The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis
1 Abstract
Background
The
nootropic neuroprotective peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro)
has proved efficient in the therapy of brain stroke; however, the
molecular mechanisms underlying its action remain obscure. Our
genome-wide study was designed to investigate the response of the
transcriptome of ischemized rat brain cortex tissues to the action of
Semax in vivo.
Results
The
gene-expression alteration caused by the action of the peptide Semax
was compared with the gene expression of the “ischemia” group animals at
3 and 24 h after permanent middle cerebral artery occlusion (pMCAO).
The peptide predominantly enhanced the expression of genes related to
the immune system. Three hours after pMCAO, Semax influenced the
expression of some genes that affect the activity of immune cells, and,
24 h after pMCAO, the action of Semax on the immune response increased
considerably. The genes implicated in this response represented over 50%
of the total number of genes that exhibited Semax-induced altered
expression. Among the immune-response genes, the expression of which was
modulated by Semax, genes that encode immunoglobulins and chemokines
formed the most notable groups.
In
response to Semax administration, 24 genes related to the vascular
system exhibited altered expression 3 h after pMCAO, whereas 12 genes
were changed 24 h after pMCAO. These genes are associated with such
processes as the development and migration of endothelial tissue, the
migration of smooth muscle cells, hematopoiesis, and vasculogenesis.
Conclusions
Semax
affects several biological processes involved in the function of
various systems. The immune response is the process most markedly
affected by the drug. Semax altered the expression of genes that
modulate the amount and mobility of immune cells and enhanced the
expression of genes that encode chemokines and immunoglobulins. In
conditions of rat brain focal ischemia, Semax influenced the expression
of genes that promote the formation and functioning of the vascular
system.
The immunomodulating effect of
the peptide discovered in our research and its impact on the vascular
system during ischemia are likely to be the key mechanisms underlying
the neuroprotective effects of the peptide.
Keywords: Semax, Pro-Gly-Pro, Focal cerebral ischemia, Expression Beadchip gene array, Gene expression, Immune cells, Immunoglobulins
Background
Ischemic
brain stroke is one of the major contributors to mortality and
disability worldwide. As the result of a critical reduction of blood
flow in the brain, it causes massive loss of neurons and leads to the
formation of the necrotic core and the penumbra zone [1].
One
of the drugs that is effectively employed currently in cerebral stroke
therapy is the Semax (Met-Glu-His-Phe-Pro-Gly-Pro), which is a synthetic
peptide consisting of a fragment of ACTH(4–7) and the C-terminal
tripeptide Pro-Gly-Pro (PGP). Studies have shown that Semax promotes the
survival of neurons during hypoxia [2] and glutamate neurotoxicity [3].
It also shows neuroprotective properties and contributes to
mitochondrial stability under stress induced by the deregulation of
calcium ion flow [3]. The action of Semax causes the inhibition of nitric oxide synthesis [4], improves the trophic supply of the brain [5], and protects the nervous system effectively against diseases of the optic nerve [6]. This peptide also possesses nootropic activity [7].
However,
the molecular mechanisms underlying the action of Semax remain unclear.
We have previously shown the effect of Semax on the expression of genes
that encode neurotrophic factors and their receptors in an experimental
model ischemia in the rat brain [8,9].
This
genome-wide study was performed to elucidate the transcriptome response
of the ischemized focal tissues of the rat brain to the action of Semax
in vivo. The main task of our study was to identify genes with an
altered expression that accounts for the positive effect exerted by
Semax in the treatment of patients with ischemic stroke [10,11].
Results
Semax-induced increase and decrease in gene expression
The
genome-wide expression changes induced by Semax in rat brain cortex
tissues damaged by focal ischemia were studied using the genome-wide
RatRef-12 Expression BeadChip (Illumina, USA), which contains 22,226
genes, according to NCBI. Data on the gene expression changes induced by
the peptide were compared with the gene expression levels in the
“ischemia” group at 3 and 24 h after pMCAO.
The largest
number of genes (96) that exhibited altered expression (cut-off 1.50)
in response to Semax administration was detected 3 h after the onset of
ischemia (Additional file 1);
moreover, the amount of the genes with decreased expression was
insignificantly larger than that of those with increased expression
(Figure 1). Semax altered the expression of 68 genes 24 h after occlusion (Additional file 2):
the expression of 51 genes was increased and the amount of genes with
decreased expression was considerably lower than that observed at 3 h
after the onset of ischemia.
Genes that were up- and downregulated.
The x-axis shows the condition of the experiment and time after pMCAO.
The y-axis represents the number of genes that exhibited changed
expression in these conditions. The cut-off of gene-expression changes
was 1.50. ...
Note
that different gene groups exhibited Semax-induced alteration of
expression at 3 h and 24 h. The overlapping group comprised only 10
genes with responses to the peptide that were contradictory (Table 1).
Comparison of genes that exhibited Semax-induced alteration of expression levels after pMCAO
Molecular functions of the protein products of genes with altered expression under Semax treatment
The
grouping of the genes according to the molecular functions of their
products and to the iReport Web tool revealed that the expression of
transcription regulator genes was predominantly enhanced, and that that
of genes encoding transmembrane receptors, transport proteins, and
various enzymes was decreased 3 h after the onset of ischemia under
Semax treatment (Figure 2A);
about 39% of the genes with altered expression encoded proteins with
molecular functions that were unrelated to the groups presented or were
not yet identified. Gene expression was increased mostly at 24 h
(Figure 2B). The largest increase in expression was observed for immunoglobulin and cytokine (chiefly chemokine) genes (Table 2). The molecular functions of 24% of the protein products of the genes that exhibited altered expression levels were unknown.
Molecular functions associated with the up- and downregulated genes.
The x-axis shows the categories of molecular functions. The y-axis
represents the number of genes associated with selected cellular
functions. The genes that were upregulated are indicated ...
Genes related to the immune system and exhibited Semax-induced alteration of expression levels
Biological processes that were significantly associated with the genes that exhibited altered expression levels in response to the administration of the peptide
We used an online program [12]
to analyze genes with altered expression in response to the
intermittent administration of Semax to ischemized animals. This led to
the identification of several biological processes that were associated
with the gene expression changes observed (Figure 3). The reliability of these processes was calculated by Fisher’s exact test.
Biological processes based on the genes that exhibited alteration in their expression levels under Semax treatment. The x-axis is the absolute value of the log transformed P-value, which means that a smaller P-value has a larger positive value on the ...
Three
hours after pMCAO, Semax exerted considerable influence on various
general biological processes (proliferation, differentiation, and
migration of cells), on vascular system processes, brain cell processes,
and on the immune system (Figure 3A).
Twenty-four hours after occlusion, similar to observed effects 3 h
after the procedure, Semax, acting in conditions of focal ischemia,
altered the expression of genes involved in cell proliferation and
migration. One day after the occlusion, however, unlike at 3 h after the
procedure, additional processes supplemented the general processes,
namely, the organization of the cytoskeleton, tissue development, and
the quantity of metal (Figure 3B).
Special attention should be drawn to the processes that were most
significantly associated with immune cell activity and calcium ion
regulation, namely, the migration and attraction of dendritic cells
(DCs), the attraction of leukocytes (Figure 3B), and the regulation of the levels of Ca2+ (Figure 3B, Table 3).
Genes related to the quantity of Ca2+ and exhibited Semax-induced alteration of expression levels (24 h)
Semax altered the expression of genes related to the immune system to a large degree (Table 2).
In conditions of experimental focal ischemia, the action of Semax
observed 3 h after the onset of ischemia influenced the expression of
several genes that are involved in the regulation of the activity of
immune cells: macrophages, neutrophils, and lymphocytes (Figure 3A).
The effect of Semax on the immune response was increased significantly
24 h after pMCAO. The genes involved in this process represented over
50% of the total amount of the genes that exhibited altered expression
levels. Semax-induced upregulation of transcripts was observed for a
majority of the immune-response genes; among these, immunoglobulin genes
formed the most prominent group, with half of them exhibiting the
highest amplitude of expression alteration among the genes for which the
level of transcripts was affected by the peptide (Table 2).
Another
remarkable group of genes with Semax-induced alteration in expression
levels consisted of genes involved in the vascular system. The
expression of 24 and 12 genes was altered 3 and 24 hours after pMCAO,
respectively (Table 4).
Genes related to the vascular system and exhibited Semax-induced alteration of expression levels
Genes that regulate the levels of Ca2+ formed a separate group of genes exhibiting a significant Semax-induced alteration of expression 24 h after occlusion (Table 3).
Discussion
Different profiles of gene expression elicited by Semax administration 3 and 24 h after pMCAO
The
dynamic state of mRNA expression in mammalian tissues changes during
pathophysiological processes and after the introduction of medicinal
peptides into the organism. In this context, we studied transcriptome
changes caused by the action of neuropeptide Semax in the ischemized rat
brain cortex. This genome-wide study showed that Semax affected the
transcript level of several dozens of genes 3 and 24 h after pMCAO;
however, the functional significance of many of them remains unknown.
Three
hours after pMCAO was used in the analysis as a time point inside the
therapeutic window of the drug and within the response time of
early-response genes [13].
At that time point, we found a considerable alteration of the
expression of genes encoding transcription factors that could set off
new signal pathways that allow the correction of the destructive
processes that developed after vascular occlusion. During the active
stage of ischemia and the response of late-response genes, i.e., 24 h
after pMCAO, we observed increased levels of transcripts encoding
transmembrane receptors and enzymes, especially cytokines and
immunoglobulins. One can presume that processes initiated by
transcription factors during the first hours of therapy of ischemized
animals were developing further. Several similar processes were observed
in the course of the associative analysis of biological processes.
Response of immune system cells to Semax administration and regulation of the expression of genes encoding chemokines and immunoglobulins
The
detailed analysis of genes that exhibited altered levels of expression 3
and 24 h after pMCAO allowed the determination of the effect of Semax
on various biological processes that were categorized under broad
subgroups, namely, general category, brain cell, immune, and vascular
processes. The neuroprotective and nootropic properties of Semax were
previously associated only with events that are directly relevant to
nervous tissues [2,7,11].
Here, we uncovered the action of Semax on the immune system for the
first time. Three hours after pMCAO, Semax acted on microglia and immune
system cells. The process of leukocyte activation was affected most
significantly (P-value = 7.6 × 10−8) in the immune response
subgroup. The processes that developed 24 h after pMCAO, which involved
leukocytes, remained significant. In addition, Semax affected DCs, the
presence of which in rat cerebral hemisphere ischemia-damaged tissues
had been reported by other researchers [14]. DCs constitute a heterogeneous class of antigen-presenting cells that are capable of immune response initiation [15] and cytokine production [16].
Both
inflammation and immune response play an important role in ischemic
stroke. It is well known that the penetration of inflammatory/immune
cells into brain tissues during the postischemia hours aggravates the
situation [17-19].
In addition, no data have been reported to date indicating the presence
of a specific cause-and-effect relationship between the penetration of
leukocytes into the damaged tissues and the pathogenesis of the ischemia
itself [20]. However, some studies support the neuroprotective abilities of immune cells [21,22].
It
should be mentioned that the most noticeable immune response to Semax
action was observed at 24 h after pMCAO. A high level of immunoglobulin
transcripts was found at that time point in the ischemized rat brain
cortex. Several studies had shown previously that intravenous
immunoglobulin (IVIG) has a strong neuroprotective effect against
ischemic impairment of the brain [23]. It is believed that IVIG application is one of the options for acute brain stroke therapy [24].
Whether or not the neuroprotective effect of Semax can be a consequence
of the enhancement of the expression of immunoglobulin should be
addressed in future studies.
Cytokines (particularly
chemokines), which are one of the most important participants in the
immune response, were also expressed actively 24 h after pMCAO under the
influence of Semax in the region of the brain where the ischemic lesion
was localized. Many reports have described chemokine expression in
astrocytes, microglia, and even neurons [25]. It is accepted that some chemokines and their receptors are involved in various neurodegenerative diseases [26], including ischemic brain damage [27].
Recent
research has shown that chemokines are a unique class of neuromediators
that ensure the cross-talk between neurons and cells from their
surrounding microenvironment [28].
In accordance with this, the division of chemokines into pro- and
anti-inflammatory factors seems to be too simplified and gives rise to
contradicting opinions regarding the neuroprotective and
neurodegenerative functions of chemokines [29].
Enhanced expression of chemokine-encoding genes is one more evidence in
favor of the possible existence of a Semax immunomodulatory effect in
conditions of focal cerebral ischemia of the brain.
Semax-induced
activation of chemokine genes presumably accounted for the altered
transcript level of genes associated with the regulation of the quantity
of Ca2+ (Figure 3B, Table 3). The ability of some chemokines to raise the level of intracellular Ca2+, which plays a messenger role in nervous tissues, has been described in several studies [30,31]. A study that used human neutrophils [32] offered experiment-based support of the effect of Semax on the Ca2+ level in cells, and showed an increase in Ca2+ levels caused by the effect of Semax on the mechanisms that regulate Ca2+-dependent channels.
It
is well known that ischemia-induced energy depletion in cells results
in disturbed operation of potential-dependent calcium channels and Na+/Ca2+ pumps, excessive intracellular accumulation of Ca2+ ions, and neuronal death [33].
However, it has been shown that Semax contributes to neuron
survivability in the conditions of glutamate neurotoxicity that
accompany ischemia [3]. Some authors have suggested that cellular death is caused by the Ca2+ influx pathway, and not by Ca2+ load [34]. Possibly, the neuroprotective effect of Semax on ischemia-damaged nervous tissues includes the impact of Ca2+ penetration into the cell on the regulatory processes. This idea is based on recent studies of the neuroprotective effect of Ca2+-activated potassium channels in conditions of brain ischemic damage [35,36].
The
opinions on the role of the immune system in the pathogenesis of
ischemia vary. Studies are available regarding the contribution of the
immune system to ischemic damages [37], the neuroprotective and healing effect of immune-cell activation [38,39], the protective role of the immune system, and its therapeutic function [40-42].
It cannot be ruled out that the observed effect of Semax on brain
stroke can be explained by its impact on protective immune mechanisms.
Some recent reports have described interactions between nervous tissues
and the immune system, which were observed after the administration of
neuropeptides. For instance, the nootropic medication cerebrolysin
favored the survival of immunocompetent cells [43]. Another preparation, the vasoactive intestinal peptide (VIP), which has neurotrophic effects, acted as an immunomodulator [44].
The possible effect of neuromodulation on the consequences of ischemia
is believed to be real, although it has not been studied sufficiently [45].
Response of the vascular system to the administration of the neuropeptide Semax
Here,
we found changes in the expression levels of several genes involved in
the functioning of the vascular system as a response to Semax
administration. The formation of new blood vessels in the ischemized
areas represents one of the approaches used in the treatment of brain
stroke [46].
It should be mentioned that the presence of immune cells in the damaged
tissues is a typical feature of postischemic revascularization [47].
Three hours after pMCAO, Semax affected the expression of genes
involved in vasculogenesis and the transcription levels of genes
associated with hematopoiesis and the migration of endothelial cells.
Some signal pathways are well known to be active in both hematopoiesis
and vasculogenesis [48].
Moreover, a large number of genes are expressed in both endothelial
cells and hematopoietic precursor cells of the adult organism [49,50].
Three hours after occlusion, Semax altered the expression of genes
associated with the artery vasodilation process as well. Our earlier
studies showed that capillary bore extension was observed as early as
15 min after the administration of the peptide [9].
As shown in Figure 3B,
24 h after occlusion, Semax affected the development of the endothelial
tissue and the migration of smooth muscle cells, which was an
indication of vessel formation and stabilization [48].
Finally, another biological process, i.e., the activation of blood
cells, was affected by Semax 24 h after pMCAO, which followed logically
after the process of the formation of blood cells induced by Semax 3 h
after the occlusion.
Thus, as
demonstrated here, the action of Semax on the expression of genes that
ensure the formation and functioning of the vascular system in ischemic
conditions also uncovered its possible vascular and regenerative
properties, in addition to its neuroprotective and vasoactive effects.
Conclusions
In
this study, we analyzed the action of the neuroprotective peptide Semax
on the transcriptome of rat brain cortical cells in conditions of
experimental focal ischemia. Although Semax has been shown to be
effective in brain stroke therapy, the molecular mechanisms underlying
its neuroprotective action remain unknown.
As shown
here, Semax influenced various biological processes that contribute to
the functioning of the different systems of the organism. The immune
response was most markedly affected by the action of Semax. The peptide
increased the amount and mobility of immune cells and enhanced the
expression of chemokine and immunoglobulin genes.
Our
data showed that Semax is likely to influence processes that accompany
the formation of new blood vessels during early ischemia cascade stages,
as well as their stabilization at later stages.
The expression of genes responsible for the intracellular level of Ca2+
was sensitive to Semax administration against the background of the
unfolding pMCAO-induced neurodegenerative processes. Our results showed
that Semax enhanced the expression of genes encoding protein products
that promote intracellular Ca2+ accumulation. Possibly, the
neuroprotective effect of Semax on ischemia-damaged nervous tissues
includes an impact on processes involved in the incorporation of Ca2+ into cells.
Thus,
the immunomodulating effects of Semax described here, as well as its
influence on the vascular system in conditions of ischemia, are likely
to be key factors in the neuroprotective effects of the peptide. It
cannot be ruled out that the large amount of genes that exhibited
changed levels of expression, the functions of which remain unknown or
not well studied, will help disclose other, hitherto unknown pathways of
Semax action on damaged brain tissues. We must state at the same time
that the baffling complexity of the multicomponent nature of cerebral
ischemia and the ability of Semax to affect a large number of biological
processes require future research to uncover the full scope of the
mechanisms of action of this peptide.
Methods
Animals
All
experimental protocol were approved by Bioethics Comission of Lomonosov
Moscow State University in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals (NIH Publ. no.
80–23, revised 1996). We used adult male Wistar rats (270–320 g)
maintained on a 12 h light/dark cycle at a temperature of 22–24°C with
free access to food and water.
Focal cerebral ischemia model
We applied the model of “focal cerebral ischemia” induced as previously described [51].
The irreversible electrical coagulation of the distal segment of the
left middle cerebral artery was performed under anesthesia with chloral
hydrate (300 mg/kg).
Focal cerebral ischemia was induced by direct pMCAO, involving craniotomy technique as previously described [52]
without occlusion of carotid artery. In detail, anesthesia was induced
by intraperitoneal administration of chloral hydrate (400 mg/kg body
weight). The left middle cerebral artery (MCA) was exposed via the
transtemporal approach. A 1.5 cm scalp incision was made at the midpoint
between the right eye and the right ear. The temporalis muscle was
separated in the plane of its fiber bundles and retracted in order to
expose the zygoma and squamosal bone. Using microsurgical technques, a
burr hole, 2 mm in diameter, was made with a dental drill 1 mm rostal to
the anterior junction of the zygoma and the squamosal bone. The dura
mater was carefully pierced with a scalpel. The exposed MCA was isolated
and occluded by short coagulation using a bipolar coagulator. The
craniotomy was covered with a small piece of gelfoam, the temporalis
muscle and overlying skin were allowed to fall back and were sutured
separately. After suturing, rats were returned to their cages until
sacrifice. The operation was last about 30 min.
Experimental groups
Animals
were divided into two groups: (1) “ischemia” and (2) “ischemia + Semax”
groups. pMCAO was performed in all animals. During the experiment,
ischemia + Semax animals were given intraperitoneal injections of Semax
(100 μg/kg), whereas ischemia animals were injected with saline. The
injections of Semax or saline were performed 15 min, 1, 4 and 8 h after
pMCAO.
The rats were decapitated under anesthesia with
ethyl ether 3 and 24 h after the operation. According to data from the
literature, significant events in the formation of a stroke area, such
as excitotoxicity, mitochondrial damage, emergence of reactive oxygen
species, and apoptosis, occur within the first 3 h after occlusion of an
artery [53],
and the expression of genes at the early stage of ischemia can be
studied at this time point. At the 24 h time point the infarction area
reaches its maximal dimensions and the formation of the penumbra is
completed [54].
Each
time point included at least five animals. We isolated the
frontoparietal cortex of the ischemic animals, in which, according to
histological analysis of our earlier research, the damaged area was
localized [51]. Total RNA was isolated from tissue samples.
Microarray data analysis
Microarray
experiments were carried out at ZAO ''Genoanalytica'', Moscow, Russia.
Total RNA was isolated from tissue samples using guanidine thiocyanate [55].
RNA integrity was assessed by comparison with the rRNA bands obtained
in agarose gel electrophoresis under denaturing conditions. RNA was
quantified using NanoDrop, and its quality was assessed using an Agilent
RNA 6000 Nano Chip. Total RNA (400 ng) was amplified using an Illumina®
TotalPrep™ RNA Amplification Kit (Ambion, USA) containing 22,523 probes
for a total of 22,228 rat genes selected primarily from the NCBI
Reference Sequence database (Illumina, USA). The Illumina RatRef-12
Expression BeadChip was used in accordance with the manufacturer’s
instructions. The BeadArray Reader was employed for data acquisition,
and the analysis was accomplished with the help of the Genome Studio
software (Illumina, USA) using the gene-expression module. The
statistical algorithm used in GenomeStudio gene expression analysis is
the Illumina Custom error model.
Functional analysis
The interactive Web-based Ingenuity iReport program [12] based on Fisher’s exact test (P-value
< 0.01) was applied to identify the molecular functions of the
products of the genes that exhibited altered expression in the
conditions established, as well as signaling pathways and statistically
significant biological processes. Ingenuity iReport helps the quick
identification of especially significant genes, signaling pathways, and
processes that are most relevant to the experimental data. Only those
genes with a change in expression of at least 1.5-fold from the baseline
value and whose P-value lower 0.05 were selected for iReport analysis.
Availability of supporting data
The
data sets supporting the results of this article are available in the
ArrayExpress repository (European Bioinformatics Institute, Cambridge,
UK) [56] with series accession number E-MTAB-1864 (https://www.ebi.ac.uk/biosamples/group/SAMEG148775).
Abbreviations
ACTH:
Adrenocorticotropic hormone; pMCAO: Permanent middle cerebral artery
occlusion; MCA: Middle cerebral artery; CNS: Central nervous system; DC:
Dendritic cells; IVIG: Intravenous immunoglobulin; VIP: Vasoactive
intestinal peptide; MHC I and II: Major histocompatibility class I and
II.
Authors’ contributions
EM
- carried out the molecular genetic studies and drafted the manuscript;
NM - synthesized Semaks and participated in the design of the study; VS
- designed the focal ischemia model; OP - performed the operations of
experimental animals; VD - isolated the frontoparietal cortex of the
ischemic animals, obtained the RNA from the tissue samples; LD – have
made substantial contributions to interpretation of data and have been
involved in revising manuscript critically for content; SL - have given
final approval of the version to be published. All authors read and
approved the final manuscript.
Supplementary Material
Additional file 1: Table S1:
List
of all genes that exhibited changed expression under Semax treatment
(3 h after pMCAO). All transcripts that showed significant difference
between the “ischemia + Semax” and “ischemia” animal groups 3 h after
pMCAO. In the table, P-values are in the form of an exponential number format. Entrez Gene is NCBI’s repository for gene-specific information.
Click here for file(65K, xls)
Additional file 2: Table S2:
List
of all genes that exhibited changed expression under Semax treatment
(24 h after pMCAO). All transcripts that showed significant difference
between the “ischemia + Semax” and “ischemia” animal groups 3 h after
pMCAO. In the table, P-values are in the form of an exponential number format. Entrez Gene is NCBI’s repository for gene-specific information.
Click here for file(56K, xls)
Acknowledgements
This
study was partially supported by grants of the Russian Foundation for
Basic Research (11-04-00843, 12-04-31528, 13-04-40083-Н), and the
“Molecular and Cell Biology” Program of the Russian Academy of Sciences,
and the Federal Program for Support of Scientific Schools of the
Russian Ministry of Science and Education.
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The peptide semax affects the expression of genes related to the immune and vasc...The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis
BMC Genomics. 2014; 15()228
- ax in acute period of ischemic stroke].[Zh Nevrol Psikhiatr Im S S Korsakova. 1999]
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- Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.[Anal Biochem. 1987]
