Red Raspberry Phenols Inhibit Angiogenesis: A Morphological and Subcellular Analysis Upon Human Endothelial Cells.

Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory, and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signaling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10 to 250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay), and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI50 = 87,64 ± 6,59 µg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signaling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. J. Cell. Biochem. 117: 1604-1612, 2016. © 2015 Wiley Periodicals, Inc.

Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia-reperfusion injury in mice.

Nitrite is protective against renal ischemia/reperfusion injury (IRI); an effect due to its reduction to nitric oxide (NO). In addition to other reductase pathways, endothelial NO synthase (eNOS) may also facilitate nitrite reduction in ischemic environments. We investigated the role of eNOS in sodium nitrite (60 microM, 10 ml/kg applied topically 1 min before reperfusion)-induced protection against renal IRI in C57/BL6 wild-type (WT) and eNOS knockout (eNOS KO) mice subjected to bilateral renal ischemia (30 min) and reperfusion (24h). Markers of renal dysfunction (plasma [creatinine] and [urea]), damage (tubular histology) and inflammation (cell recruitment) were elevated following IRI in WT mice; effects significantly reduced following nitrite treatment. Chemiluminescence analysis of cortical and medullary sections of the kidney demonstrated rapid (within 1 min) distribution of nitrite following application. Whilst IRI caused a significant (albeit substantially reduced compared to WT mice) elevation of markers of renal dysfunction and damage in eNOS KO mice, the beneficial effects of nitrite were absent or reduced, respectively. Moreover, nitrite treatment enhanced renal dysfunction in the form of increased plasma [creatinine] in eNOS KO mice. Confirmation of nitrite reductase activity of eNOS was provided by demonstration of nitrite (100 microM)-derived NO production by kidney homogenates of WT mice, that was significantly reduced by L-NMMA. L-NMMA was without effect using kidney homogenates of eNOS KO mice. These results support a role for eNOS in the pathways activated during renal IRI and also identify eNOS as a nitrite reductase in ischemic conditions; activity which in part underlies the protective effects of nitrite.

Exercise training decreases proinflammatory profile in Zucker diabetic (type 2) fatty rats.

OBJECTIVE: In the present study we evaluated the effect of exercise on the plasma levels of proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and the anti-inflammatory molecule uric acid in the Zucker diabetic fatty (ZDF) rats that are more prone to develop type 2 diabetes mellitus. METHODS: Sixteen obese ZDF (Gmi fa/fa) rats (8 wk old, 228.40 +/- 4.05 g) were randomly assigned to one of two groups (n = 8 each): an exercise-trained group and a sedentary one. In addition, 16 lean ZDF (Gmi +/+) rats (8 wk old, 199.00 +/- 3.50 g) were subjected to identical sedentary and exercise conditioning (n = 8 each). Initially, rats swam 15 min/d (5 d/wk) in a 36 degrees C bath. The exercise protocol was gradually increased by 15 min/d until a swimming period of 1 h/d (1 wk) was attained. Thereafter, rats swam 1 h/d, 3 d/wk, for an additional period of 11 wk. Rats were sacrificed 48 h after the last training period and the blood and pancreas were collected. Circulating levels of glucose, glycosylated hemoglobin, total cholesterol, triglycerides, insulin, uric acid, IL-6, and TNF-alpha were assessed. The concentrations of proinflammatory cytokines in the pancreas were also evaluated. RESULTS: In the diabetic ZDF (fa/fa) rats, exercise decreased hyperuricemia (-37.3%) and IL-6 and TNF-alpha levels (-16.9% and -12.7% respectively) and maintained the weight of the pancreas at near normal. Immunohistochemistry revealed a marked decrease in the expression of TNF-alpha and IL-6 in the pancreatic islet cells of ZDF (fa/fa) rats. CONCLUSION: These results indicate that aerobic exercise is anti-inflammatory in nature.

Effects of diethyldithiocarbamate (DETC) on liver injury induced by ischemia-reperfusion in rats.

PURPOSE: In organ transplantation, ischemia-reperfusion injury (I/R) is a constant occurrence. Dithiocarbamates, a class of antioxidants, have been reported to inhibit nuclear factor-kappaB (NF-kappaB), a critical transcription factor in the intracellular inflammatory cascade. We studied the effects of diethyldithiocarbamate (DETC) on liver injury induced by I/R. MATERIALS AND METHODS: Male Wistar rats were pretreated with DETC (100 mg/kg IV) 10 minutes before hepatic ischemia, which was followed by 2 hours reperfusion, or 10 minutes prior to the reperfusion. Blood samples were obtained at the end of the reperfusion period to determine the biochemical markers of liver injury. Results were compared using the one-way analysis of variance (ANOVA), followed by the Bonferroni posttest, and presented as mean values +/- SEM. RESULTS: I/R produced significant increases in aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (gamma-GT) serum levels compared with sham-operated rats. Administration of DETC prior to the onset of reperfusion significantly reduced the liver injury. However, DETC administered before the ischemic period failed to protect the liver from an I/R injury. CONCLUSION: DETC, administered just before the reperfusion period, resulted in a significant decrease in the I/R injury to the liver, an observation that may have therapeutic implications.

Protective effects of hydroxytyrosol-supplemented refined olive oil in animal models of acute inflammation and rheumatoid arthritis.

Virgin olive oil is the primary source of fat in the Mediterranean diet, and its beneficial health effects have been related with oleic acid and phenolic compounds content. Hydroxytyrosol, a typical virgin olive oil phenolic compound, has beneficial antioxidant and anti-inflammatory properties as previously reported. The aim of this study was to evaluate the effect of hydroxytyrosol-supplemented refined olive oil at 0.5 and 5 mg/kg in a rodent model of rheumatoid arthritis. Rheumatoid arthritis was induced by intradermic administration, in male Wistar rats, of Freund's adjuvant with collagen type II on days 1 and 21. Hydroxytyrosol-supplemented refined olive oils were administrated by gavage from day 23 until day 35. The treatment at 5-mg/kg dose significantly decreased paw edema (P<.01), histological damage, cyclooxygenase-2 and inducible nitric oxide synthase expression, and markedly reduced the degree of bone resorption, soft tissue swelling and osteophyte formation, improving articular function in treated animals. Acute inflammation, induced by carrageenan, was also evaluated for hydroxytyrosol-supplemented refined olive oils at 0.5 and 5 mg/kg. Both doses significantly reduced paw edema (P<.001). Our results suggest that the supplementation of refined olive oil with hydroxytyrosol may be advantageous in rheumatoid arthritis with significant impact not only on chronic inflammation but also on acute inflammatory processes.

Tempol, an intracelullar free radical scavenger, reduces liver injury in hepatic ischemia-reperfusion in the rat.

Liver ischemia is of clinical interest because of its role in liver failure and also hepatic graft rejection. The generation of reactive oxygen species contributes to the injury that follows ischemia-reperfusion. One therapy utilizes the administration of antioxidants; however, only limited experience suggests a potential benefit of systemic administration of these compounds. To overcome the limitations of these compounds, small molecules with improved cell membrane permeability characteristics and higher potency, such as tempol, are being tested in vivo. Tempol, a membrane-permeable radical scavenger, interferes with the formation or the effects of many radicals, including superoxide anions, hydroxyl radicals, and peroxynitrite. The aim of this study was to investigate the effects of tempol in an in vivo rat model of liver ischemia-reperfusion injury. Male Wistar rats were pretreated with tempol (30 mg/kg, i.v.) 5 minutes prior to liver ischemia (for 30 minutes) and reperfusion (for 2 hours). The liver injury was assessed by measuring serum levels of transaminases, lactate dehydrogenase, and gamma-glutamyl transferase. Tempol significantly mitigated the increase in transaminases, lactate dehydrogenase, and gamma-glutamyl transferase following liver ischemia-reperfusion, suggesting an improvement in liver function and resistance to injury.

Chemical characterization of a red raspberry fruit extract and evaluation of its pharmacological effects in experimental models of acute inflammation and collagen-induced arthritis.

Berries are an important dietary source of fibres, vitamins, minerals and some biologically active non-nutrients. A red raspberry fruit extract was characterized in terms of phenolic content and the anti-inflammatory properties and protective effects were evaluated in two experimental models of inflammation. The antioxidant potential of the extract, the cellular antioxidant activity and the effects over neutrophils' oxidative burst were also studied to provide a mechanistic insight for the anti-inflammatory effects observed. The extract was administered in a dose of 15 mg kg(-1), i.p. and significantly inhibited paw oedema formation in the rat. The same dose was administered via i.p. and p.o. routes in the collagen-induced arthritis model in the rat. The extract showed pharmacological activity and was able to significantly reduce the development of clinical signs of arthritis and markedly reduce the degree of bone resorption, soft tissue swelling and osteophyte formation, preventing articular destruction in treated animals.

Evaluation of cardiovascular protective effect of different apple varieties - Correlation of response with composition.

Epidemiological evidence supports the concept that diets rich in fruits and vegetables promote health and attenuate or delay the onset of cardiovascular disease (CVD). In particular, a reduced risk of CVD has been associated with apple consumption, probably due to the cholesterol-lowering effect of the main bioactive compounds, namely fibre and polyphenols. In this work, the effect of diet supplementation with 20% of three Portuguese apple cultivars (Bravo de Esmolfe, Malápio Serra and Golden), containing distinct phenolic and fibre concentrations, on serum lipid profile and oxLDL of male Wistar rats fed a cholesterol-enriched diet (2%) was evaluated. After 30 days, only Bravo de Esmolfe apple was able to decrease significantly serum levels of triglycerides, total and LDL cholesterol concentrations (reductions of 27.2%, 21.0% and 20.4%, respectively, in relation to the cholesterol-enriched diet group, P<0.05). The levels of oxLDL were also significantly improved with the consumption of this apple variety (reductions of 20.0% and 11.9%, in relation to the cholesterol-enriched diet group and control group, respectively, P>0.05) as well as with Malapio da Serra apple (reductions of 9.8% in relation to the cholesterol-enriched diet group, P<0.05). Correlation of the bioactive response with chemical composition showed that catechin, epicatechin, procyanidin B1 and ß-carotene are the major phytocompounds responsible for the cholesterol lowering ability of apples. The antioxidant potential may have also contributed to this beneficial effect.

Alginate films containing Lactobacillus plantarum as wound dressing for prevention of burn infection.

The topical application of lactobacilli is receiving attention for the prevention of superficial skin and burn wound infections. We studied the properties of Lactobacillus plantarum immobilized in calcium alginate films and investigated the antibacterial activity of these films in a model burn wound in rats. A multiresistant clinical isolate, VIM-2-metallo-ß-lactamase-producing Pseudomonas aeruginosa, was used as the indicator strain. Films incorporating L. plantarum at cell concentrations of 10(8)cfu/mL caused a reduction of 5-6 log(10) in P. aeruginosa in the model burn wounds. L. plantarum immobilized in freeze-dried calcium alginate films remained viable during six months of storage at 4 °C.

In vivo anti-inflammatory effect and toxicological screening of Maytenus heterophylla and Maytenus senegalensis extracts.

Maytenus heterophylla (Eckl & Zeyh.) Robson and Maytenus senegalensis (Lam). Exell are two African medicinal plants used to treat painful and inflammatory diseases. We evaluated the in vivo (per os) anti-inflammatory activity of M. heterophylla leaf, stem and root extracts and of M. senegalensis leaf and stem extracts. Additionally, we assessed their in vivo acute and sub-acute toxicities. Anti-inflammatory activities of ethanol extracts were determined in Wistar albino rats, by the carrageenan-induced paw oedema method. Acute and sub-acute toxicity screening of the extracts was evaluated in adult male CD-6 mice. Leaf extracts of M. heterophylla and M. senegalensis exhibited significant anti-inflammatory activity (120 mg/kg, per os), reducing oedema by 51% and 35%, respectively. While M. heterophylla extracts at 1200 mg/kg have shown to be non-toxic, M. senegalensis extracts indicated some toxicity. Our results show a significant anti-inflammatory effect of both M. heterophylla and M. senegalensis leaf extracts in a local model of acute inflammation and suggest the absence of acute and sub-acute toxicity signs of the M. heterophylla leaf extract (but not of M. senegalensis). Ongoing studies will surely shed some light into the mechanism of action of this active extract and establish its chemical fingerprint.