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Short peptides vesugen and D-7 have stimulated proliferation-associated protein Ki-67 decreased during aging in tissue-specific cell cultures received from young and old animals and in dissociated vascular endothelial cell cultures. Peptides vesugen and D-7 have interacted with promoter region of MKI67 gene coding protein Ki-67 that was obtained using methods of molecular docking. Both peptides have contacted with core promoter 5'-agcctcaaccatcaggaaaacaagagt-3' located in MKI67 gene from -14 to +12 base pairs relative to the transcriptional initiation site through sequence CATC(ENSG00000148773). Thus, vasoprotective effect of peptide vesugen revealed previously in elder people could be realized through epigenetic regulation of Ki-67 gene expression.

Cytokines CCL11 (eotaxin) and HMGB1 (alarmin 1) are molecular markers of ageing and neurological, cardiovascular, and immune diseases. Created at the St. Petersburg Institute of Bioregulation and Gerontology, these short peptides are known to regulate gene expression and protein synthesis. They promote decreased mortality and deceleration of pathology in the elderly. The review suggests that the dipeptide vilon (Lys-Glu) and tetrapeptide epitalon (Ala-Glu-Asp-Gly) are involved in regulation of the CCL11 and HMGB1 genes as activators of their expression. The geroprotective effect of vilon and epitalon is probably due to the suppression of these genes.

Polypeptide complex isolated from calf kidneys stimulates the processes of cell renewal in organotypic kidney tissue cultures from young and old rats. The polypeptide complex enhances expression of proliferation marker Ki-67 and reduces expression of proapoptotic peptide p53 in kidney explants obtained from young and old animals. Short peptides T-31 (AED) and T-35 (EDL) also stimulate proliferation and reduce apoptosis of the kidney cells, but to a lesser degree than the polypeptide complex. The results provide the basis for further investigation of the polypeptide complex as a preparation for the therapy of kidney diseases, including age-related pathologies.

Data on endothelial signaling molecules involved in homeostasis regulation and development of age-associated disorders are summarized. Regulation of the endothelium function is implemented by a system of signaling molecules: NO, NO synthase, prostacyclin, thrombomodulin, thromboxane, antithrombin, endothelins, fibronectin, von Willebrand factor, etc. Changes in their synthesis can cause arterial hypertension, atherosclerosis, ischemic heart disease, myocardial infarction, and other age-related disorders. Study of the molecular mechanisms mediating endothelial functioning is essential for developing new methods of diagnostics and a treatment for age-associated cardiovascular diseases.

Irisin produced by muscles during exercise and promoting fat burning also exhibits geroprotective effect and induces telomere elongation in normal somatic cells. Special attention is paid to studies of the role of peptides Lys-Glu, Glu-Asp-Arg, and Ala-Glu-Asp-Gly in epigenetic regulation of irisin content. The data suggest that the immunomodulatory peptide Lys-Glu and neuroprotective peptide Glu-Asp-Arg modulate the life span by modulating irisin gene expression.

In primary culture of mouse hippocampal neurons, peptide EDR (200 ng/ml) under conditions of amyloid synaptotoxicity (a model of Alzheimer’s disease) increased the number of mushroom spines by 71% and returned this parameter to the normal level. Under the same conditions, tripeptide KED (200 ng/ml) increased the number of mushroom spines in hippocampal neurons by 20%. Tripeptide EDR can be recommended for further experimental study as a candidate neuroprotective agent for prevention and treatment of Alzheimer’s disease.

Peptides Glu-Asp-Arg and Lys-Glu-Asp stimulate serotonin expression in aging cultures of brain cortex cells. Peptide regulation of 5-tryptophan hydroxylase gene encoding the enzyme involved in serotonin synthesis was demonstrated by the molecular docking method. The CCTGCC nucleotide sequence in 5-tryptophan hydroxylase gene was found to be complementary to these peptides. Hence, Glu-Asp-Arg and Lys-Glu-Asp peptides epigenetically regulate serotonin synthesis in the brain cortex, which indicates their neuro- and geroprotective activities.

In this work, it was shown that short peptides, such as vilon, thymogen, crystagen and R-1, possess different age-associated immunoprotecting properties in the spleen. Both the R-1 and vilon peptides were found to activate T-helper cells. T-Helper activation by vilon was based on the inhibition of cellular apoptosis, whereas R-1 acted through positive stimulation of cellular proliferation and differentiation. Thymogen was found to be an activator of B-lymphocytes and acted through the inhibition of apoptosis, as well as by the stimulation of spleen cells proliferation. Crystagen was also found to activate the B-cell immunity system but had no effect on the processes of cellular renewal in the spleen during aging.

Peptide bioregulators promote restoration of the physiological activity of the retina in retinitis pigmentosa in older adults and in animal models. The molecular mechanism of the physiological activity of peptides is associated with their ability to epigenetically regulate the synthesis of protein markers of the differentiation of retinal neurons and pigment epithelium.

Data on various aspects of the molecular and cellular mechanism of the biological activity of short peptides used as potential drugs were analyzed. Based on the published data and our own results, a possible mechanism of the penetration of short peptides into the cytoplasm and cell nucleus is considered. In addition, the possibility of the involvement of short peptides in the mechanisms of the epigenetic regulation of gene expression via their complimentary interaction with promoter regions of genes in DNA is substantiated.

The review contains different aspects indicating the participation of cytokines in aging-associated diseases. Authors reveal the role of short peptides in molecular mechanisms of homeostatic regulation in immune, cardiovascular, haemostatic systems which is based on the literature data and own experimental data. It is established that short peptides realize their geroprotective effect by changing gene expression of immune and proinflammatory cytokines and g-interferon.

In this study, the molecular mechanisms of pancreoprotective action of the tetrapeptide H-Lys-Glu-Asp-Trp-NH2 in aging human pancreatic cells have been investigated. It has been established that the tetrapeptide under study increases the expression of matrix metallopreteinases MMP2 and MMP9, serotonin, glycoprotein CD79α, antiapoptotic protein Mcl-1, and proliferation markers PCNA and Ki67, as well as decreases the expression of proapoptotic protein p53 in aged pancreatic cell cultures. Thus, the clinical effect of the tetrapeptide observed in elderly patients with type-2 diabetes mellitus and pancreatitis may be due to its ability to activate the expression of signaling molecules, i.e., markers of functional activity of pancreatic cells.

The decrease of lymphocyte interferon gamma expression during aging is one of the main mechanisms leading to the immunodeficiency state in the elderly. Cell penetrating geroprotective peptide Ala-Glu-Asp-Gly has the capability to activate the proliferation of lymphocytes in thymus during its aging. The nucleotide sequence which is complementary contacted with peptide Ala-Glu-Asp-Gly was found in promoter region of interferon gamma gene. Thus, the immune protection of this peptide can be explained by its activation of the interferon gamma production in T-cells.

The influence of individual amino acids and their combinations on the growth of cultures of Escherichia coli O75 and the proliferation of the culture of organotypic tissues was studied. Based on their effect on the colony size, all amino acids were divided into neutral, stimulating, and inhibiting with regard to growth. The biological effect of amino acids was associated with the concentration of amino acids and was dependent on the heterogeneity of the bacterial population. The activity of two amino acids, which occur in pairs, was different from the activity of individual compounds. Some combinations of amino acids, which cause recognizable effects in bacterial cultures, also had an effect on the tissue cultures of mammals. It was shown that the combinations of amino acids that constitute peptides were less active than the original peptide.

Analysis of the main parameters of molecular mechanics (number of hydrogen bonds, hydrophobic and electrostatic interactions, DNA-peptide complex minimization energy) provided the data to validate the previously proposed qualitative models of peptide-DNA interactions and to evaluate their quantitative characteristics. Based on these estimations, a three-dimensional model of Lys-Glu and Ala-Glu-Asp-Gly peptide interactions with DNA sites (GCAG and ATTTC) located in the promoter zones of genes encoding CD5, IL-2, MMP2, and Tram1 signal molecules.

We compared the expression of signal molecules in the culture of human endothelial cells under normal conditions and in atherosclerosis and restenosis. Expression of connexin-37 and sirtuin-1 in atherosclerosis and restenosis surpassed the normal by 2 and 5 times, respectively, and expression of endothelin-1 3-fold surpassed the normal. In restenosis, changes in the expression connexin-37 and endothelin-1 became more pronounced in comparison with atherosclerosis. Connexin-37 and endothelin-1 can serve as predictive markers for prognosis of post-stenting complications in patients with atherosclerosis.

Tripeptide Lys-Glu-Asp stimulates proliferation and inhibits apoptosis in organotypic cultures of neuroimmunoendocrine system cells. Lys-Glu-Asp accelerates cell renewal processes (decrease of apoptosis marker p53 and increase of proliferation marker Ki-67) in the pineal gland; this effect is more pronounced in cultures derived from old animals than in young cultures. The tripeptide induces the expression of low-differentiated lymphocyte marker CD5 and macrophage marker CD68, but in "old" cultures this effect is less pronounced than in "young" ones. Thus, in tissue culture Lys-Glu-Asp primarily affects the nervous and endocrine tissues during aging and produces a less pronounced effect on the nervous tissue. Physiological activity of the tripeptide consists in modulation of associative learning of honey bee in the model of short-term and the long-term memory.

The effects of epithalone and vilone peptides on the synthesis of melatonin and factors involved in this process, arylalkylamine-N-acetyltransferase (AANAT) enzyme and pCREB transcription protein, were studied in rat pinealocyte culture. Epithalone stimulated AANAT and pCREB synthesis and increased melatonin level in culture medium. Simultaneous addition of norepinephrine and peptides into the culture potentiated the expression of AANAT and pCREB.

Short peptides constitute the system of signal molecules regulating the functions of the organism at the molecular, genetic, subcellular, cellular, and tissue levels. One short peptide can regulate dozens of genes, but the molecular mechanism of this process remains unclear. We suppose that short peptides penetrate through the cytoplasmic and nuclear membrane and bind to DNA. Spatial models of DNA-peptide complexes are constructed for 19 short peptides by the docking method. Some peptides have the same binding sites. Peptides KE and EDP bind agat sequence, peptides KEDW and AED to acct sequence, and peptides AEDL and EDL to ctcc sequence.

The effect peptides KE, KED, AED and AEDG on proliferation (Ki-67), regeneration and aging (CD98hc), apoptosis (caspase-3), and extracellular matrix remodeling (MMP-9) in skin fibroblasts during their aging in culture were studied by immunofluorescent confocal microscopy. All studied peptides inhibited MMP-9 synthesis that increases during aging of skin fibroblasts and enhanced the expression of Ki-67 and CD98hc that are less intensively synthesized during cell aging. Peptides AED and AEDG suppressed caspase-dependent apoptosis that increases during aging of cell cultures.

Expression levels of genes encoding specific transcription factors and other functionally important proteins vary upon aging of pancreatic and bronchial epithelium cell cultures. The peptides KEDW and AEDL tissue-specifically affect gene expression in pancreatic and bronchial cell cultures, respectively. It is established in this work that the DNA methylation patterns of the PDX1, PAX6, NGN3, NKX2-1, and SCGB1A1 gene promoter regions change upon aging in pancreatic and bronchial cell cultures in correlation with variations in their expression levels. Thus, stable changes in gene expression upon aging of cell cultures could be caused by changes in their promoter methylation patterns. The methylation patterns of the PAX4 gene in pancreatic cells as well as those of the FOXA1, SCGB3A2, and SFTPA1 genes in bronchial cells do not change upon aging and are unaffected by peptides, whereas their expression levels change in both cases. The promoter region of the FOXA2 gene in pancreatic cells contains a small number of methylated CpG sites, their methylation levels being affected by cell culture aging and KEDW, though without any correlation with gene expression levels. The promoter region of the FOXA2 gene is completely unmethylated in bronchial cells irrespective of cell culture age and AEDL action. Changes in promoter methylation might be the cause of age- and peptide-induced variations in expression levels of the PDX1, PAX6, and NGN3 genes in pancreatic cells and NKX2-1 and SCGB1A1 genes in bronchial cells. Expression levels of the PAX4 and FOXA2 genes in pancreatic cells and FOXA1, FOXA2, SCGB3A2, and SFTPA1 genes in bronchial cells seem to be controlled by some other mechanisms.

On the page 553 Acknowledgements should be as follows: The study was supported by the Russian Science Foundation (grant 14-25-00024-P).

The effects of polypeptide complex from the kidney and short peptides AED, EDL, and AEDG on renal functions were studied in rats with cisplatin-induced acute renal failure. AED peptide decreased protein excretion and electrolyte concentration in the urine. Polypeptide complex from the kidney and peptides EDL and AEDG normalized diuresis, creatinine concentration in the urine and its excretion, glomerular filtration rate, and absolute resorption of sodium ions and reduced protein concentration in the urine and its excretion, the concentrations of sodium and potassium ions in the urine, and other parameters. EDL peptide produced was potent nephroprotective effect. It is known that the polypeptide complex of the kidney and short peptides restore the expression of signal molecules (marker of functional state of the kidneys), so these peptide substances can have nephroprotective effect during various renal pathologies.

Some studies have shown that peptides have high treatment potential due to their biological activity, harmlessness, and tissue-specific action. Tetrapeptide Ala-Asp-Glu-Leu (ADEL) was effective on models of acute bacterial lung inflammation, fibrosis, and toxic lung damage in several studies. We measured Ki67, Mcl-1, p53, CD79, and NOS-3 protein levels in the 1st, 7th, and 14th passages of bronchoepithelial human embryonic cell cultures. Gene expression of NKX2-1, SCGB1A1, SCGB3A2, FOXA1, FOXA2, MUC4, MUC5AC, and SFTPA1 was measured by real-time polymerase chain reaction. Using the methods of spectrophotometry, viscometry, and circular dichroism, we studied the ADEL–DNA interaction in vitro. Peptide ADEL regulates the levels of Ki67, Mcl-1, p53, CD79, and NOS-3 proteins in cell cultures of human bronchial epithelium in various passages. The strongest activating effect of peptide ADEL on bronchial epithelial cell proliferation through Ki67 and Mcl-1 was observed in “old” cell cultures. ADEL regulates the expression of genes involved in bronchial epithelium differentiation: NKX2-1, SCGB1A1, SCGB3A2, FOXA1, and FOXA2. ADEL also activates several genes, which reduced expression correlated with pathological lung development: MUC4, MUC5AC, and SFTPA1. Spectrophotometry, viscometry, and circular dichroism showed ADEL–DNA interaction, with a binding region in the major groove (N7 guanine). ADEL can bind to specific DNA regions and regulate gene expression and synthesis of proteins involved in the differentiation and maintenance of functional activity of the bronchial epithelium. Through activation of some specific gene expression, peptide ADEL may protect the bronchial epithelium from pulmonary pathology. ADEL also may have a geroprotective effect on bronchial tissue.

We studied the effect of polypeptide complex isolated from calf kidney and short peptides T-31 (AED) and T-35 (EDL) on the expression of signaling molecules, markers of cell renewal (Ki-67, p53), remodeling of the extracellular matrix (MMP-14), and immune response (IL-8) in primary kidney cell cultures during aging. The complex of renal polypeptides and T-31 peptide activate cell renewal processes during aging of the renal epithelium, while gelatinase MMP-14 is the target of T-35 peptide.

We studied molecular mechanisms of the vasoprotective effects of tripeptide T-38 and dipeptide RR-1. Short peptides T-38 and the RR-1 activate the processes of cell renewal in organotypic and dissociated cultures of vascular cells during aging by increasing the expression of Ki-67 and reducing the synthesis of p53 protein. T-38 and RR-1 reduce the synthesis of E-selectin, adhesion molecule involved in the formation of atherosclerotic plaques.

We performed a comparative analysis of biological activity of Lys-Glu peptide and its amino acid constituents. It was established that Lys-Glu stimulated proliferation of splenic cells in organotypic culture, while the mixture of glutamic acid and lysine inhibited culture growth. Using the method of molecular docking, we showed that glutamic acid, lysine, and Lys-Glu peptide can interact with different DNA sequences. The energy of interaction and the most beneficial localization of glutamic acid, lysine, and Lys-Glu peptide in DNA molecule was calculated. We demonstrated the interaction of the peptide and amino acids with DNA along the minor groove. The energy of DNA interaction with the peptide is higher than with individual amino acids. The peptide bonds increase the interaction of Lys-Glu peptide with DNA, which potentiates the biological effect on cell proliferation in organotypic culture of splenic cells.

We studied Molecular mechanisms of the retinoprotective effect of short peptides AEDG and KE. The peptides stimulate differentiation of neurons and retinal pigment epithelium cells and therefore can be considered as potential retinoprotective preparations for the treatment of age-related degenerative changes in the retina.

We studied molecular mechanisms of immunoprotective effects of two dipeptides, AB-O and R-1, on cultured human and rat thymic cells. Both dipeptides were shown to increase the expression of lymphocyte differentiation marker CD5 in thymic cells. Dipeptide AB-O induced T-cells precursor differentiation towards CD4(+)T-helpers and its effect was weaker than that of dipeptide R-1. Dipeptide R-1 stimulates differentiation of CD5(+) cells to mature T-helpers and cytotoxic CD8(+) T cells and hence can be considered as a bioactive substance possessing immunomodulator and antiallergic activity.

Expression of differentiation markers was found to be reduced during ageing of pancreatic cells. Tetrapeptide pancragen stimulates the expression of differentiation factors of acinar (Pdx1, Ptfl a) and islet of Langerhans (Pdx1, Pax6, Pax4, Foxa2, NKx2.2) cells in “young” and “aged” cultures. Differentiation of acinar and islet pancreatic cells induced by pancragen can be a mechanism underlying its anti-diabetic and anti-inflammatory effects. Thus, transcription factors that regulate differentiation of pancreatic cells are a pharmacological target for pancragen, which allows considering it as an effective tool in the treatment of diabetes mellitus and pancreatitis.

We developed a method of culturing and phenotyping of a monolayer of cells of the retinal tissue, thymus and spleen on the basis of organotypic culture. All characteristic types of neurons and fibroblasts were found in their microenvironment in the retinal cell monolayer. Lymphocytes, macrophages, and fibroblasts were verified in the monolayer of thymus and spleen cells. Histological staining, immunocytochemistry, and electron microscopy demonstrated the possibility of assessing the differentiation degree and functional activity of the cell monolayer. The developed technique preserves cell-cell interactions and a variety of cell types characteristic of the examined organ in the monolayer. This opens up new prospects for its application in basic research and in screening of different physiologically active substances.

The processes of differentiation, proliferation, and apoptosis were studied in a cell culture of human cortical thymocytes under the influence of short peptides T-32 (Glu-Asp-Ala) and T-38 (Lys-Glu-Asp). Peptides T-32 and T-38 amplified cortical thymocytes differentiation towards regulatory T cells, increased their proliferative activity, and decreased the level of apoptosis. Moreover, peptides under study stimulated proliferative and antiapoptotic activity of the mature regulatory T cells.