The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis

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 .

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.

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. Figure 1 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 ). Table 1 Comparison of genes that exhibited Semax-induced alteration of expression levels after pMCAO Gene symbol Ischemia + semax 3 h Ischemia + semax 24 h ENTREZ GENE_ID lg (Ratio) P -value lg (Ratio) P -value RGD1562905 0.58 1.1E-04 −0.53 2.1E-34 292539 Adamts1 0.34 5.2E-11 −0.25 7.5E-09 79252 Zfp36 0.24 5.5E-03 −0.16 2.0E-04 79426 Ptprcap −0.28 2.3E-03 0.29 9.8E-08 499300 Ccdc53 −0.29 1.3E-06 0.18 4.6E-03 299707 RT1-Ba −0.29 3.1E-07 0.36 1.5E-15 309621 Cd74 −0.30 1.6E-09 0.21 5.3E-06 25599 RT1-A1 −0.30 7.6E-10 0.29 2.8E-11 24973 H2-Ea −0.31 4.1E-10 0.15 7.3E-04 294269 Igh-1a −0.79 2.6E-30 0.47 3.5E-26 299352 The table shows the genes that exhibited significantly changed expression under Semax treatment at both time points (3 and 24 h). The logarithmic transformed value visually represents the symmetric changes in expression levels: negative value indicates decrease and positive value – increase of the gene expression level. The cut-off of gene-expression changes was 1.40. Ratio – relative expression level.

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. Figure 3 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 x-axis. Significance was determined using Fisher’s exact test ( P -value < 0.01). A value of 2 on the x-axis is equivalent to a P -value of 0.01. The y-axis shows the categories of biological processes that were related to lists of genes that exhibited changed expression in the experimental conditions. A . Data obtained 3 h after pMCAO for the ischemic rat cortex under Semax treatment; B . Data obtained 24 h after pMCAO for the ischemic rat cortex under Semax treatment. 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 3 A). 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 3 B). 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 3 B), and the regulation of the levels of Ca 2+ (Figure 3 B, Table 3 ). Table 3 Genes related to the quantity of Ca 2+ and exhibited Semax-induced alteration of expression levels (24 h) Definition (12 genes) Gene symbol ENTREZ GENE_ID Fold change P-value Transthyretin (Ttr), mRNA. Ttr 24856 20.55 2.1E-34 Chemokine (C-X-C motif) ligand 13, mRNA. Cxcl13 498335 4.12 1.6E-08 Chemokine (C-X-C motif) ligand 9, mRNA. Cxcl9 246759 2.42 1.8E-14 Chemokine (C-X-C motif) ligand 10, mRNA. Cxcl10 245920 2.32 1.9E-03 Chemokine (C-C motif) ligand 5, mRNA. Ccl5 81780 1.98 6.5E-05 Thyrotropin releasing hormone, mRNA. Trh 25569 1.95 8.5E-08 Chemokine (C-X-C motif) ligand 11, mRNA. Cxcl11 305236 1.85 2.1E-03 Chemokine (C-C motif) ligand 7, mRNA. Ccl7 287561 1.78 8.3E-04 Chemokine (C-C motif) ligand 19, mRNA. Ccl19 362506 1.72 3.4E-05 Secreted phosphoprotein 1, mRNA. Spp1 25353 1.58 3.4E-06 Gastrin releasing peptide, mRNA. Grp 171101 1.53 1.3E-03 Chemokine (C-C motif) ligand 20, mRNA. Ccl20 29538 0.44 3.4E-05 Entrez Gene is NCBI's repository for gene-specific information. In the table, P -values are in the form of an exponential number format. 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 3 A). 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 ). Table 4 Genes related to the vascular system and exhibited Semax-induced alteration of expression levels Genes related to the vascular system (24 genes): Gene symbol ENTREZ GENE_ID Fold change P-value 3h Cysteine-rich, angiogenic inducer, 61, mRNA. Cyr61 83476 2.43 1.5E-07 Activating transcription factor 3, mRNA. Atf3 25389 2.24 3.9E-03 Kruppel-like factor 4 (gut), mRNA. Klf4 114505 2.23 2.0E-03 A disintegrin-like and metallopeptidse (reprolysin type) with thrombospondin type 1 motif, 1, mRNA. Adamts1 79252 2.18 5.2E-11 FBJ murine osteosarcoma viral oncogene homolog, mRNA. Fos 314322 2.11 2.0E-04 Jun B proto-oncogene, mRNA. Junb 24517 2.0

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 Ca 2+ (Figure 3 B, Table 3 ). The ability of some chemokines to raise the level of intracellular Ca 2+ , 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 Ca 2+ level in cells, and showed an increase in Ca 2+ levels caused by the effect of Semax on the mechanisms that regulate Ca 2+ -dependent channels. It is well known that ischemia-induced energy depletion in cells results in disturbed operation of potential-dependent calcium channels and Na + /Ca 2+ pumps, excessive intracellular accumulation of Ca 2+ 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 Ca 2+ influx pathway, and not by Ca 2+ load [ 34 ]. Possibly, the neuroprotective effect of Semax on ischemia-damaged nervous tissues includes the impact of Ca 2+ penetration into the cell on the regulatory processes. This idea is based on recent studies of the neuroprotective effect of Ca 2+ -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. Studi

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 Ca 2+ 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 Ca 2+ accumulation. Possibly, the neuroprotective effect of Semax on ischemia-damaged nervous tissues includes an impact on processes involved in the incorporation of Ca 2+ 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.

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.

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.

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 ).

The authors declare that they have no competing interests.

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 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