Transcriptomic response of rat hippocampus and spleen cells to single and chronic administration of the peptide selank

Abstract

A new direction in designing new drugs able to effectively reduce anxiety without having side effects is the use of endogenous regulatory peptides. Research� ers of the Institute of Molecular Genetics, Russian Academy of Sciences, and the Zakusov Research Institute of Pharmacology, Russian Academy of Med� ical Sciences, have created the preparation selank, the effective substance of which is a synthetic peptide, an analogue of the short fragment Thr–Lys–Pro–Arg of the heavy chain of human immunoglobulin G, elon� gated at the C terminus with the tripeptide Pro–Gly– Pro. It was shown that selank has a stable nootropic and anxiolytic effects, facilitate brain cell survival in hypoxia, and exhibits an antiviral effect [3, 10].

Full text

ISSN 16076729, Doklady Biochemistry and Biophysics, 2010, Vol. 430, pp. 5–6. © Pleiades Publishing, Ltd., 2010.
Origi nal Russian Text © T. A. Kolomin , M.I. Shadr ina, Ya.V. A gniullin, S.I. Shram, P.A. Slominskii, S.A. Lim borska, N. F. Myasoed ov, 2010, published in Doklady Akademii Nauk,
2010, Vol. 430, No. 1, pp. 127–129.
5
A new direction in designing new drugs able to
effectively reduce anxiety without having side effects is
the use of endogenous regulatory peptides. Research
ers of the Institute of Molecular Genetics, Russian
Academy of Sciences, and the Zakusov Research
Institute of Pharmacology, Russian Academy of Med
ical Sciences, have created the preparation selank, the
effective substance of which is a synthetic peptide, an
analogue of the short fragment Thr–Lys–Pro–Arg of
the heavy chain of human immunoglobulin G, elon
gated at the
C
terminus with the tripeptide Pro–Gly–
Pro. It was shown that selank has a stable nootropic
and anxiolytic effects, facilitate brain cell survival in
hypoxia, and exhibits an antiviral effect [3, 10].
Recent studies showed that many peptides, semax
in particular, can modulate genome expression [1, 13].
Since the peptide selank also belongs to the regulatory
peptide family, it was of interest to assess its effect on
the genome expression. For this purpose, we per
formed a search for the genes whose expression in the
rat hippocampus and spleen changes under the influ
ence of this peptide.
Experiments were performed with male Wistar
rats weighing, on average, 260 g. The animals were
divided into three groups (eight animals in each): the
control group (C), the group with single administra
tion (SA) of selank, and the group with chronic
administration (CA) of selank. Rats of two groups, C
and SA, were intranasally administered with water
once a day for five days, and the animals of group CA
were intranasally administered with aqueous selank
solution (200
µ
g/kg body weight). On 6 day, the rats
of group SA were intranasally administered with
aqueous selank solution (200
µ
g/kg). One hour later,
the animals were decapitated. Total RNA was iso
lated from the hippocampus and spleen using the
RNAgents
TM
Total RNA Isolation System (Promega,
United States), which was then used to synthesize the
first strand of cDNA with the RevertAid
TM
H Minus
First Strand cDNA Synthesis Kit (Fermentas,
Lithuania).
The effect of selank of gene expression in rat hip
pocampus was studied using the SBC–R–RC–100–
13 microtemplate comprising 12000 genes (Shanghai
Biochip
TM
, China). The level of expression of certain
genes was quantitated by realtime PCR in an
Mx3000P
TM
RealTime QPCR System (Stratagene
Equipment, United States) using the SYBR Green I
dye (Sintol, Russia) and RT
2
qPCR Primer Assay
SYBR
®
Green primers (Super Array, United States).
The obtained values of the threshold reaction cycles
(
Ct
) were normalized relative to the
Ct
housekeeping
gene encoding the ribosomal protein L3. The results
were statistically processed using the Relative Expres
sion Software Tool384, version 2 software [8].
At the first stage of the study, we hybridized the hip
pocampal RNA on the microtemplate. The results of
these experiments showed that both single and chronic
administration of selank caused a twofold change in
the expression of five genes (table). Taking into
account the fact that selank exhibits a pronounced
antiviral activity, the study of the mechanism of action
of this peptide on the expression of these five genes in
the rat spleen was of particular interest.
The quantitative assessment showed that the effect of
selank on the expression of the five selected genes in the
spleen is much stronger than in the hippocampus. In the
spleen, an increase in the expression of all five genes was
observed. The most significant increase (by a factor of
over 4.5) was observed after a single administration of
selank. In the case of chronic administration, the effect
of the peptide was less pronounced: the expression of the
selected genes increased at most twice (table).
It should be noted that the direction of the effect of
selank on the expression of
ACTN1
and
CX3CR1
genes
in rat hippocampus and spleen was opposite. In the
hippocampus, the
ACTN1
expression after a single and
chronic selank administration and the
CX3CR1
expression after a single selank administration signifi
Transcriptomic Response of Rat Hippocampus and Spleen Cells
to Single and Chronic Administration of the Peptide Selank
T. A. Kolomin, M. I. Shadrina, Ya. V. Agniullin, S. I. Shram, P. A. Slominskii,
S. A. Limborska, and
Academician
N. F. Myasoedov
Received June 2, 2009
DOI:
10.1134/S1607672910010023
Institute of Molecular Genetics,
Russian Academy of Sciences, pl. Akademika Kurchatova 46,
D182, Moscow, 123182 Russia
BIOCHEMISTRY, BIOPHYSICS
AND MOLECULAR BIOLOGY

6
DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 430 2010
KOLOMIN et al.
cantly decreased. In the spleen, conversely, the expres
sion of these genes increased, especially after a single
selank administration.
The greatest change in gene expression was
observed in the spleen for three genes—
PTPRN2,
ACTN1
, and
CX3CR1
after a single selank administra
tion. The maximum (70fold) increase in expression
was detected for the
PTPRN2
gene. This gene encodes
an integral glycoprotein involved in the regulation of
transmembrane signaling [4]. The
PTPRN2
gene is a
key autoantigen in insulindependent diabetes melli
tus and may have a pathogenic role in the development
of this disease [11].
The expression of
ACTN1
and
CX3CR1
genes
increased 16 times after a single selank administration.
The
ACTN1
gene encodes the calciumsensitive protein
that crosslinks Factin fibrils and plays a key role in the
maintenance of the cytoplasm viscosity and elasticity
required for the preservation of the integrity of macro
molecules associated with the plasma membrane [12].
The discovered change in the
CX3CR1
expression is
of special interest because this gene is involved in the
regulation of inflammatory processes. The
CX3CR1
gene encodes the specific serpentinetype fractalkine
receptor that is involved in leukocyte maturation,
transfer, and recirculation as well as in the initiation of
local inflammation as a result of involvement of
inflammatory cells in chemotaxis [5, 9]. The interac
tion of fractalkine with CX3CR1 may serve as a regu
lator of the relationship between neurons and micro
glial and be involved in microglia activation and
migration [7]. There are data indicating that CX3CR1
functions as a neuroprotector and can inhibit apopto
sis [6]. In addition, it was shown that CX3CR1 may
function as a coreceptor for HIV1 penetration into
the cell [2].
Our results indicate that selank may participate in
the regulation of inflammatory processes in the body.
The complex biological effect of selank on the body at
least partially may be determined by the systemic
effect of this peptide on the genome expression. This
mechanism of action of peptides opens new vistas for
directional changes of transcriptional profile under
the influence of oligopeptides, homologues of natural
biologically active peptides. However, further studies
of the mechanisms of action of peptides, including
selank, on various systems of the body and the pro
cesses in them are required.
ACKNOWLEDGMENTS
This study was supported by the Russian Founda
tion for Basic Research (project no. 090401237a),
the program “Molecular and Cellular Biology” of the
Russian Academy of Sciences, and the State Contract
no. 02.512.11.2245.
REFERENCES
1. Agapova, T.Y., Agniullin, Y.V., Shadrina, M.I., et al.,
Neurosci. Lett.
, 2007, vol. 417, no. 2, pp. 201–205.
2. Combadiere, C., Salzwedel, K., Smith, E.D., et al.,
J. Biol. Chem.
, 1998, vol. 273, no. 37, pp. 23799–
23804.
3. CzabakGarbacz, R., Cygan, B., Wolanski, L., and
Kozlovsky, I.,
Pharmacol. Rept.
, 2006, vol. 58,
pp. 562–567.
4. Fitzgerald, L.R., Walton, K.M., Dixon, J.E., and Lar
gent, B.L.,
J. Neurochem.
, 1997, vol. 68, no. 5,
pp. 1820–1829.
5. Imai, T., Hieshima, K., Haskell, C., et al.,
Cell
, 1997,
vol. 91, pp. 521–530.
6. Meucci, O., Fatatis, A., Simen, A.A., and Miller, R.J.,
Proc. Natl. Acad. Sci. USA
, 2000, vol. 97, no. 14,
pp. 8075–8080.
7. Nishiyoria, A., Minamia, M., Ohtania, Y., et al.,
FEBS
Lett.
, 1998, vol. 429, pp. 167–172.
8. Pfaffl, M.W., Tichopad, A., Prgomet, C., and Neuvi
ans, T.P.,
Biotechnol. Lett.
, 2004, vol. 26, no. 6,
pp. 509–515.
9. Ruth, J.H., Volin, M.V., Haines, K.G., et al.,
Arthritis
Rheum.
, 2001, vol. 44, no. 7, pp. 1568–1581.
10. Semenova, T.P., Kozlovskaya, M.M., Zuikov, A.V.,
et al.,
Neurosci. Behav. Physiol.
, 2008, vol. 38.
11. Torii1, S., Saito, N., Kawano, A., et al.,
Traffic
, 2005,
vol. 6, pp. 1213–1224.
12. Uribe, R. and Jay, D.,
Mol. Biol. Rept.
, 2009, vol. 36,
pp. 121–125.
13. Zolotarev, Y.A., Dolotov, O.V., Inozemtseva, L.S.,
et al.,
Amino Acids
, 2006, vol. 30, no. 4, pp. 403–408.
Relative changes in gene expression in the rat hippocampus and spleen caused by single (SA) and chronic (CA) adminis
tration of the peptide selank relative to the control
Gene Gene name Hippocampus Spleen
SA CA SA CA
ACTN1
α
Actinin 1 0.42* 0.49* 15.96** 1.97**
CX3CR1
Chemokine (CX
3
C) receptor 1 0.46* 2.77* 15.93** 1.87**
FGF7
Fibroblast growth factor 7 2.35* 2.23* 8.15** 2.08**
PTPRN2
Receptorlike protein tyrosine phosphatase N2 2.61* 3.01* 70.36** 1.09
XTRP3
Sodium and chloridedependent transporter XTRP3 3.98* 2.13* 4.47* 1.19
*
p
< 0.05; **
p
< 0.01.