[Antioxidant activity of inhibitors of free radical reactions in the dressing materials for wound healing].

Antioxidant activity (AA) of inhibitors of free radical reactions (FRR) (dieton, mexidol, trypsin), aplied to the dressing material for wound healing was studied. In our work we used a model system containing suspension of laminated liposome, formed from fraction of total chicken yolk phospholipids. Lipid peroxidation (LPO) of liposome membranes was initiated by addition of Fe2+ ions. The kinetics of FRR was followed by coumarine-enhanced chemiluminescence (CL). It was found that AA of the inhibitors was determined by their ability to intersept aqueous and hydrofobic free radicals and chelate Fe2+ ions. Their ability to intersept radicals reduced in the following order: dieton > trypsin > mexidol. In addition we discovered unknown ability of mexidol to interact with Fe2+, that resulted in elemination of FRR catalyst. Investigating AA of the FRR inhibitors in the two-components mixture, consisting of dieton and mexidol, we observed the effect of multifunctionality: dieton, increased the duration of latent period of CL by intersepting lipid peroxyl radicals, while mexidol, decreased its value by interacting with Fe2+, i.e. mexidol masked the action of dieton. Investigating AA of two-components mixture, consisting of mexidol and trypsine, we observed the same effect of multifunctionality. In the two-component mixture, consisting of trypsine and dieton, the action of the inhibitors was found to be synergistic. All antioxidant properties of these FRR inhibitors were also preserved in the three component mixture. Hence, mixture components, dieton, mexidol and trypsin, possess high AA, that validates their use in dressing materials employed for wound healing.

[Comparison of the effects of laser and light-emitting diodes on lipid peroxidation in rat wound exudate].

The effect of laser and light-emitting diode radiation on lipid peroxidation in rat wound exudate was studied with the aim to compare the efficiency of coherent laser and incoherent light-emitting diode radiations. A model of aseptic wound in rat suggested by L.I. Slutskii was used. A He-Ne laser (632 nm) and a U-332B light-emitting diode were used in this study. The intensity of lipid peroxidation was estimated by the TBA assay. The antioxidative capacity of rat wound fluid was evaluated by means of chemiluminescent assays in two model systems: a) aqueous system with ABAP and luminol and b) in phospholipid liposome suspension with Fe2+ and cumarin. It was shown that exposure of rat wounds to both laser and light-emitting diode radiation decreased the concentration of TBA products and increased the antioxidative capacity of wound exudates, compared with the control group (without irradiation). The results obtained show that exposure of wounds to both laser and light-emitting diode irradiation causes a decrease in the oxidative stress in the rat wound fluid. No significant quantitative difference between the effects of laser and light-emitting diode irradiation was found.

[A comparison of the effects of laser and light-emitting diodes on superoxide dismutase activity and nitric oxide production in rat wound fluid].

The action of laser and light-emitting diode radiation in the visible region on the content of reactive nitrogen species and activity of superoxide dismutase in rat wound fluid was studied, and efficiency of action of coherent laser and incoherent light emitting diode radiations in the red region of the spectrum on the parameters under study was compared. A model of incised aseptic wounds in rats proposed by L.I. Slutskiy was used. A He-Ne laser (632 nm) and a Y-332B light emitting diode served as radiation sources. It was shown that (1) exposure of wounds to the visible light of both laser and light-emitting diodes causes dose-dependent changes in superoxide dismutase activity and production of nitrites and (2) the radiation coherence does not play any significant role in the changes of superoxide dismutase activity or nitrogen oxide formation by wound fluid phagocytes.

[A comparative study of the effects of laser and light-emitting diode irradiation on the wound healing and functional activity of wound exudate leukocytes].

The effects of coherent He-Ne laser and non-coherent light-emitting diode radiation on rat skin wound healing and functional activity of wound excudate leukocytes were compared. A comparative pathomorphological analysis showed that the He-Ne laser and light-emitting diode irradiation stimulated the transition of the inflammatory phase of the wound healing into the reparative (proliferative) and scarring phases sequentially. It was also detected that the functional activity of leucocytes changed in a dose-dependent manner. The leukocyte activity was found to be similar in the groups with laser and light-emitting diode irradiation. Thus, we can conclude that coherent laser and non-coherent light-emitting diode radiation have very close effects on wound healing and activity of wound exudate leukocytes, and coherence is not required for this activity.

[The role of blood plasma porphyrins in the effects of He-Ne-laser on human leukocytes].

The He-Ne-laser induced effects in human blood leukocytes in the presence of autologic plasma were investigated. Experiments were performed in two ways: (1) He-Ne-laser irradiation of cells in the presence of autologic plasma or (2) laser irradiation and subsequent addition of autologic plasma to the cell suspension. The concentration dependencies of plasma additions were evaluated. To obtain different concentrations of porphyrins in plasma samples, we either diluted the samples with PBS or selected patients with different porphyrin plasma content. The effects of He-Ne-laser irradiation were characterized by the maximum effect dose (Dmax) of irradiation and the degree of maximum cell activation (Amax, priming index). In the first series of experiments, we irradiated leukocytes in the presence of autologic plasma taken from patients with pneumonia and bronchial asthma. It was found that Dmax decreased with increasing porphyrin concentration in plasma. It was observed that, at low porphyrin concentrations, Amax increased severalfold with increasing photosensitizer concentration. At a porphyrin concentration of 0.46 pmol a decrease in Amax was detected as the porhyrin concentration increased. The same effects were revealed at high doses of laser irradiation. Very similar effects were found in experiments with the addition of irradiated plasma to cells. However, the Amax value was considerably less compared to that after irradiation in the presence of plasma (160% vs. 230 - 270% upon combined irradiation of cells and plasma). The Dmax value was higher in the series of experiments in which plasma was irradiated separately. The results suggest that laser-induced leukocyte activation can be mediated by blood plasma porphyrins and the products of lipid peroxidation formed as a result of porphyrin-photosensitized lipid oxidation.

[The role of lipid peroxidation products in the effect of He-Ne laser on human blood leukocytes].

The role of lipid peroxidation products formed in membranes of human blood leukocytes after irradiation with He-Ne laser was studied. It was found that low-intensity laser irradiation (0.3-1.6 J/cm2) leads to both cell activation and an increase in the content of lipid peroxidation products. The intensity of lipid peroxidation was analyzed by estimating the amount of TBA reactive products and lipid diene conjugates. Irradiation in the presence of an exogenous photosensitizer (protoporphyrin IX) enhanced the phenomena observed. The use of antioxidants (tocopherol and ionol) completely eliminated the laser-induced effects (changes in leukocyte activity and accumulation of lipid peroxidation products). These results can be explained by the fact that laser irradiation leads to the activation of lipid peroxidation in leukocyte membranes, which in turn enhances the response of cells to the stimulus (priming).

Changes in superoxide dismutase activity and peroxynitrite content in rat peritoneal macrophages exposed to He-Ne laser radiation.

The formation of reactive oxygen and nitrogen species by rat peritoneal macrophages induced by a low-intensity He-Ne laser radiation (LR) was studied in this work. It was found that the formation of reactive oxygen species, nitric oxide, and peroxynitrite as well as changes in the activity of superoxide dismutase (SOD) depended to a large extent on the LR dose. In particular, it was found that activation of SOD at low LR doses was accompanied by nitric oxide level increase, while the level of peroxynitrite showed no significant changes. On the other hand, an enhanced LR dose inhibited the enzyme, and this was accompanied by peroxynitrite accumulation. All the measurements were carried out the day after LR treatment. The revealed regularities consequently demonstrate the existence of a deferred LR action on macrophages associated with the production of reactive oxygen and nitrogen species.

[Registration of surface fluorescence of endogenous porphyrins during low-level laser therapy of gastric and duodenal ulcers]. / Izmerenie poverkhnostei fluorestsentsii éndogennykh porfirinov v protsesse lazeroterapii iazv zheludka i dvenadtsetiperstnoi kishki.

With the use of the developed opto-fiber spectroscope and gastroscope, the intensity of fluorescence from the ulcer surface into the gastrointestinal tract was measured in the range of 670-690 nm under the He-Ne laser (= 632.8 nm) illumination. Surface fluorescence data were collected with the use of a special diagnostic fiber with a total diameter of about 2 mm, which combines some illuminating and receiving fibers. To do the measurements, the diagnostic fiber was brought into a slight contact with the mucosa surface. The quantitative results were calculated with the use of the special fluorescent contrast coefficient (Kf), which takes into account the backscattered light as well. After the fluorescent diagnostic procedure, the low-level laser therapy was applied to all patients. It was found that: first of all, during the laser medical cure the registered fluorescence for the patients with a good dynamics of treatment had an evident tendency to a decrease, and the coefficient Kf for observed ulcers tended to approach the value obtained from normal mucosa (Kf), which indicates the normalization of the porphyrin content in the tissue. Second, the retrospective analysis of the efficacy of laser therapy versus initial values of Kf showed that, for the patients with the initial values Kf = 0.2, the efficiency of the laser treatment was the highest: the relation between the patients with good treatment results and bad ones was 4:1 (more than 80%). For the initial Kf = 0.2-0.3, this relation was 3:1 but for Kf = 0.4-0.5 it was 2:3. The differences in the efficiency of the low-level laser therapy, except the cases of the cancer in the ulcers, may be due to different content of porphyrin in the tissue: at high concentrations of the photosensitizer in the ulcer, the effect of the overdosing could be realized. In this case, another technology of laser illumination is needed. The results are discussed in terms of the free-radical conception of the stimulating effect of laser radiation.

[The use of flavonoid antioxidants in the complex treatment of patients with peripheral vitreo-chorioretinal dystrophies and dystrophic retinal detachment]. / Primenenie flavonoidnykh antioksidantov v kompleksnom lechenii bol'nykh s perifericheskimi vitreokhorioretinal'nymi distrofiiami i distroficheskoi otsloikoi setchatki.

Parameters of lipid peroxidation (LP) and antioxidant activity (AOA) of tear and blood plasma were examined in 22 healthy subjects (44 eyes) as well as in 33 patients with peripheral vitreochoreoretinal dystrophies (PVCRD--60 eyes), in 32 patients with non-operated dystrophic retinal detachment (DRD--34 eyes) and in 135 patients with operated retinal detachment, stable visual functions and with the postoperative period ranging from 4 months to 10 years (137 eyes). The results denoted a lower tear AOA (on the average by 35%) in patients with DRD and PVCRD, whereas, the blood plasma AOA or LP products' content remained unchanged. It is indicative of a local nature of metabolic impairments, specifically, of impairments in the system of antioxidant protection of the eye. Flavonoid antioxidants (dikvertin and ginkgo biloba) reduced the content of LP products, and induced the AOA in tear and blood plasma in patients with PVCRD and retinal detachment; they also improved the visual functions in patients with operated retinal detachment. Therefore, the flavonoid antioxidants can be recommended for adding to the complex treatment of PVCRD and DRD for the purpose of improving and stabilizing the visual functions and for neuroprotection.

[Molecular and cellular mechanisms of the low intensity laser radiation effect]. / Molekuliarno-kletochnye mekhanizmy deistviia nizkointensivnogo lazernogo izlucheniia.

The main aspects of the free radical conception of the molecular and cellular mechanisms of the stimulating action of low-intensity radiation in the red region of the spectrum were considered. These are: (1) Primary acceptors of incident radiation are endogenous porphyrins, which may act as photosensitizers giving initiator-radicals for secondary free radical reactions. (2) Target cells for light irradiation during quantum therapy may be blood leukocytes, fibroblasts, keratinocytes, endotheliocytes, etc. (3) The initiation of the secondary free radical reactions due to lipid peroxidation of cell membranes (in particular, of leukocytes) brings about an increase in ion permeability including that for calcium. The increase in intracellular calcium concentration leads to phagocytes priming, i.e., to increased production of reactive oxygen species (ROS) under subsequent stimulation of the cell. (4) Photosensitized generation of ROS in the cytoplasm of some cells induces a free-radical activation of synthesis of proteins, the most significant in the light of the present concept being the de novo synthesis of inducible NO-synthase, superoxide dismutase, and various cytokines. The experimental evidence for the basic statements of the conception of free radical mechanisms for the stimulating action of low-intensity laser and noncoherent radiations is presented. A relation between the primary mechanisms of the stimulating action of light and the secondary effects that determine the sanative effect of quantum therapy in the process of wound healing (bactericidity, cell proliferation, and improved microcirculation) was established. Moreover, it was shown that nitrosyl complexes of heme proteins, such as hemoglobin and cytochrome c, are the primary chromophores of laser radiation. Upon irradiation, they can easily dissociate to produce free nitric oxide. In turn, released nitric oxide may be responsible for blood vessel relaxation and activation of mitochondrial respiration. This phenomenon is just observed during phototherapy by means of low-intensity laser radiation.

Photobiological principles of therapeutic applications of laser radiation.

Laser therapy based on the stimulating and healing action of light of low-intensity lasers (LIL), along with laser surgery and photodynamic therapy, has been lately widely applied in the irradiation of human tissues in the absence of exogenous photosensitizers. Besides LIL, light-emitting diodes are used in phototherapy (photobiostimulation) whose action, like that of LIL, depends on the radiation wavelength, dose, and distribution of light intensity in time but, according to all available data, does not depend on the coherence of radiation.

Comparative study of antioxidant properties of immunoregulatory peptides.

We studied antioxidant properties of immunofan, bursin, cyclobursin, thymopoietin II fragment, glycine, and Siberian ginseng. Experiments were performed in 2 model systems: Fe(2+)-induced oxidation of multilamellar phospholipid liposomes in a heterogeneous water-lipid system and oxidation of luminol induced by alpha,alpha'-azo-bis(isobutyramidine dihydrochloride) in a homogenous aqueous system. By the ability to entrap lipid peroxyl radicals, antioxidant activity of substances decreased in the following order: Siberian ginseng extract>bursin>cyclobursin>thymopoietin II fragment>immunofan, glycine. Siberian ginseng extract and thymopoietin II fragment interacted with Fe(2+), which contributed to elimination of catalyst of lipid peroxidation from the system. The ability of substances to interact with aqueous peroxyl radicals and luminol radicals decreased in the following order: Siberian ginseng extract>thymopoietin II fragment>immunofan>glycine, cyclobursin, bursin. Substances with high antioxidant activity improved the state of the endogenous antioxidant system and protected cells from oxidative stress. They entrapped reactive oxygen species formed in the cytoplasm, modulated free radical processes, and regulated the synthesis of bioactive molecules.

[Effect of low intensity laser light in the red range on macrophage superoxide dismutase activity]. / Vliianie nizkointensivnogo lazernogo izlucheniia krasnogo diapazona na aktivnost' superoksiddismutazy makrofagov.

The effect of lipopolysaccharides (LPS) and low-power laser radiation on nitric oxide (NO) production and the activity of superoxide dismutase (SOD) in rat peritoneal exudate macrophages was investigated. It was found that LPS increased NO production and SOD activity in macrophages in a concentration-dependent manner. The maximal activity of SOD was more than 100 times greater than in the control and was achieved at an LPS concentration of less than 10 ng/ml. The maximal production of NO was more than 250% of the control and was achieved at an LPS concentration of 100 ng/ml. The presence of cycloheximide, a transcriptional synthesis inhibitor, in conditioned media almost completely abolished SOD activation and NO production. This implies that the LPS-induced increase in SOD activity and NO production were determined by the activation of de novo protein synthesis (SOD and inducible NO synthase). The irradiation of macrophages by red light resulted in a dose-dependent increase in NO production and SOD activity. The incubation of irradiated cells in the presence of 10 microM cycloheximide abolished the increase. The presence of antioxidants (mexidol and ascorbate) also significantly inhibited the laser-induced activity of macrophages. Thus, laser irradiation of cells in the red range activates the synthesis of SOD and inducible NO-synthase de novo due to photosensitized initiation of free radical reactions.

[Antioxidant properties of 3-oxypyridine analogues: mexidol, emoxipin, proxipin]. / Antioksidantnye svoistva proizvodnykh 3-oksipiridina: meksidola, émoksipina i proksipina.

Using three chemiluminescent model systems of oxidation (suspension of phospholipid liposomes, a geous solution of haemoglobin-hydrogen peroxide-luminol and a geous solution 2,2'-azo-bis-(2-methylpropionamidine)dihydrochloride-luminol) the antioxidant activity and mechanism of antioxidant action of three 3-oxypyridine analogues: (mexidol, emoxipin and proxipin) were studied. These compounds were shown: a) to interact with catalitically active two valency iron ions (Fe2+), that causes elimination of ions from the model system; b) to scavenge reactive oxygen species and/or luminol radicals produced in the model systems. Their activity reduced in the following order: mexidol > emoxipin > proxipin. The antioxidant activity of 3-oxypyridines may underline known clinical effects of these compounds.

Mechanism of therapeutic effect of low-intensity infrared laser radiation.

The effect of infrared low-intensity laser irradiation on functional activity of blood polymorphonuclear leukocytes was studied in vitro. A dose-dependent priming of polymorphonuclear leukocytes induced by infrared low-intensity laser irradiation was demonstrated. Similar effects were also observed in the presence of the photosensitizer photosense.

Effect of the combined action of flavonoids, ascorbate and alpha-tocopherol on peroxidation of phospholipid liposomes induced by Fe2+ ions.

The effect of alpha-tocopherol, ascorbate, rutin and dihydroquercetin on chemiluminescence (CL) accompanying the Fe2+-induced peroxidation of unsaturated fatty acids in phospholipid liposomes has been investigated. The amplitude of CL decreased and the latent period increased in the presence of alpha-tocopherol, rutin and dihydroquercetin which is typical of peroxide radical traps. Ascorbate also reduced the CL amplitude but only at small concentrations up to about 4 microM. A further increase of ascorbate concentration had a negligible effect on the amplitude. At the same time, the latent period in CL development increased with the growth of ascorbate concentration, apparently, as a result of recycling of divalent iron oxidized in the course of lipid peroxidation. The effects of rutin and dihydroquercetin on the liposomal CL in the presence of alpha-tocopherol and ascorbate in all experiments were almost the same as when these compounds were added individually. The antioxidant effects were merely summed up without any mutual enhancement or inhibition of each other's action.

[Serum antioxidative activity]. / Antioksidatnaia aktivnost' syvorotki krovi.

Model systems used in the determination of serum antioxidative activity (AOA), which differ both in the way of generating free radicals and in the mode of their detection, are clinically analyzed. The specific features and potentialities of the model systems developed at the authors' laboratory are characterized. These included yolk lipoprotein suspensions, liposomal suspensions formed from total phospholipid fraction, the hemoglobin-hydrogen peroxide-luminol system. The investigations show that most model systems for determining serum AOA contribute to the water soluble interceptors of free radicals (ascorbate, urate, plasma proteins, etc.), chelating and oxidative agents of catalytically active Fe2+ (ceruloplasmin, transferrin, albumin, etc.). The serum AOA levels measured with different model systems vary with the body's status. To determine serum AOA and the contribution of major endogenous antioxidants and inhibitors of free radical reactions may be a basis for the goal-oriented use of exogenous antioxidants in the therapy of a great variety of diseases.

[Free radical mechanisms of low-intensive laser radiation]. / Svobodnoradikal'nye mekhanizmy stimuliruiushchego deistviia nizkointensivnogo lazernogo izlucheniia.

A hypothesis of free radical mechanisms of stimulating action of low-intensive laser radiation (LILR) used for therapy of a variety of inflammatory diseases is formulated. The main points of the above hypothesis are as follows. Endogenous porphins are a LILR chromofor in the red band (lambda = 632.8 nm). Light absorption induces the production of initiating radicals that are involved in subsequent free radical reactions, in lipid peroxidation in particular. Modified lipid peroxidation in the cell membranes causes an increase in ion permeability, including that for Ca2+. The higher levels of Ca2+ in the leukocytic cytosol result in Ca(2+)-dependent cellular priming, which appeared as the increased cell functional potential and which is seen in subsequent leukocytic stimulation of the greater production of prooxidants and other biologically active products. These products include nitric oxide and a number of cytokines involved in the regulation of microcirculation. The paper presents experimental findings that can be regarded as evidence for some points of the above hypothesis which are used to provide a chain of events underlying the free radical mechanisms of stimulating action of low-intensive laser radiation.

[The modulation of phagocyte oxygen metabolism by recombinant cytokines and a complex of natural cytokines]. / Moduliatsiia kislorodnogo metabolizma fagotsitov rekombinantnymi tsitokinami i kompleksom prirodnykh tsitokinov.

The data on the modulating function of cytokines on the oxygen-producing function of peritoneal exudate cells of rats are presented. As priming agents, recombinant cytokines IL1 beta and TFR beta 1, as well as the natural complex of cytokines, were used. The priming action of cytokines was studied by changing in the production of active forms of oxygen by peritoneal exudate cells of rats, stimulated with opsonized zymosan, by the method of luminol-dependent chemiluminescence. The study revealed that IL1 beta and the natural complex of cytokines primed peritoneal exudate cells for the production of active forms of oxygen. The maximum value of the prestimulation index was 1.9 +/- 0.1 and 2.95 +/- 0.27 respectively. The preincubation of peritoneal exudate cells of rats with TFR beta 1 led to the pronounced inhibition of the intensity of the chemiluminescent response of cells. The prestimulation index did not exceed 1.06 +/- 0.1. Moreover, as revealed with the use of the probe Fura-2/AM, in the process the prestimulation of phagocytes with the natural complex of cytokines the intracellular concentration of calcium increased from 0.86 +/- 0.15 to 1.86 +/- 0.2 microM/ml. The mechanism of the prestimulation of peritoneal exudate cells of rats cytokines seems to be calcium-dependent.