MgCl2 and 1.5 U of Taq DNA polymerase («iTaqTM»,
«Bio-Rad», USA).
The PCR amplifications consisted of the initial dena-
turing step of 95 oC for 3 min, followed by 35 (28 for
Actb gene was used as internal control of reaction due to
its constitutive expression) cycles of 95 oC for 45 s, the
annealing step (with the optimal annealing temperature):
Cckbr (184 b. p., 59 oC – 45 s), Gast (310 b. p., 52 oC –
40 s), Reg1a (608 b. p., 48 oC – 45 s) and Actb (521 b. p.,
49 oC– 40 s); the extending step at 72 oC for 1 min 15 s
(for Cckbr, Reg1a) or 1 min (for Gast and Actb). The fi-
nal extension step was performed upon 72 oC for 5 min.
The following primer sequences were used for re-
actions:
for Cckbr –
forward – GCAAGCACGAGTATGGCAAA;
reverse – TAGCACGGACCAGGTTTGTT;
for Gast –
forward – GCCCAGCCTCTCATCATC;
reverse – GGGGACAGGGCTGAAGTG;
for Reg1a –
forward – AGCCTGCAGAGATTGTTGAC;
reverse – CCATAGGGCAGTGAGGCAAG;
for Actb –
forward – TGGGACGATATGGAGAAGAT;
reverse – ATTGCCGATAGTGATGACCT.
Reproducibility of the amplification results was eva-
luated in the parallel experiments by the PCR reactions
repetition with all animals and each primer at least three
times. The separation of PCR products was performed
electrophoretically in 1.6 % agarose gel with 0.5 � TBE
366
DRANITSINA A. S. ET AL.
367
EXPRESSION OF Gast, Cckbr, Reg1a GENES IN RAT DUODENAL EPITHELIAL CELLS
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Gast
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Actb
521
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Level of Gast (A), Cckbr (B)
and Reg1a (C) genes mRNA
in rat duodenum upon long-
term hypoacidity and with
the multiprobiotic administ-
ration: a – RT-PCR electro-
phoregram (Ì – molecular
mass marker – «GeneRu-
ler
TM
100 bp DNA Ladder»,
«Fermentas», Lithuania; N-
PCR – negative PCR cont-
rol); b – relative expression
histogram (* – p �0.05 in re-
lation to control; # – p �0.05
compared to the animals trea-
ted with omeprazole;� – p �
0.05; villi in comparison with
crypts). A, B, C: villus epi-
thelial cells: 1 – control; 2 –
Symbiter; 3 – omeprazole;
4 – omeprazole + Symbiter;
crypt epithelial cells: 5 – con-
trol; 6 – Symbiter; 7 – ome-
prazole; 8 – omeprazole +
Symbiter
buffer as described in Sambrook et al. [18]. Based on
densitometry results, the ImageJ 1.45s program was used
for semi-quantitative analysis of amplicon expression.
The indexes of mRNA expression for each sample were
calculated following Konturek et al. [19].
The statistical processing of the experimental data
was performed using GraphPad Prism 5.04 («Graph
Pad Software Inc.», USA). The normal Gaussian distri-
bution of the data was verified by the Shapiro-Wilk
normality test. Two-way analysis of variance (two-way
ANOVA) and Bonferroni post tests were performed
with the data obtained. The statistical significance was set
at p � 0.05. The reviewed data are expressed as means
and standard deviations.
Results and discussion. mRNA of Gast gene was de-
tected in the rat's duodenal villus and crypt epithelial cells
in the control groups of investigated animals (Figure, A).
As seen in Table, the level of Gast gene expression
was 1.9 times higher than the control value in villi and
5.9 times higher than in crypts of the animals treated
only with omeprazole for 28 days (p � 0.05). Upon si-
multaneous administration of the multiprobiotic «Sym-
biter» this parameter was 1.5 and 4.9 times lower com-
pared to the third experimental group (p � 0.05). In the
rats treated only with «Symbiter» this value was similar
to the control in both villus and crypt epitheliocytes of
duodenum (Figure, A, Table).
As we have already mentioned, gastrin exerts a tro-
phic impact on certain GIT cells [4, 8, 9]. In our experi-
ments in hypoacidic conditions we demonstrated the
elevation of the Gast mRNA level in villus epithelial
cells as well as high expression level of this gene in im-
mature crypts. The results obtained may be a sign of pa-
thological hypertrophy in duodenal cells, which could
lead to dystrophic, necrotic and sclerotic irreversible or-
gan changes, accompanied by chronic inflammation [13,
20]. As it is known from the literature data, overexpres-
sion and «adventive» expression of gastrin may be as-
sociated with the development of gastrinoma; it is a com-
mon feature of Zollinger-Ellison syndrome [8].
We demonstrated the Gast mRNA overexpression
in duodenum upon the same hypoacidic conditions con-
firms our previous results concerning the Gast gene ex-
pression in hypoacidic rat pancreas [21]. Considering
this fact, we can also assume a possible existence of neo-
plasia in duodenal epithelial cells at the later stages of
the pathology development upon long-term use of PPI.
The mRNA of Cckbr gene was detected in the rat
duodenal villus and crypt epithelial cells in the control
groups of investigated animals (Figure, B).
As seen from Table, the level of the Cckbr gene ex-
pression was 1.5 times higher compared to the control
values in villi and 4 times higher than in crypts upon
long-term hypoacidity (p � 0.05). In case of the simulta-
neous administration of the multiprobiotic «Symbiter»
the parameter was respectively 1.4 and about 2 times
lower than in the third group of animals (p � 0.05). In
rats treated only with «Symbiter» there was no change
of this value in both villus and crypt epitheliocytes of
duodenum in comparison to the control one.
368
DRANITSINA A. S. ET AL.
Groups of animals Epithelial cell types Relative expression Gast/Actb Relative expression Cckbr/Actb Relative expression Reg1a/Actb
Control Villi 0.072 � 0.0086� 0.051 � 0.0078� 0
Crypts 0.044 � 0.0056� 0.036 � 0.0078� 0.305 � 0.0110�
Symbiter Villi 0.091 � 0.0145� 0.047 � 0.0058 0
Crypts 0.061 � 0.0080� 0.036 � 0.0091 0.314 ± 0.0231�
Omeprazole Villi 0.134 ± 0.0155*� 0.074 ± 0.0107*� 0.622 ± 0.0247*�
Crypts 0.259 ± 0.0150*� 0.143 ± 0.0136*� 0.540 ± 0.0403*�
Omeprazole + Symbiter Villi 0.092 ± 0.0135#� 0.053 ± 0.0084#� 0.289 ± 0.0351#
Crypts 0.053 ± 0.0100#� 0.073 ± 0.0091*#� 0.292 ± 0.0135#
N o t e s. SD – standard deviation; * – p �0.05 related to control; # – p �0.05 in comparison with animals treated with omeprazole; �– p �0.05
villi in comparison with crypts.
The levels of Gast, Cckbr, Reg1a genes mRNA in rat duodenal cells upon long-term hypoacidity and with the multiprobiotic administration
(m ± SD, n = 6)
The majority of reports suggest that the normal duo-
denum cells appear to express the Cckbr gene at low le-
vels [8, 22]. To date, the role of cholecystokinin as a small
bowel and colon growth stimulator is not yet establi-
shed [9]. So, we demonstrated the overexpression of
Cckbr gene in villus and particularly in crypt epithelial
cells upon long-term hypoacidic conditions. The appa-
rent high level of Cckbr mRNA may be caused by the in-
tensification of its expression by gastrin, because the
latter, in addition to gastric secretion, is known to regu-
late proliferation of the cells expressing receptors for
this ligand. Such regulation may be performed by sti-
mulating the expression of early response genes as well
as other growth-related genes [9, 22]. The abundant ex-
pression of Cckbr may be also associated with the pa-
thological hypertrophy in duodenal cells and the inten-
sification of duodenum inflammation [13]. On the other
hand, a high expression level of the CCKBR protein is
found in esophageal adenocarcinoma as well as in ileal
carcinoid [9]. The results obtained by us this time ap-
peared to be in agreement with our previous data about
Cckbr expression in rat pancreas upon hypoacidic con-
ditions [21]. Thus, we can also suggest a possible neo-
plasia in duodenal epithelial cells at the later stages of
the pathology development upon long-term omeprazo-
le administration.
mRNA of Reg1a gene was detected in the rat duo-
denal crypts of the control group, although the mRNA
of this gene was not found in this group villous epithe-
lium (Figure, C).
So, in the functional villous epithelium of duodenum
upon long-term hypoacidity we found the Reg1a gene
mRNA. At the same time, the level of this gene in the
fourth experimental group of rats was 2.2 times lower
compared to the animals injected with omeprazole (p �
0.05).
In crypt epithelium of the animals treated only with
omeprazole for 28 days the level of Reg1a mRNA was
1.8 times higher than the control values (p � 0,05).
Upon the simultaneous administration of the multipro-
biotic «Symbiter» this parameter was approximately
1.9 times lower in comparison to the third group of ani-
mals (p � 0.05). In rats treated only with «Symbiter» this
value was similar to the control (Figure, C, Table).
It is well known, that the main function of crypts is
the cell reproduction control during intestinal epithelial
tissue regeneration. Moreover, it is known, that the crypt
base columnar cells can only proliferate. The mid-crypt
columnar cells have the ability to both proliferation and
differentiation, whereas the crypt top columnar cells ne-
ver divide, but continue to differentiate and then move
along the villi lines [4, 12]. The Reg1a gene mRNA ex-
pression was observed in Paneth cells, in the glands of
Lieberkuhn (crypts) and nonmature columnar cells of
the human intestinal crypts, but never in the functional
mature villous epithelium [13].
In our experiment we demonstrated the elevation of
the Reg1a mRNA level in crypt epithelial cells upon hy-
poacidic conditions (Figure, C, Table). Based on these
results it can be assumed that the inflammatory process
in duodenum intensifies [13, 20]. The appearance of
Reg1a gene expression in duodenal villous cells of the
rats treated only with omeprazole (Figure, C, Table)
may be related to a possible neoplasia in epithelial cells
at the later stages of the pathological process develop-
ment [11–13, 20]. Indeed, the Reg1a gene expression
in response to gastrin has been reported in ECL cells
(enterochromaffin-like cells) and may be involved not
only in the gastric mucosal cell growth, but also in the
tumor development [9].
Thus, the obtained changes in expression of Gast,
Cckbr, Reg1a gene in rat duodenal villus and crypts epi-
thelial cells should point out a possible development of
teratoid displacements in duodenal tissue upon hypoaci-
dic conditions due to the development of dysbiosis, which
makes promoting influence on the gastrin secretion in
rat's stomach and evaluates the effect of hypergastrine-
mia [4–9, 11–13, 15, 20, 21], according to the results of
different studies of patients treated with omeprazole [2,
6, 7]. Different rates of alterations of the above mentio-
ned gene expression in villus and crypts epithelial cells
appear to be determined by their structural and functio-
nal characteristics [4]. According to the literature data,
a well-established early feature of some inflammatory
diseases, including pathological intestine disorders, is
the formation of crypt abscesses, containing neutro-
phils migrating across the epithelium into the crypt lu-
men [4].
The first among possible mechanisms of the «Sym-
biter» action on gene expression in rat duodenal cells,
may be based on the ability of this multiprobiotic to li-
quidate dysbiosis and GIT bacterial colonization. As a
369
EXPRESSION OF Gast, Cckbr, Reg1a GENES IN RAT DUODENAL EPITHELIAL CELLS
result of this action, the burden of pathogenic microbio-
ta is removed from GIT and associated organs [14, 15].
Furthermore, the multicomponent probiotic «Symbi-
ter» is able to increase the reduced glutathione synthe-
sis de novo (the main low-molecular cellular antioxi-
dant), which results in its higher content in both GIT and
duodenum. Moreover, the «Symbiter» bacterial vital
products (vitamins, exopolysaccharides, short chain fat
acids, immune-response modulating agents, etc.) have
certain antioxidant properties, so they can delay the oxi-
dative stress development and decrease the intensity of
inflammation and other pathological processes in duo-
denal cells [14, 15, 23]. From the results of correspon-
ding studies, it may be suggested that the observed «Sym-
biter» effects can be linked not only to normalization of
GIT microbiota, but also to restriction of hypergastrine-
mia effects [5, 15, 21]. However, the final acceptance or
rejection of this hypothesis requires further real-time
PCR analysis and other research techniques along with
a usage of selective antagonists of gastrin receptor. It will
allow us to clarify the consequences of hypergastrine-
mia and GIT bacterial colonization.
Conclusions. In summary, we have shown, that long-
term experimental hypoacidity is accompanied by the
changes of the Gast, Cckbr and Reg1a genes expression
in the rat duodenal epithelial cells. The level of studied
gene expression upon the simultaneous administration
of the multiprobiotic «Symbiter» in most cases did not
change in comparison to the control values. Based on the
obtained data, it might be assumed, that the analyzed ge-
nes are involved in the development of the pathological
processes in duodenum, so there may be a certain poten-
tial risk of duodenal carcinogenesis upon long-term use
of omeprazole as well as, probably, of other PPI.
Åêñïðåñ³ÿ ãåí³â Gast, Cckbr, Reg1a â åï³òåë³îöèòàõ
äâàíàäöÿòèïàëî¿ êèøêè ùóð³â çà óìîâ òðèâàëî¿ øëóíêîâî¿
ã³ïîàöèäíîñò³ òà çà ââåäåííÿ ìóëüòèïðîá³îòèêà
À. Ñ. Äðàíèöèíà, Ó. Â. Ñàâêî, Ê. Î. Äâîðùåíêî, Ë. ². Îñòàï÷åíêî
Ðåçþìå
Ìåòà. Äîñë³äèòè åêñïðåñ³þ ãåí³â Cckbr, Gast ³ Reg1a ó äâàíàäöÿ-
òèïàë³é êèøö³ ùóð³â çà óìîâ òðèâàëî¿ ã³ïîàöèäíîñò³ òà çà ââåäåí-
íÿ ìóëüòèïðîá³îòèêà Ñèìá³òåð. Ìåòîäè. Äîñë³äè âèêîíàíî íà á³-
ëèõ íåë³í³éíèõ ùóðàõ-ñàìöÿõ. óïîàöèäíèé ñòàí ìîäåëþâàëè ³í-
òðàïåðèòîíåàëüíèì ââåäåííÿì îìåïðàçîëó ïðîòÿãîì 28 ä³á. г-
âåíü åêñïðåñ³¿ ãåí³â âèçíà÷àëè íàï³âê³ëüê³ñíèì àíàë³çîì ðåçóëüòà-
ò³â ÇÒ-ÏËÐ. Ðåçóëüòàòè. Ïîêàçàíî çðîñòàííÿ ð³âíÿ ìÐÍÊ ãåí³â
Cckbr ³ Gast â åï³òåë³îöèòàõ âîðñèíîê ³ êðèïò äâàíàäöÿòèïàëî¿
êèøêè ùóð³â; ï³äâèùåííÿ ð³âíÿ åêñïðåñ³¿ ãåíà Reg1a â åï³òåë³îöè-
òàõ êðèïò, à òàêîæ ïîÿâó åêñïðåñ³¿ öüîãî ãåíà â åï³òåë³àëüíèõ êë³-
òèíàõ âîðñèíîê çà ã³ïîàöèäíîãî ñòàíó. Ïðè ââåäåíí³ ìóëüòèïðî-
á³îòèêà Ñèìá³òåð çà òèõ æå óìîâ âì³ñò ìÐÍÊ äîñë³äæóâàíèõ
ãåí³â çìåíøóâàâñÿ àáî áóâ íà ð³âí³ êîíòðîëüíèõ çíà÷åíü. Âèñíîâ-
êè. Ñòàí òðèâàëî¿ øëóíêîâî¿ ã³ïîàöèäíîñò³ ñóïðîâîäæóºòüñÿ
çì³íîþ åêñïðåñ³¿ ãåí³â Cckbr, Gast ³ Reg1a ó äâàíàäöÿòèïàë³é êèø-
ö³ ùóð³â, òîä³ ÿê çà ââåäåííÿ ìóëüòèïðîá³îòèêà Ñèìá³òåð ïàòåðí
åêñïðåñ³¿ öèõ ãåí³â ó á³ëüøîñò³ âèïàäê³â ïîä³áíèé äî êîíòðîëþ.
Êëþ÷îâ³ ñëîâà: øëóíêîâà ã³ïîàöèäí³ñòü, äâàíàäöÿòèïàëà
êèøêà, åêñïðåñ³ÿ ãåí³â Cckbr, Gast, Reg1a, ìóëüòèïðîá³îòèêè.
Ýêñïðåññèÿ ãåíîâ Gast, Cckbr, Reg1a â ýïèòåëèîöèòàõ
äâåíàäöàòèïåðñòíîé êèøêè êðûñ â óñëîâèÿõ äëèòåëüíîé
æåëóäî÷íîé ãèïîàöèäíîñòè è ïðè ââåäåíèè ìóëüòèïðîáèîòèêà
À. Ñ. Äðàíèöèíà, Ó. Â. Ñàâêî, Å. À. Äâîðùåíêî, Ë. È.Îñòàï÷åíêî
Ðåçþìå
Öåëü. Èññëåäîâàòü ýêñïðåññèþ ãåíîâ Cckbr, Gast è Reg1a â ýïèòå-
ëèîöèòàõ äâåíàäöàòèïåðñòíîé êèøêè êðûñ â óñëîâèÿõ äëèòåëü-
íîé ãèïîàöèäíîñòè, à òàêæå ïðè ââåäåíèè ìóëüòèïðîáèîòèêà
Ñèìáèòåð. Ìåòîäû. Èññëåäîâàíèÿ ïðîâåäåíû íà áåëûõ íåëèíåé-
íûõ êðûñàõ-ñàìöàõ. Ñîñòîÿíèå ãèïîàöèäíîñòè æåëóäêà ìîäåëè-
ðîâàëè èíòðàïåðèòîíåàëüíûì ââåäåíèåì îìåïðàçîëà â òå÷åíèå
28 äíåé. Óðîâåíü ýêñïðåññèè ãåíîâ îïðåäåëÿëè ïîëóêîëè÷åñòâåí-
íûì àíàëèçîì ðåçóëüòàòîâ ÎÒ-ÏÖÐ. Ðåçóëüòàòû. Ïîêàçàíî ïî-
âûøåíèå óðîâíÿ ìÐÍÊ ãåíîâ Cckbr è Gast â ýïèòåëèîöèòàõ âîðñè-
íîê è êðèïò äâåíàäöàòèïåðñòíîé êèøêè êðûñ; óâåëè÷åíèå óðîâíÿ
ýêñïðåññèè ãåíà Reg1a â ýïèòåëèîöèòàõ êðèïò, à òàêæå ïîÿâëå-
íèå ýêñïðåññèè ýòîãî ãåíà â ýïèòåëèàëüíûõ êëåòêàõ âîðñèíîê ïðè
ãèïîàöèäíîñòè. Ïðè ââåäåíèè ìóëüòèïðîáèîòèêà Ñèìáèòåð â òåõ
æå óñëîâèÿõ ñîäåðæàíèå ìÐÍÊ ïðîàíàëèçèðîâàííûõ ãåíîâ óìåíü-
øàëîñü èëè áûëî íà óðîâíå êîíòðîëüíûõ çíà÷åíèé. Âûâîäû. Ñî-
ñòîÿíèå äëèòåëüíîé æåëóäî÷íîé ãèïîàöèäíîñòè ñîïðîâîæäàåò-
ñÿ èçìåíåíèåì ýêñïðåññèè ãåíîâ Cckbr, Gast è Reg1a â äâåíàäöàòè-
ïåðñòíîé êèøêå, òîãäà êàê ïðè ââåäåíèè ìóëüòèïðîáèîòèêà Ñèì-
áèòåð ïàòòåðí ýêñïðåññèè èññëåäîâàííûõ ãåíîâ â áîëüøèíñòâå
ñëó÷àåâ áëèçîê ê êîíòðîëþ.
Êëþ÷åâûå ñëîâà: æåëóäî÷íàÿ ãèïîàöèäíîñòü, äâåíàäöàòè-
ïåðñòíàÿ êèøêa, ýêñïðåññèÿ ãåíîâ Cckbr, Gast, Reg1a, ìóëüòè-
ïðîáèîòèêè.
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