Darja Svirina

Patterns of inflammatory responses in large and small airways in smokers with and without chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is characterised by progressive and irreversible airway obstruction. Smoking causes persistent inflammation in lung tissue. However, differences in inflammatory responses between the large and small airways have not been systematically explored among smokers with and without COPD. Objectives: The aim of our research was to characterise the expression and localisation of NF-ĸBp65 and histone deacetylase 2 (HDAC2) as well as inflammatory cell (macrophages, lymphocytes, neutrophils) distribution in large and small airways, in nonsmokers and in smokers with and without COPD. Methods: Nineteen nonsmokers, 20 smokers with normal lung ventilation function and 20 smokers with moderate COPD, undergoing lung resection for a solitary peripheral carcinoma, were enrolled in the study. Immunohistochemical methods were used to evaluate NF-ĸBp65 and HDAC2 expression and identify inflammatory cells in airways. Results: COPD patients had increased NF-ĸBp65 expression compared to nonsmokers and smokers without COPD, in both large and small airways, which corresponded to increased numbers of macrophages, CD8+ T lymphocytes and neutrophils. COPD patients had more macrophages in large compared to small airways and more CD8+ T lymphocytes and neutrophils in small compared to large airways. HDAC2 expression was significantly downregulated in smokers with COPD in small compared to large airways. Conclusions: Our findings indicate a nonuniform distribution of inflammatory cells throughout the bronchial tree. However, in both smokers with and without COPD, similar patterns of inflammatory processes occur in both large and small airways. The difference between smokers with and without COPD is only quantitative.

Influence of metformin on GLUT1 gene and protein expression in rat streptozotocin diabetes mellitus model.

Context: Metformin improves hyperglycaemia via mechanisms which include activation of AMP-activated protein kinase (AMPK). Recent findings indicate that some metabolic actions of metformin occur also by AMPK-independent mechanisms. Objective: To study the action of metformin on expression of GLUT1 glucose transporter in rat streptozotocin model of diabetes mellitus. Materials and methods: Streptozotocin-induced rats were treated with metformin while monitoring parameters of carbohydrate and lipid metabolism. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time quantitative RT-PCR and immunohistochemistry correspondingly. Results: Metformin treatment decreased glucose concentration, glycated haemoglobin % and improved glucose tolerance. Streptozotocin diabetes provoked increase of both GLUT1 gene and protein expression in kidneys, metformin treatment produced normalization of the GLUT1 expression levels. In the liver, diabetes triggered an increase in GLUT1 protein expression, which was normalized by metformin. Conclusion: Metformin is prospective for treatment of diabetic nephropathy.

Neuroprotective properties of mildronate, a mitochondria-targeted small molecule.

Mildronate, a representative of the aza-butyrobetaine class of drugs with proven cardioprotective efficacy, was recently found to prevent dysfunction of complex I in rat liver mitochondria. The present study demonstrates that mildronate also acts as a neuroprotective agent. In a mouse model of azidothymidine (anti-HIV drug) neurotoxicity, mildronate reduced the azidothymidine-induced alterations in mouse brain tissue: it normalized the increase in caspase-3, cellular apoptosis susceptibility protein (CAS) and iNOS expression assessed by quantitative and semi-quantitative analysis. Mildronate also normalized the changes in cytochrome c oxidase (COX) expression, reduced the expression of glial fibrillary acidic protein (GFAP) and cellular infiltration. The present results show that the neuroprotective action of mildronate results at least partially from anti-neurodegenerative (anti-apoptotic) and anti-inflammatory mechanisms. It might be suggested that the molecular conformation of mildronate can facilitate its easy binding to mitochondria, and regulate the expression of different signal molecules, hence maintaining cellular signaling and survival.

Correction of glycaemia and GLUT1 level by mildronate in rat streptozotocin diabetes mellitus model.

Anti‐ischaemic drug mildronate suppresses fatty acid metabolism and increases glucose utilization in myocardium. It was proposed that it could produce a favourable effect on metabolic parameters and glucose transport in diabetic animals. Rats with streptozotocin diabetes mellitus were treated with mildronate (100 mg/kg daily, per os, 6 weeks). Therapeutic effect of mildronate was monitored by measuring animal weight, concentrations of blood glucose, insulin, blood triglycerides, free fatty acids, blood ketone bodies and cholesterol, glycated haemoglobin per cent (HbA1c%) and glucose tolerance. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time RT‐PCR and immunohistochemistry correspondingly. In the streptozotocin + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose, cholesterol, free fatty acid and HbA1c concentrations and improved glucose tolerance. Induction of streptozotocin diabetes mellitus provoked increase of both GLUT1 gene and protein expression in kidneys, heart and muscle, mildronate treatment produced normalization of the GLUT1 expression levels. In the liver a similar effect was observed for GLUT1 protein expression, while GLUT1 gene expression was increased by mildronate. Mildronate produces therapeutic effect in streptozotocin diabetes model. Mildronate normalizes the GLUT1 expression up‐regulated by streptozotocin diabetes mellitus in kidneys, heart, muscle and liver. Copyright

Effects of kaempferol and myricetin on inducible nitric oxide synthase expression and nitric oxide production in rats.

When administered as drugs or consumed as food components, polyphenolic compounds synthesized in plants interfere with intracellular signal transduction pathways, including pathways of nitric oxide synthase expression. However, effects of these compounds in vivo do not always correlate with nitric oxide synthase-inhibiting activities revealed in experiments with cultured cells. The initial goal of this work was to compare effects of flavonoids kaempferol and myricetin on inducible nitric oxide synthase mRNA and protein expression monitored by real-time RT-PCR and immunohistochemistry and to evaluate the impact of these effects on nitric oxide production in rat organs measured by means of electron paramagnetic resonance spectroscopy. Kaempferol and myricetin attenuated the lipopolysaccharide-induced outburst of inducible nitric oxide synthase gene expression; kaempferol also significantly decreased the lipopolysaccharide-induced outburst of inducible nitric oxide synthase protein expression in the liver. Myricetin decreased nitric oxide production in intact rat liver. Kaempferol did not decrease nitric oxide production neither in intact rats nor in the lipopolysaccharide-treated animals. Kaempferol even enhanced the lipopolysaccharide-induced increase of nitric oxide production in blood. Myricetin did not interfere with lipopolysaccharide effects. As both kaempferol and myricetin are known as inhibitors of inducible nitric oxide synthase expression, our results suggest that modifications of nitric oxide level in tissues by these compounds cannot be predicted from data about its effects on nitric oxide synthase expression or activity.

Neuroprotective Properties of Mildronate, a Small Molecule, in a Rat Model of Parkinson’s Disease

Previously, we have found that mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a small molecule with charged nitrogen and oxygen atoms, protects mitochondrial metabolism that is altered by inhibitors of complex I and has neuroprotective effects in an azidothymidine-neurotoxicity mouse model. In the present study, we investigated the effects of mildronate in a rat model of Parkinson’s disease (PD) that was generated via a unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). We assessed the expression of cell biomarkers that are involved in signaling cascades and provide neural and glial integration: the neuronal marker TH (tyrosine hydroxylase); ubiquitin (a regulatory peptide involved in the ubiquitin-proteasome degradation system); Notch-3 (a marker of progenitor cells); IBA-1 (a marker of microglial cells); glial fibrillary acidic protein, GFAP (a marker of astrocytes); and inducible nitric oxide synthase, iNOS (a marker of inflammation). The data show that in the 6-OHDA-lesioned striatum, mildronate completely prevented the loss of TH, stimulated Notch-3 expression and decreased the expression of ubiquitin, GFAP and iNOS. These results provide evidence for the ability of mildronate to control the expression of an array of cellular proteins and, thus, impart multi-faceted homeostatic mechanisms in neurons and glial cells in a rat model of PD. We suggest that the use of mildronate provides a protective effect during the early stages of PD that can delay or halt the progression of this neurodegenerative disease.

Effects of lycopene, indole-3-carbinol, and luteolin on nitric oxide production and iNOS expression are organ-specific in rats.

Effects of Lycopene, Indole-3-Carbinol, and Luteolin on Nitric Oxide Production and iNOS Expression are Organ-Specific in Rats Natural compounds are known to modify NO content in tissues; however, the biological activity of polyphenol-rich food often does not correspond to the effects of individual polyphenols on NO synthase activity. The aim of this study was to see how natural compounds luteolin, indole-3-carbinol, and lycopene modify NO production in rat tissues and change the expression of the iNOS gene and protein. Indole-3-carbinol produced multiple effects on the NO level; it significantly decreased NO concentration in blood, lungs, and skeletal muscles and increased it in the liver. Indole-3-carbinol enhanced lipopolyssaccharide (LPS)-induced NO production in all rat organs. It decreased iNOS gene expression in the brain cortex of animals that did not receive LPS and up-regulated it in the LPS-treated animals. Lycopene increased the iNOS gene transcription rate in the brain cortex of LPS-treated animals. Luteolin did not modify NO production in any organ of LPS-untreated rats, nor did it affect gene expression in the liver. In the brain it slightly decreased iNOS gene expression. Luteolin decreased NO production in the blood of LPS-treated animals and the number of iNOS-positive cells in these animals. Our results suggest that changes in tissue NO levels caused by natural compounds cannot be predicted from their effect on NOS expression or activity obtained in model systems. This stresses the importance of direct measurements of NO and NOS expression in animal tissues. Djelovanje likopena, indol-3-karbinola i luteolina na stvaranje dušikova monoksida i ekspresiju iNOS-gena u štakora ovisi o organu Zna se da prirodni spojevi utječu na sadržaj NO u pojedinome tkivu. Ipak, biološka aktivnost hrane bogate polifenolima često ne odgovara djelovanju pojedinih polifenola na aktivnost NO-sintaze. Cilj je ovoga ispitivanja bio utvrditi kako prirodni spojevi luteolin, indol-3-karbinol i likopen utječu na stvaranje NO u tkivu štakora te kako utječu na ekspresiju gena i razine inducibilne sintaze dušikova monoksida (iNOS). Indol-3-karbinol višestruko je utjecao na razinu NO; značajno je snizio njegovu koncentraciju u krvi, plućima i skeletnim mišićima, ali ju je povisio u jetri. Također je povećao njegovo stvaranje u svim organima štakora koji su primili lipopolisaharide (LPS). U kori mozga životinja koje nisu primile LPS doveo je do smanjenja ekspresije iNOS-gena, a u onih koje su primile LPS do njegova povećanja. Likopen je doveo do ubrzane transkripcije iNOS-gena u kori mozga životinja koje su primile LPS, ali nije utjecao na stvaranje NO u životinja koje nisu primile LPS, odnosno na ekspresiju gena u jetri. U mozgu životinja koje nisu primile LPS doveo je do laganoga smanjenja ekspresije iNOS-gena. Luteolin je povećao stvaranje NO u krvi životinja koje su primile LPS te njihov broj iNOS-pozitivnih stanica. Naši rezultati upućuju na to da se promjene razina NO uzrokovane prirodnim spojevima ne daju predvidjeti na temelju ekspresije gena iNOS odnosno aktivnosti toga enzima dobivene na modelu. Stoga je veoma važno izravno mjeriti razine NO i ekspresiju NO-sintaze u tkivu životinja.

CXC chemokine ligand 4 (CXCL4) is predictor of tumour angiogenic activity and prognostic biomarker in non-small cell lung cancer (NSCLC) patients undergoing surgical treatment

Abstract Objective: To evaluate the association of CXC chemokine ligand 4 (CXCL4) plasma levels with tumour angiogenesis in non-small cell lung cancer (NSCLC) and to assess association of CXCL4 with clinical outcomes. Patients and methods: Fifty patients with early stage NSCLC who underwent pulmonary resection. CXCL4 levels were analysed by ELISA. Angiogenesis was assessed by immunohistochemistry, and microvessel density (MVD) count. Results: There was positive correlation between MVD and CXCL4 levels. Patients with higher CXCL4 levels had worse overall and disease-free survival. Conclusions: Plasma levels of CXCL4 are associated with tumour vascularity. Increased CXCL4 levels in NSCLC patients undergoing treatment may indicate active cancer-induced angiogenesis associated with relapse and worse outcome.