Nicotinamide prevention in diabetes-induced alterations in the rat liver.

Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.

Matrix Metalloproteinase-9 is Involved in the Fibrotic Process in Denervated Muscles after Sciatic Nerve Trauma and Recovery.

Fibrosis of the injured muscles is a problem of recovery from trauma and denervation. The aim of the work was to investigate the interconnection of matrix metalloproteinase-9 (ММР-9) activity in denervated muscles with fibrosis and to estimate its role in nerve restoration by the epineurial suture, fibrin-based glue, and polyethylene glycol hydrogel. The activity of matrix metalloproteinases was estimated by gelatin zymography. Collagen density in muscles was determined histochemically. An increased level of the active MMP-9 is associated with the fibrous changes in the denervated skeletal muscles and after an epineurial suture. The use of fibrin glue and polyethylene glycol hydrogel resulted in a lower level of collagen and ММР-9 activity, which may be a therapeutic target in the treatment of neuromuscular lesions, and has value in fibrosis analysis following microsurgical intervention for peripheral nerve reconstruction.

Modulatory effects of vitamin B3 and its derivative on the levels of apoptotic and vascular regulators and cytoskeletal proteins in diabetic rat brain as signs of neuroprotection.

BACKGROUND: Beneficial effects of nicotinamide (NAm) and its derivates have been earlier shown in animal models of diabetes mellitus (DM), but the mechanisms of their neuroprotective activities are still largely unknown. The aim of the present study was to investigate if NAm and conjugate of nicotinic acid with gamma-aminobutyric acid (N-GABA) are able to modulate expression levels of apoptosis regulators, angiogenesis-related molecules, and specific cytoskeletal proteins in diabetic rat brain. METHODS: After six weeks of streptozotocin induced type 1 DM, rats were daily administered either by NAm (100 mg/kg) or N-GABA (55 mg/kg) intraperitoneally for two weeks. Protein levels were assessed by western blot and immunohistochemistry. RESULTS: Both NAm and N-GABA down-regulated NF-κB and Bax levels in diabetic rat brain, suggesting their anti-apoptotic activities. Tested compounds normalized VEGF and nNOS contents improving pro-angiogenic signaling reduced by hyperglycemia. Western blot showed marked up-regulation of astroglial marker GFAP and lowering neurofilament protein levels in DM group, confirmed immunohistochemically, indicating the development of reactive astrogliosis as a major response to diabetes-induced neurodegeneration. NAm had no effects on GFAP and Nf-L levels in the diabetic brain, while N-GABA increased their expression. Inversely, NAm and N-GABA dramatically reduced enhanced levels of GFAP and Nf-L found in the blood serum of diabetic rats, providing for the first time strong evidence for preserving blood-brain barrier integrity by studied compounds. CONCLUSION: Thus, NAm and N-GABA may exert neuroprotective effects by decreasing pro-apoptotic regulators levels and improving expression of angiogenic and cytoskeletal proteins impaired by hyperglycemia in rat brain.

The parp-1 and bax genes as potential targets for treatment of the heart functioning impairments induced by type 1 diabetes mellitus.

Objectives. The present study was designed to assess whether apoptosis-related genes as parp-1 and bax could be targets for treatment of diabetes mellitus and whether vitamin D may exert beneficial effects. Methods. Vitamin D3 treatment for 4 weeks, starting after 4 weeks of the diabetes duration. The expression of parp-1 and bax genes was estimated on mRNA levels using real time quantitative polymerase chain reaction. Results. After 8 weeks, diabetic rats had weight loss, while blood glucose was increased about 4.9-fold compared to control group. Vitamin D3 administration to diabetic animals had no effect on these parameters. It was found that total serum alkaline phosphatase activity was significantly elevated in diabetic rats as compared to control animals and was restored by vitamin D3. Diabetes was accompanied by reduction of nicotinamidadenindinucleotide, a substrate of poly-ADP-ribosylation, level by 31.7% as compared to control rats, which was not reversed in response to vitamin D3 treatment. In diabetic hearts, the mRNA expression level of parp-1 gene was 2.8-fold higher compared to control rats and partially decreased by vitamin D3 treatment. Less significant alterations were observed in diabetic hearts for the mRNA expression level of bax gene that was 2.0-fold higher compared to control animals and vitamin D3 normalized it. These results indicate that cardiomyocytes have a tendency to apoptosis. Conclusions. The findings suggest that investigated genes can be targets at the transcriptional level for vitamin D action that may be contributed to the improving metabolic/signaling pathways induced by diabetes mellitus.

The study of leukocyte phagocytic activity in the presence of herpetic infection and stroke.

OBJECTIVE: Inrtoduction: Post-stroke complications are one of the urgent and insufficiently resolved problems. According to different literature data 23% to 65% of patients suffer from the post-stroke development of an infectious process. Herpes simplex virus type 1 and 2 can also be etiological factors of stroke development, however their reactivation is seldom mentioned in clinical observations. The development of immune suppression is considered to be the cause of these complications. The aim: The current study aims at determining post-stroke changes in leukocyte component of the immunity and in the presence of concomitant herpetic infection as well as at finding changes in phagocytosis parameters during antiviral treatment. PATIENTS AND METHODS: Materials and methods: The experiments were carried out on mice of the Balb/с line. The animals were infected with the herpes simplex virus type I, and 30 days later hemorrhagic stroke was simulated by administering 0.1 ml of autoblood into the right hemisphere. Following the acute stroke some animals were given acyclovir, proteflazid or altabor. From the animals' blood leukocytes were obtained and phagocytic activity and production of reactive oxygen species of granulocytes and agranulocytes in relation to fluorescent E.coli bacteria were studied by flow cytometry. RESULTS: Results: The experiment revealed significant changes in the redistribution between two major types of leukocytes in mice with stroke (an increased number of agranulocytes by 19.9%) and decreased phagocytosis activity, in the animals infected with herpes simplex virus type І in particular. Ischemic brain damage had an immunosuppressive effect on blood leukocytes. For comparison a significant increase in phagocyte count in leukocytes was found in the case of viral infection. The use of drugs with antiviral effects did not affect the activity of granulocytes / agranulocytes. CONCLUSION: Conclusion: Stroke can be the cause of latent herpes virus infection reactivation and has essential negative effect on immune characteristics of leukocytes that remain unchanged with the use of antiviral agents.

Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors Reduce Reactive Gliosis and Improve Angiostatin Levels in Retina of Diabetic Rats.

Diabetic retinopathy (DR) is a multifactorial disease characterized by reactive gliosis and disbalance of angiogenesis regulators, contributing to endothelial dysfunction and microvascular complications. This study was organized to elucidate whether poly(ADP-ribose) polymerase-1 (PARP-1) inhibition could attenuate diabetes-induced damage to macroglia and correct angiogenic disbalance in diabetic rat retina. After 8 weeks of streptozotocin (STZ)-induced diabetes, Wistar male rats were treated with PARP-1 inhibitors, nicotinamide (NAm) or 3-aminobenzamide (3-AB) (100 and 30 mg/kg/daily i.p., respectively), for 14 days. After the 10-weeks experiment period, retinas were undergone an immunohistochemical staining for glial fibrillary acidic protein (GFAP), while western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of PARP-1, poly(ADP-ribosyl)ated proteins (PARs), GFAP, and angiostatin isoforms. Diabetes induced significant up-regulation and activation of retinal PARP-1, reactive gliosis development, and GFAP overexpression compared to non-diabetic control. Moreover, extensive fragmentation of both PARP-1 and GFAP (hallmarks of apoptosis and macroglia reactivation, respectively) in diabetic retina was also observed. Levels of angiostatin isoforms were dramatically decreased in diabetic retina, sustaining aberrant pro-angiogenic condition. Both NAm and 3-AB markedly attenuated damage to macroglia, evidenced by down-regulation of PARP-1, PARs and total GFAP compared to diabetic non-treated group. PARP-1-inhibitory therapy prevented formation of PARP-1 and GFAP cleavage-derived products. In retinas of anti-PARP-treated diabetic animals, partial restoration of angiostatin's levels was shown. Therefore, PARP-1 inhibitors counteract diabetes-induced injuries and manifest retinoprotective effects, including attenuation of reactive gliosis and improvement of angiogenic status, thus, such agents could be considered as promising candidates for DR management.

Facile fabrication of luminescent organic dots by thermolysis of citric acid in urea melt, and their use for cell staining and polyelectrolyte microcapsule labelling.

Luminescent organic dots (O-dots) were synthesized via a one-pot, solvent-free thermolysis of citric acid in urea melt. The influence of the ratio of the precursors and the duration of the process on the properties of the O-dots was established and a mechanism of their formation was hypothesized. The multicolour luminescence tunability and toxicity of synthesized O-dots were extensively studied. The possible applications of O-dots for alive/fixed cell staining and labelling of layer-by-layer polyelectrolyte microcapsules were evaluated.