Bruno Sepodes

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin.

Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the β common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58–82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia–reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPOs trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPOs three-dimensional structure.

Recombinant human erythropoietin protects the liver from hepatic ischemia-reperfusion injury in the rat

Recently, erythropoietin was shown to have both hematopoietic as well as tissue‐protective properties. Erythropoietin (EPO) had a protective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, myocardial infarction, and ischemia‐reperfusion (I/R) injury of the kidney. It is not known whether EPO protects the liver against I/R injury. Using a rat model of liver I/R injury, we aimed to determine the effect of the administration of human recombinant erythropoietin (rhEPO) on liver injury. Rats were subjected to 30 min of liver ischemia followed by 2 h of reperfusion. When compared with the sham‐operated rats, I/R resulted in significant rises in the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, gamma‐glutamyl transferase, tissue lipid peroxidation, caspase‐3 activity and altered histology. Administration of rhEPO 5 min before ischemia was able to reduce the biochemical evidence of liver injury; however, this protection was not evident when rhEPO was administered 5 min before reperfusion. Mechanistically, early administration of rhEPO was able to reduce the oxidative stress and caspase‐3 activation, suggesting the subsequent reduction of apoptosis. This study provides the first evidence that rhEPO causes a substantial reduction of the liver injury induced by I/R in the rat.

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.

Anti-inflammatory effect of lycopene on carrageenan-induced paw oedema and hepatic ischaemia–reperfusion in the rat

The regular intake of tomatoes or its products has been associated with a reduced risk of chronic diseases and these effects have been mainly attributed to lycopene. Here, we evaluated the anti-inflammatory properties of lycopene and its protective effects on organ injury in two experimental models of inflammation. In order to study the effects of lycopene in local inflammation, a carrageenan-induced paw oedema model in rats was performed. Lycopene was administered as an acute (1, 10, 25 or 50 mg/kg, intraperitoneally, 15 min before carrageenan injection) and chronic treatment (25 or 50 mg/kg per d, 14 d). Inflammation was assessed by the measurement of paw volume increase after 6 h. Lycopene significantly inhibited paw oedema formation at two doses (25 and 50 mg/kg) in both acute and repeated administration. The effect of lycopene on liver inflammation was evaluated in a liver ischaemia-reperfusion (I/R) model. Rats were subjected to 45 min of ischaemia of three-quarters of the liver followed by 2 h of reperfusion. In this model, lycopene was administered daily at two doses (25 and 50 mg/kg) during the 14 d that preceded the experiments. Repeated administration of lycopene reduced liver injury induced by I/R, as demonstrated by the reduction of the increase in liver injury markers (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and gamma-glutamyl transferase) and attenuation of liver tissue lipoperoxidation was evidenced by a decrease in malondialdehyde production. The present results show that lycopene exhibited local anti-inflammatory activity and also attenuated liver injury induced by I/R. We speculate that lycopene administration might be useful in the pharmacological modulation of inflammatory events.

Antihyperglycaemic and protective effects of flavonoids on streptozotocin-induced diabetic rats.

The antihyperglycaemic effect of eight standard flavonoids, previously identified in the ethanol extract of the claimed antidiabetic plant Genista tenera, was evaluated on streptozotocin (STZ)‐induced diabetic Wistar rats. The aglycones apigenin, chrysoeriol and genistein, the monoglucosides apigenin 7‐O‐glucoside, luteolin 7‐O‐glucoside and genistein 7‐O‐glucoside and the diglycosides rutin and luteolin 7,3′‐di‐O‐glucoside were administered i.p. for 7 days (4 mg/kg b.w./day). The protective effect of these compounds over liver and kidneys of STZ–diabetic models was also evaluated by the determination of seric AST, ALT and urea levels. After 7 days of treatment, apigenin, chrysoeriol and genistein significantly lowered the blood glucose levels of diabetic animals; this effect was more pronounced (P < 0.01) in the oral glucose tolerance test. Glucose tolerance was also significantly improved in the rutin (P < 0.01) and in the genistein 7–O–glucoside (P < 0.05) treated groups. In addition, almost all the tested compounds effectively protected the liver and kidneys against STZ‐induced damage in rats. Copyright

Anti-inflammatory effect of rosmarinic acid and an extract of Rosmarinus officinalis in rat models of local and systemic inflammation.

Rosmarinic acid is a polyphenolic compound and main constituent of Rosmarinus officinalis and has been shown to possess antioxidant and anti‐inflammatory properties. We aimed to evaluate the anti‐inflammatory properties of rosmarinic acid and of an extract of R. officinalis in local inflammation (carrageenin‐induced paw oedema model in the rat), and further evaluate the protective effect of rosmarinic acid in rat models of systemic inflammation: liver ischaemia–reperfusion (I/R) and thermal injury models. In the local inflammation model, rosmarinic acid was administered at 10, 25 and 50 mg/kg (p.o.), and the extract was administered at 10 and 25 mg/kg (equivalent doses to rosmarinic acid groups) to male Wistar rats. Administration of rosmarinic acid and extract at the dose of 25 mg/kg reduced paw oedema at 6 hr by over 60%, exhibiting a dose–response effect, suggesting that rosmarinic was the main contributor to the anti‐inflammatory effect. In the liver I/R model, rosmarinic acid was administered at 25 mg/kg (i.v.) 30 min. prior to the induction of ischaemia and led to the significant reduction in the serum concentration of transaminases (AST and ALT) and LDH. In the thermal injury model, rosmarinic acid was administered at 25 mg/kg (i.v.) 5 min. prior to the induction of injury and significantly reduced multi‐organ dysfunction markers (liver, kidney, lung) by modulating NF‐κB and metalloproteinase‐9. For the first time, the anti‐inflammatory potential of rosmarinic acid has been identified, as it causes a substantial reduction in inflammation, and we speculate that it might be useful in the pharmacological modulation of injuries associated to inflammation.

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.

Protective Role of Peroxisome Proliferator–activated Receptor-β/δ in Septic Shock

RATIONALE Peroxisome proliferator-activated receptor (PPAR)-β/δ is a transcription factor that belongs to the PPAR nuclear hormone receptor family, but the role of PPAR-β/δ in sepsis is unknown. OBJECTIVES We investigated the role of PPAR-β/δ in murine models of LPS-induced organ injury and dysfunction and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. METHODS Wild-type (WT) and PPAR-β/δ knockout (KO) mice and C57BL/6 mice were subjected to LPS for 16 hours. C57BL/6 mice received the PPAR-β/δ agonist GW0742 (0.03 mg/kg intravenously, 1 h after LPS) or GW0742 plus the PPAR-β/δ antagonist GSK0660 (0.1 mg/kg intravenously, 30 min before LPS). CD-1 mice subjected to CLP received GW0742 or GW0742 plus GSK0660. MEASUREMENTS AND MAIN RESULTS In PPAR-β/δ KO mice, endotoxemia exacerbated organ injury and dysfunction (cardiac, renal, and hepatic) and inflammation (lung) compared with WT mice. In C57BL/6 mice subjected to endotoxemia, GW0742 significantly (1) attenuated organ (cardiac and renal) dysfunction and inflammation (lung); (2) increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3β; (3) attenuated the increase in extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT)-3 phosphorylation; and (4) attenuated the activation of nuclear factor (NF)-κB and the expression of inducible nitric oxide synthase (iNOS). In CD-1 mice subjected to CLP, GW0742 improved 10-day survival. All the observed beneficial effects of GW0742 were attenuated by the PPAR-β/δ antagonist GSK0660. CONCLUSIONS PPAR-β/δ protects against multiple organ injury and dysfunction, and inflammation caused by endotoxic shock and improves survival in polymicrobial sepsis by a mechanism that may involve activation of Akt and inhibition of GSK-3β and NF-κB.

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 β-carotene are the major phytocompounds responsible for the cholesterol lowering ability of apples. The antioxidant potential may have also contributed to this beneficial effect.

Lysophosphatidylcholine reduces the organ injury and dysfunction in rodent models of Gram‐negative and Gram‐positive shock

1 Lysophosphatidylcholine (LPC) modulates the inflammatory response and reduces mortality in animal models of sepsis. Here, we investigate the effects of LPC from synthetic (sLPC) and natural, soy bean derived LPC, (nLPC) sources on the organ injury/dysfunction caused by systemic administration of lipopolysaccharide (LPS) or peptidoglycan (PepG) and lipoteichoic acid (LTA). 2 Rats were subjected to (i) endotoxaemia (LPS 6 mg kg–1 i.v.) and treated with sLPC (1–100 mg kg−1), (ii) endotoxaemia and treated with nLPC (10 mg kg−1) or (iii) Gram‐positive shock (PepG 10 mg kg–1 and LTA 3 mg kg–1 i.v.) and treated with sLPC (10 mg kg−1). 3 Endotoxaemia or Gram‐positive shock for 6 h resulted in increases in serum makers of renal dysfunction and liver, pancreatic and neuromuscular injury. 4 Administration of sLPC, at 1 or 2 h after LPS, dose dependently (1–10 mg kg−1) reduced the organ injury/dysfunction. High doses of sLPC (30 and 100 mg kg−1) were shown to be detrimental in endotoxaemia. sLPC also afforded protection against the organ injury/dysfunction caused by Gram‐positive shock. nLPC was found to be protective in endotoxaemic animals. 5 The beneficial effects of sLPC were associated with an attenuation in circulating levels of interleukin‐1β (IL‐1β). 6 In conclusion, LPC dose and time dependently reduces the organ injury and circulating IL‐1β levels caused by Gram‐negative or Gram‐positive shock in the rat. Thus, we speculate that appropriate doses of LPC may be useful in reducing the degree of organ injury and dysfunction associated with shock of various aetiologies.