Proof of stimulating effect of dalargin of the process of cell division through opiate receptors

The role of opiate receptors on cell division in corneal epithelium during administration of dalargin was analysed. Naloxone injection/200 micrograms/kg/decreased MI two times, DNA-synthesis 1.4 times over 24 hours. Naloxone prevented dalargin effect on cell proliferation. Another testimonies of dalargin opiate-binding mitogenic effect were the results of the study with dalargin analogues. They are agonists of opiate receptors too. These drugs, as well as dalargin, in a dose 10 micrograms/kg increased DNA-synthesis 1.5 times, MI and MIK 2.2 times. It turned out, that the administration of another two analogues of dalargin, which are not ligands of opiate receptors, probably do not cause an adequate increase of DNA-synthesis and mitotic index.     

Effect of a stable analog of leu-enkephalin, dalargin, on the cell division of the corneal epithelium in white rats

The effect of Leu-enkephalin analog--dalargin--on the corneal epithelium proliferation has been studied in white rats. 10 microliter dalargin per 1 kg body weight were administered intraperitoneally at 8 a.m. The mitotic index (MI), DNA synthesis cell index and label intensity (LI) were determined every 4 hours over a 24-hour period. The results obtained demonstrate that dalargin stimulates DNA synthesis in cells throughout the entire period of action. MI increased only 4, 8, 12 hours after dalargin administration. Mean daily DNA synthesis cell index and MI increased 2.1-fold and 3.1-fold, respectively after dalargin administration. It is suggested that dalargin activates the cell division processes by speeding up mitosis, shortening the premitotic period, accelerating the speed of the DNA synthesis and increasing cell proliferation pool.     

Effect of the hexapeptide dalargin on ornithine decarboxylase activity in the duodenal mucosa of rats in experimental duodenal ulcer

The effect of an opioid antiulcerogenic hexapeptide dalargin on ornithine decarboxylase activity of duodenal mucosa has been studied in rats with experimental duodenal ulcers induced by cysteamine. The intraperitoneal injection of 12.5 micrograms/kg of dalargin inhibited ulcerogenesis and activated the enzyme. The effect of the peptide was antagonized by an opiate antagonist naloxone. 5000 micrograms/kg of dalargin failed to inhibit the ulcer formation or to activate ornithine decarboxylase. Since ornithine decarboxylase activation is a marker of intensified cell proliferation and tissue regeneration, our results suggest that the antiulcerogenic effect of dalargin is due to the enhancement of duodenal mucosa regeneration.     

Effects of opioid receptor ligands on DNA synthesis and histamine contents in the gastric mucosa and blood of white rats

Using autoradiographic study with 3H-thymidine and spectrofluorometric method the authors studied the influence of opioid receptors' ligands on the DNA synthesis in the stomach epithelium histamine content and the blood. Leu-encephalin, B-endorphin, dalargin, napoxone were administered intraperitoneally to male rats. The dose of injection was 0.1 ml per 100 g body weight. It was observed that B-endorphin and dalargin 1.5-1.6 fold increased the quantity of DNA-synthesised nuclei in the epithelium of fundal stomach section. Leu-encephalin and dalargin increased the histamine concentration in the stomach, at the same time dalargin caused a rapid decrease of histamine concentration in the blood. Naloxone also decreased histamine concentration in the stomach. The obtained results are being discussed in connection with dalargin therapy of ulcerous diseases.     

Oxygen tension affects terminal differentiation of corneal limbal epithelial cells

Oxygen concentration has been shown to be crucial in the proliferation and differentiation of various types of cells, while the impact of oxygen tension on the lineage commitment of epithelial cells remains elusive. In this study, we investigated the effect of hypoxia on the differentiation of corneal limbal epithelium using an ex vivo squamous metaplasia model. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of human corneal limbal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The Notch signaling pathway was activated in hypoxic cultures, whereas the p38 MAPK signaling pathway was downregulated. The addition of Notch inhibitor under hypoxic conditions restored the activation of p38 MAPK and resulted in the recidivation of limbal epithelial cells to epidermal-like differentiation. Moreover, the epidermal-like differentiation of rabbit limbal epithelial cells was also blocked under hypoxic conditions in corneal epithelial cell sheets engineered ex vivo. We concluded that hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal limbal epithelial cells. Hypoxia coupled with air exposure can be used in the tissue engineering of corneal limbal epithelium.     

Effects of dalargin on the proliferative processes of the gastric epithelium under repeated action of different stressors]

Using radiography with H-thymidine method we studied the synthesis of DNA process in pyloric parts of stomach epithelium in white rats, which have been five-fold effected by different kinds of stressors against a background of dalargin injections. In the first hour after animals were stressed, DNA synthesis was depressed. Dalargin injections caused DNA synthesis normalization in the first hour after hypoxia and hyperthermia. Since 24 hours after hyperthermia and immobilization against a background of dalargin injections the normalization of DNA synthesis took place, and after hypoxia the post-stressing IMN activation was growing week. One of the mechanisms of dalargin correction of DNA synthesis breach under the influence of stressors is a stabilization of noradrenaline and histamine concentration in tissue of the stomach.     

Effects of dalargin on hemodynamics of anesthesized rats during truncal vagotomy

I/v dalargin injection (20-25 g/kg) to narcotized rats in case of total myoplegia effectively protect hemodynamic changes under nociceptive stimulation. Bilateral truncal vagotomy partially decrease the protective effect of dalargin. Protective effect of the medicine results in activation of central and peripheral opioid receptors.     

Activation of cell division and nucleic acid synthesis in the corneal epithelium of white rats undergoing repeated exposure to stress

A study was made of the influence of single and repeated stress on the mitotic activity and synthesis of DNA and RNA in the corneal epithelium of albino rats. The stressors used were fixation on the back during one hour, the influence of 1.5-h hyperthermia (41.5 degrees C), and the influence of hypoxia (4h, 9000 m). Single stress induced the depression of the mitotic activity. The autography tests of DNA synthesis were made constantly. Repeated stress (5 exposures) entailed activation of the synthesis of the nucleic acids and cell division, evidence of a structural trace of stress, in the epithelial tissues.     

Comparative effect of opioid receptor ligands on cell division in the epithelium of the tongue in white rats

Beta-endorphin, leu-enkephalin, dalargin and naloxone influences on cell division have been studied in tongue epithelium of white rats. The preparations were administered at a dose of 0.1 ml per 100 g body weight as a 2.10(-9) M solution. Cell division was studied 24 hours after administration. beta-endorphin, leu-enkephalin, dalargin and naloxone caused a 1.5-1.7-fold increase in the number of DNA-synthesizing nuclei, which was accompanied by an adequate rise in mitotic index in experiments with dalargin.     

Molecular mechanisms of antioxidant action of dalargin on the liver in experimental cholestasis]

Changes of antioxidant activity of dalargin in the liver after naloxone (100 micrograms/kg) administration were examined in experiment on 144 rats with cholestasis. It was found that dalargin inhibited the activity of xanthine oxidase by 32-37% in different time periods after the injection. Dalargin and naloxone, when used in combination, had no effect on the enzyme activity. Glutathione-S-transferase activity rose by 38.0% and 21.8% on hour 1 and 3 after the injection, respectively, while simultaneous injection of dalargin and naloxone induced no changes in the enzyme activity after 1 hour, though decreased it by 36.8% and 26.4% on hour 3 and 5, respectively. Dalargin inhibited lipid peroxidation by 29-35%, simultaneous injection of dalargin and naloxone raised lipid peroxidation by 109.2%, 80.7% and 25.7% after 1, 3 and 5 hours, respectively. Dalargin injection elucidated a marked tendency to lowering of blood release of the liver-specific enzymes histidase and urokaninase in line with enhancement of their activity in the liver. A combined injection of dalargin and naloxone promoted high release of histidase and urokaninase in blood and did not change histidase activity in the liver in all cases. Urokanidase activity elevated in 5 hours. It was noticed that dalargin raised leu-enkephalin levels in the liver 3.5-fold 1 h after the injection. The reduced dalargin antioxidant effect coupled with naloxone pretreatment demonstrated indirect action of the neuropeptide on the liver via neuron receptors of the liver.     

Selenium Compound Protects Corneal Epithelium against Oxidative Stress

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.     

Participation of central and peripheral mu- and delta opiate receptors in anti-arrhythmia action of enkephalins]

It was found, that injection of delta-receptors agonist dalargin before the occlusion of left anterior coronary artery in rats prevented the decrease of ventricular fibrillation threshold (VFT). An injection of naloxone in dose 0.5 mg/kg (for the blockade of mu-receptors only) had no influence on the VFT. Naloxone in dose 1 mg/kg (for the blockade peripheric mu- and delta-receptors) decreased VFT. An intraventricular infusion of dalargin (10 mkg) induced bradycardia and an increase of VFT. It was assumed that anti-arrhythmic effects of enkephalins in acute myocardial ischemia could be realized by an activation of peripheric delta-receptors and central mu-receptors.     

The normalizing effect of dalargin on the glucocorticoid and opioid levels of the blood in CBA and C57Bl/6 mice undergoing footshock stress]

Radioreceptor investigations showed that it is impossible to interpret changes in the activity of opioid receptor ligands of mu and delta types in plasma of mice under footshock stress (FSS) as activation or depletion of opioid system (OS). There occurred qualitative changes characterized by interstrain differences (ID). These are also typical for dynamics of development of FSS effects on blood corticosterone levels. Administration of dalargin diminishes qualitative and quantitative changes in OS function at different periods after FSS preventing early rise of corticosterone levels after the exposure. In spite of common features in dalargin action on opioid and steroid metabolism in mice of both strains, ID are also present.     

Modulating effect of opioid peptides on hemopoiesis in stress

The influence of leu-enkephalin and dalargin on the blood system was studied during immobilization stress in mice. The early transmitted reactions of the peripheral blood were shown to decrease upon single drug infusions after immobilization. At later terms the activation of bone marrow hematopoiesis was not registered in mice receiving opioid peptides in contrast to the control animals. It correlates with drug-induced decrease in the mitotic activity of bone-marrow cells. Suppressive effect of opioids on hematopoiesis during stress was connected with their decreasing effect on corticosteroid level in the animal plasma. The latter can suggest indirect influence of enkephalins on bone marrow hematopoiesis in immobilization stress.     

Ultrastructure of cardiomyocytes in the peri-infarct zone during the treatment of experimental myocardial infarct in rats using the hexapeptide dalargin

The experiments on white rats with induced myocardial infarction have studied the influence of dalargin on the infarction size and peri-infarction zone ultrastructure. 24 hours later the decrease in the infarction zone size was detected in rats who had received dalargin in a dose of 50 and 100 micrograms/kg. In the peri-infarction zone the increase in glycogen quantity, the lower degree of lipid infiltration, the increase in mitochondrial number and mitochondrial energy effectiveness coefficient were noted, as compared to control animals. Sarcolemma of cardiomyocytes from the peri-infarction zone in rats on dalargin was impermeable for colloidal lanthanum. The decrease in the infarction size under the effect of dalargin is explained by its influence on the survival of cardiomyocytes in the peri-infarction zone.     

Pharmacological correction of experimental alcoholic embryopathy

In the experiments on the progeny of ethanol-exposed rats it was shown that consumption of 15% alcohol instead of water during pregnancy resulted in the worsening of shuttlebox avoidance learning and decrease in succinate dehydrogenase activity in the visual and sensorimotor cortex. These data are indicative of cerebral hypoxia during embryogenesis. The injection of synthetic opioid peptide dalargin during critical periods of development (at the end of embryogenesis and early ontogeny) prevented partially the disturbances of higher nervous activity and tissue breathing which were induced by alcohol intoxication. Possible mechanisms of positive dalargin effect are discussed.