Whole transcriptome expression array analysis of human colon fibroblasts culture treated with Helichrysum italicum supports its use in traditional medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Helichrysum italicum (HI) is a Mediterranean plant with well-reported use in traditional medicine for a wide range of applications, including digestive and liver disorders, intestinal parasitic infections, wound healing, stomach ache and asthma. However, little is known about the global mechanism behind its pleiotropic activity. AIM OF THE STUDY: The aim of this study was to explain the mechanism behind the previously demonstrated effects of HI and to justify its use in traditional medicine. MATERIALS AND METHODS: A microarray-based transcriptome analysis was used to discover the global transcriptional alterations in primary colon fibroblasts after exposure to HI infusion for 6 h and 24 h. In addition, quantitative real-time PCR was used to verify the microarray results. RESULTS: Altogether we identified 217 differentially expressed genes compared to non-treated cells, and only 8 were common to both treatments. Gene ontology analysis revealed that 24 h treatment with HI infusion altered the expression of genes involved in cytoskeletal rearrangement and cell growth, whereas pathway analysis further showed the importance of interleukin signaling and transcriptional regulation by TP53. For the 6 h treatment only the process of hemostasis appeared in the results of both enrichment analyses. In functional assays, HI infusion increased cell migration and decreased blood clotting and prothrombin time. CONCLUSIONS: With the careful evaluation of the role of individual genes, especially SERPING1, ARHGAP1, IL33 and CDKN1A, represented in the enriched pathways and processes, we propose the main mode of HI action, which is wound healing. In addition to its indirect prevention of diseases resulting from the impaired barrier integrity, HI also effects inflammation and metabolic processes directly, as it regulates genes such as LRPPRC, LIPA, ABCA12, PRKAR1A and ANXA6.

High-Protein Bar as a Meal Replacement in Elite Sports Nutrition: A Pilot Study.

This study was focused on the creation of high-protein bars formulated using whey protein isolate (24%) and soy protein isolate (6%) as the sources of proteins; oat flakes and inulin, both abundant in dietary fibres, and creatine monohydrate and other minor ingredients (vitamin and mineral mixture, potassium sorbate) to achieve the requirements for a meal replacement formula for physically active people. The nutritional profile of the high-protein bar was examined (energy 1215 kJ/288 kcal; protein 34.1 ± 0.20 g, fat 6.01 ± 0.13 g of which was saturated 3.12 ± 0.08 g, fibre 3.10 ± 0.17 g carbohydrate 23.0 ± 0.16 g of which sugars 1.50 ± 0.19 g and starch 21.5 ± 0.11 g in 100 g), and sensory properties with instrumental parameters (texture and colour) were determined and compared with bars commercially available on the market. The created high-protein bar was sensorily acceptable in comparison to other commercially available bars. The dietary intervention study was conducted on elite athletes (professional handball players) to evaluate effects of created versus control bar consumption on their metabolic parameters. The baseline characteristics (mean age, body mass index (BMI), fat mass, muscle mass, lean mass and fat percentage) of the athletes (8) were determined at the start of the study. The cross-over intervention study was organized in two successive phases (5 days each) with a seven-day long washout period between phases. Bars were consumed after the afternoon training unit. Blood samples were collected at the start and the end of the intervention study to analyse the metabolic profiles of the athletes. Serum levels of high-density cholesterol (HDL), low-density cholesterol (LDL) and total cholesterol (HOL), glucose, triacylglycerides (TAG), total and direct bilirubin, creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. The results showed that bar consumption significantly decreased serum aspartate transaminase (AST) and lactate dehydrogenase (LDH) and increased total and direct bilirubin levels, suggesting lower exercise-induced muscle damage and increased antioxidative response, respectively. Therefore, it can be concluded that the consumption of the created high-protein bar was able to improve physiological adaptation after training.

Polymorphism rs2073618 of the osteoprotegerin gene as a potential marker of subclinical carotid atherosclerosis in Caucasians with type 2 diabetes mellitus.

BACKGROUND: The OPG/RANKL/RANK (osteoprotegerin/receptor-activator of nuclear factor κB ligand/receptor-activator of nuclear factor κB) axis has been recently linked to the development of atherosclerosis and plaque destabilization. We have investigated whether polymorphism rs2073618 of the OPG gene is associated with subclinical markers of carotid atherosclerosis in subjects with type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS: 595 subjects with T2DM were enrolled in the cross-sectional study. Subclinical markers of carotid atherosclerosis (carotid intima media thickness, plaque thickness, and plaques presence) were assessed with ultrasound at the time of recruitment. Genotyping for rs2073618 (a missense variant located in exon I of the OPG gene) was performed, and OPG serum levels were determined by ELISA. RESULTS: Compared to the GG genotype, the CC genotype of the rs2073618 polymorphism had a significantly increased risk for the presence of carotid plaque (OR = 2.54, 95 % CI = 1.22-5.28, p = 0.01). No statistically significant difference could be detected (p = 0.68) upon comparing median values of serum OPG levels among studied genotype groups in subjects with T2DM. Multivariable linear regression analyses in T2DM subjects demonstrated that GC and CC genotypes (p = 0.03 and p = 0.003), together with statin therapy (p = 0.009), were independent predictors of the number of carotid segments with plaques. CONCLUSIONS: Despite the fact that OPG rs2073618 genotypes failed to predict the serum OPG levels as there was no statistical difference among compared genotypes, our results demonstrate that the rs2073618 polymorphism could be a possible genetic marker for the prediction of increased risk for carotid plaque burden as a measure of advanced subclinical atherosclerosis in T2DM subjects.

Total Serum Antioxidant Capacity in Healthy Normal Weight and Asymptomatic Overweight Adults.

Obesity and overweight are major contributors to the burden of chronic disease. Both are defined as abnormal or excessive fat accumulation and by increased production of free radicals leading to oxidative stress. The aim of the present study was to evaluate whether overweight and fat accumulation is associated with serum total antioxidant capacity (TAC) in men and women, irrespective of nutritional habits, nutrient intakes, physical activity, smoking, and other confounders, which may be responsible for modifying the association between serum TAC and overweight/obesity measures. This cross-sectional study was conducted on 60 normal weight and 60 overweight adults aged 25-49. All participants underwent standard anthromorphological measurements of body composition, blood pressure and biochemical measurements, aerobic capabilities assessment and dietary intake evaluation. TAC was measured by using the photochemioluminescence method. All data were analysed with SPSS software. Men had higher values of TAC than women and concentrations of TAC were significantly higher in overweight subjects compared to normal weight subjects. In the present study TAC tended to be increased by various metabolic risk factors, especially overweight/obesity parameters (body mass index, body fat), inflammation and increased serum levels of Cysteine, irrespective of nutritional habits, nutrient intakes, physical activity and smoking. Overweight and obesity at an early stage may stimulate TAC. Therefore, the elevation of TAC in overweight adults may be a compensatory response to oxidative stress, generated by reactive oxygen species.

A neurotoxic secretory phospholipase A2 induces apoptosis in motoneuron-like cells.

Ammodytoxin A (AtxA) is a presynaptically neurotoxic secretory phospholipase A(2) from snake venom. The aim of this study was to investigate the mechanism of its cytotoxicity expressed against mouse motoneuronal NSC34 cells. AtxA displayed a potent dose- and time-dependent cytotoxicity that was associated with apoptosis and not necrosis, as revealed by a reduction of mitochondrial membrane potential, activation of caspase-3, and by the absence of propidium iodide staining. The cytotoxic- and apoptosis-inducing effects of AtxA were specific for the motoneuronal cells; human embryonic kidney (HEK293) and mouse myoblast (C2C12) cells were shown to be resistant to the toxin.

A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol.

The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.