The probable mechanism of reparative effect of eukaryotic DNA oligonucleotides as agonists Toll-like receptors 9 on the model of drug Derinat

A. A. Klimovich , Institute of Experimental Biology and Medicine VGMA them. NN Burdenko

Enhancing the natural reparative effect in damaged tissues, is probably much depends on the activity of macrophages, and has a wide phenotypic plasticity. It can be assumed that the possibility of this effect on activity by agonists of Toll-like receptors 9 (TLR 9). In the role of TLR agonists may act 9 fragments of eukaryotic DNA oligonucleotides [5] terminate unmethylated motif CpG.

Key words: Toll-like receptors 9, unmethylated CpG motifs of eukaryotic DNA, phenotypic plasticity macrophages, TGF-β, PDGF, angiogenesis.

A leading contemporary reparations hypothesis is that the immune system is involved in switching between the regeneration and fibrous fibroid degeneration. Reparative processes unfolding as the attenuation of the acute phase of inflammation, reduced to regeneration and fibroplasia. Fibroplasia - a growth of fibrous tissue replacement defect. Regenerative process - is to replace damaged cells by cells of the same type. Proliferation is the final phase of inflammation, which provides reparative tissue regeneration at the site of alteration focus (damage). Proliferation is developing from the beginning of inflammation together with signs of alteration and exudation. At the reparative processes cell regeneration or fibroplasia is achieved as activation of proliferation and apoptosis restriction (programmed cell death) of cells.

Full development, proliferation of connective tissue and organ, cellular elements is achieved after "clean" areas of damage from cellular detritus and pathogens inflammation of tissue macrophages and neutrophils, and rare. This process may be stimulated by exposure to 9 TLR (Toll-like receptor 9) macrophages via to its specific ligand unmethylated motif CpG, oligonucleotides [6] contained in eukaryotic DNA, which includes of a commercial drag Derinat ®.

Unmethylated CpG motifs are found in eukaryotic DNA primarily in CpG - islands. The total content of unmethylated CpG motifs is in which more than 50%. All house-keeping genes (the genes that are always active) and 40% tissue-specific genes (tissue-specific genes) contain CpG - islands.

Molecule Derinat® (sodium salt of deoxyribonucleic acid) is an oligodeoxynucleotide (ODN) eukaryotic origin with a molecular weight up to 500 kDa, derived from salmon milt of fish species. Given that the mass of one nucleotide is about 345 Daltons, the number of nucleotides in the molecule of the starting substance is evaluated as 1500. Considering that the length of most common ODN measured in base pairs, the pairs 20-30 and is then Derinat ® molecule consists of an average of 750 base pairs. In the available literature suggests that the mechanism of action of the drug Derinat® on immunocompetent cells may be mediated by TLR 9. Drag Derinat® is preferably includes short and medium chain DNA ending on average, at least 50% motif CpG. Macrophages activated by Derinat® is rapidly cleared from the surface of the wound from detritus and infectious agents. The proliferation precedes the formation of neutrophilic and monocytic barrier, which are formed at the periphery of the zone of alteration. This process is also accelerated by stimulation of macrophages TLR 9, by Derinat® [1, 2, 3]

Recovery and replacing damaged tissue begins with exit from the blood vessels molecules of fibrinogen and the formation of a fibrin, which forms a kind of grid, for the subsequent frame cell multiplication. Already on this frame are distributed in the hearth repair quickly formed fibroblasts. Division, growth and migration of fibroblasts are possible only after binding to fibrin or collagen fibers. Later in the emerging connective tissue begin to grow blood vessels, is a process of neoangiogenesis. The most active and universal mediator of fibrosis and angiogenesis, is a transforming growth factor β (TGF-β), which is synthesized by macrophages M2 activated through TLR9, motives CpG, contained in Derinat®,

Macrophages, depending on the stimulation and the presence, or absence, of an infectious environment can excrete as pro- and anti-inflammatory cytokines. In these conditions the stimulation of resident macrophages and blood monocytes by drag Derinat® leads to macrophage phenotype polarization to the direction of M2. TGF-β is an anti-inflammatory cytokine. Macrophages start to secrete it when there is no stimulation of scavenger receptors and lectin receptors, there is not stimulation of macrophage by infectious agents. In an aspect of the repair and regeneration, this means that the factor TGF-β released by macrophages in a "clean wounds." In a "clean wound" TLR9 stimulation of macrophages is results in the production of TGF-β, the target cells which, in particular, are fibroblasts. Expression of the high affinity receptor for TGF-β is widespread in various tissues, therefore increase of its synthesis by macrophages resident in various organs amplify the repair and regeneration processes in practically all tissues.

TGF-β stimulates fibrogenesis (stimulation of collagen synthesis by fibroblasts), stimulates the synthesis of fibronectin, angiogenesis. TGF-β is achemoattractant fibroblasts and inhibitor of proteolysis. At low a concentration he is stimulates proliferation through the induction of platelet derived growth factor (PDGF).

Macrophages stimulated via Derinat® by TLR 9 also is synthesized transforming growth factor α (TGF-α), epidermal growth factor (EGF), endothelial cell growth factor (VEGF), platelet factor (PDGF), hepatocyte growth factor (HGF). Target cells for these various factors are epithelial cells, hepatocytes, fibroblasts derived from skin, smooth muscle cells, synoviocytes, and vascular endothelium.

Activated macrophages by TLR 9 also are secreting granulocyte-monocyte colony stimulating factor (GM- CSF) and granulocyte colony stimulating factor (G-CSF). Synthesis of these factors is explained Derinat® hematopoietic effect in the treatment of leukopenia result various factors myelotoxic (ionizing radiation, chemicals - benzene, toluene, arsenic and chemotherapeutic agents).


Repair and regeneration is dependent

1. The rate of wound cleansing of detritus.
2. on the degree fibrosis activation factors, angiographic, epiteliogeneza.

Key cells in both cases are resident (tissue) M1 or M2 macrophages. Action Derinat® effect on both phases of wound healing process. Acting on TLR9 of macrophages in wound cleansing phase, is amplified phagocytosis by macrophages of tissue detritus, infectious agents (bacteria). After cleansing the wound, when the impact of the environment on infectious macrophages by PAMP (pathogen-associated membrane patterns) via scavenger receptors and lectin receptors is absent, stimulation of TLR9 by Derinat® leads to increased synthesis of TGF-β, anti-inflammatory factor and the most powerful stimulator of repair and regeneration. In applying Derinat® on stage wound cleansing, is amplified phagocytic activity of macrophages that promotes the preparation of the wound repair process. In case of a clean wound or metabolically inhibited process repair and regeneration, are such as diabetes or hypoxic tissue in post-thrombotic syndrome, application Derinat® amplifies synthesis macrophages of TGF-β and accelerating the process of repair and regeneration.


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