Elisa Benetti

Anti-apoptotic and anti-inflammatory effects of hydrogen sulfide in a rat model of regional myocardial I/R.

Hydrogen sulfide (H2S) is a novel gaseous mediator produced by cystathionine-&bgr;-synthase and cystathionine-&ggr;-lyase in the cardiovascular system, including the heart. Using a rat model of regional myocardial ischemia/reperfusion, we investigated the effects of an H2S donor (sodium hydrogen sulfide [NaHS]) on the infarct size and apoptosis caused by ischemia (25 min) and reperfusion (2 h). Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by NaHS. Specifically, we demonstrate that NaHS (1) attenuates the increase in caspase 9 activity observed in cardiac myocytes isolated from the area at risk (AAR) of hearts subjected in vivo to regional myocardial I/R and (2) ameliorates the decrease in expression of Bcl-2 within the AAR obtained from rat hearts subjected to regional myocardial I/R. The cardioprotective effects of NaHS were abolished by 5-hydroxydeconoate, a putative mitochondrial adenosine triphosphate-sensitive potassium channel blocker. Furthermore, NaHS attenuated the increase in the I/R-induced (1) phosphorylation of p38 mitogen-activated protein kinase and Jun N-terminal kinase, (2) translocation from the cytosol to the nucleus of the p65 subunit of nuclear factor-&kgr;B, (3) intercellular adhesion molecule 1 expression, (4) polymorphonuclear leukocyte accumulation, (5) myeloperoxidase activity, (6) malondialdehyde levels, and (7) nitrotyrosine staining determined in the AAR obtained from rat hearts subjected to regional myocardial I/R. In conclusion, we demonstrate that the cardioprotective effect of NaHS is secondary to a combination of antiapoptotic and anti-inflammatory effects. The antiapoptotic effect of NaHS may be in part due to the opening of the putative mitochondrial adenosine triphosphate-sensitive potassium channels.

Generation of endogenous hydrogen sulfide by cystathionine γ-lyase limits renal ischemia/reperfusion injury and dysfunction

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine γ-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-κB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-κB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

The NLRP3 Inflammasome as a novel player of the intercellular crosstalk in metabolic disorders.

The combination of obesity and type 2 diabetes is a serious health problem, which is projected to afflict 300 million people worldwide by 2020. Both clinical and translational laboratory studies have demonstrated that chronic inflammation is associated with obesity and obesity-related conditions such as insulin resistance. However, the precise etiopathogenetic mechanisms linking obesity to diabetes remain to be elucidated, and the pathways that mediate this phenomenon are not fully characterized. One of the most recently identified signaling pathways, whose activation seems to affect many metabolic disorders, is the “inflammasome,” a multiprotein complex composed of NLRP3 (nucleotide-binding domain and leucine-rich repeat protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and procaspase-1. NLRP3 inflammasome activation leads to the processing and secretion of the proinflammatory cytokines interleukin- (IL-) 1β and IL-18. The goal of this paper is to review new insights on the effects of the NLRP3 inflammasome activation in the complex mechanisms of crosstalk between different organs, for a better understanding of the role of chronic inflammation in metabolic disease pathogenesis. We will provide here a perspective on the current research on NLRP3 inflammasome, which may represent an innovative therapeutic target to reverse the detrimental metabolic consequences of the metabolic inflammation.

TREATMENT WITH THE GLYCOGEN SYNTHASE KINASE-3β INHIBITOR, TDZD-8, AFFECTS TRANSIENT CEREBRAL ISCHEMIA/REPERFUSION INJURY IN THE RAT HIPPOCAMPUS

The serine/threonine glycogen synthase kinase 3&bgr; (GSK-3&bgr;) is abundant in the central nervous system, particularly in the hippocampus, and plays a pivotal role in the pathophysiology of a number of diseases, including neurodegeneration. This study was designed to investigate the effects of GSK-3&bgr; inhibition against I/R injury in the rat hippocampus. Transient cerebral ischemia (30 min) followed by 1 h of reperfusion significantly increased generation of reactive oxygen species and modulated superoxide dismutase activity; 24 h of reperfusion evoked apoptosis (determined as mitochondrial cytochrome c release and Bcl-2 and caspase-9 expression), resulted in high plasma levels of TNF-&agr; and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and intercellular adhesion molecule-1. The selective GSK-3&bgr; inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administered before and after ischemia or during reperfusion alone to assess its potential as prophylactic or therapeutic strategy. Prophylactic or therapeutic administration of TDZD-8 caused the phosphorylation (Ser9) and hence inactivation of GSK-3&bgr;. Infarct volume and levels of S100B protein, a marker of cerebral injury, were reduced by TDZD-8. This was associated with a significant reduction in markers of oxidative stress, apoptosis, and the inflammatory response resulting from cerebral I/R. These beneficial effects were associated with a reduction of I/R-induced activation of the mitogen-activated protein kinases JNK1/2 and p38 and nuclear factor-&kgr;B. The present study demonstrates that TDZD-8 protects the brain against I/R injury by inhibiting GSK-3&bgr; activity. Collectively, our data may contribute to focus the role of GSK-3&bgr; in cerebral I/R.

Acute treatment with relaxin protects the kidney against ischaemia/reperfusion injury

Although recent preclinical and clinical studies have demonstrated that recombinant human relaxin (rhRLX) may have important therapeutic potential in acute heart failure and chronic kidney diseases, the effects of acute rhRLX administration against renal ischaemia/reperfusion (I/R) injury have never been investigated. Using a rat model of 1‐hr bilateral renal artery occlusion followed by 6‐hr reperfusion, we investigated the effects of rhRLX (5 μg/Kg i.v.) given both at the beginning and after 3 hrs of reperfusion. Acute rhRLX administration attenuated the functional renal injury (increase in serum urea and creatinine), glomerular dysfunction (decrease in creatinine clearance) and tubular dysfunction (increase in urinary excretion of N‐acetyl‐β‐glucosaminidase) evoked by renal I/R. These beneficial effects were accompanied by a significant reduction in local lipid peroxidation, free radical‐induced DNA damage and increase in the expression/activity of the endogenous antioxidant enzymes Mn‐ and CuZn‐superoxide dismutases (SOD). Furthermore, rhRLX administration attenuated the increase in leucocyte activation, as suggested by inhibition of myeloperoxidase activity, intercellular‐adhesion‐molecule‐1 expression, interleukin (IL)‐1β, IL‐18 and tumour necrosis factor‐α production as well as increase in IL‐10 production. Interestingly, the reduced oxidative stress status and neutrophil activation here reported were associated with rhRLX‐induced activation of endothelial nitric oxide synthase and up‐regulation of inducible nitric oxide synthase, possibly secondary to activation of Akt and the extracellular signal‐regulated protein kinase (ERK) 1/2, respectively. Thus, we report herein that rhRLX protects the kidney against I/R injury by a mechanism that involves changes in nitric oxide signalling pathway.

Celecoxib decreases expression of the adhesion molecules ICAM‐1 and VCAM‐1 in a colon cancer cell line (HT29)

We investigated the ability of celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, to modulate expression of ICAM‐1 and VCAM‐1 in the colon cancer cell line HT29.

Peroxisome proliferator-activated receptor β/δ agonism protects the kidney against ischemia/reperfusion injury in diabetic rats

Diabetes is an important risk factor for ischemic acute kidney injury, whose pharmacological treatment remains an unmet medical need. The peroxisome proliferator-activated receptor (PPAR) β/δ is highly expressed in the kidney, although its role has not yet been elucidated. Here, we used an in vivo model of renal ischemia/reperfusion (I/R) in streptozotocin-induced diabetic rats (i) to evaluate whether diabetes increases kidney susceptibility to I/R injury and (ii) to investigate the effects of PPARβ/δ activation. The degree of renal injury (1h ischemia/6h reperfusion) was significantly increased in diabetic rats compared with nondiabetic littermates. PPARβ/δ expression was increased after I/R, with the highest levels in diabetic rats. Administration of the selective PPARβ/δ agonist GW0742 attenuated the renal dysfunction, leukocyte infiltration, and formation of interleukin-6 and tumor necrosis factor-α. These effects were accompanied by an increased expression of the suppressor of cytokine signaling (SOCS)-3, which plays a critical role in the cytokine-activated signaling pathway. The beneficial effects of GW0742 were attenuated by the selective PPARβ/δ antagonist GSK0660. Thus, we report herein that PPARβ/δ activation protects the diabetic kidney against I/R injury by a mechanism that may involve changes in renal expression of SOCS-3 resulting in a reduced local inflammatory response.

Substance P-induced cyclooxygenase-2 expression in human umbilical vein endothelial cells

Substance P (SP) is a neuropeptide involved in neurogenic inflammation and an agonist for NK1, NK2, and NK3 receptors. SP induces prostaglandin (PG) production in various cell types, and these eicosanoids are responsible for numerous inflammatory and vascular effects. Cyclooxygenase (COX) are needed to convert arachidonic acid to PGs. The study evaluated the effect of SP on COX expression in human umbilical vein endothelial cells (HUVEC). COX‐2 protein expression was upregulated by SP with a peak at 100 nM and at 20 h; in the same experimental conditions COX‐1 protein expression was unchanged. A correlation between COX‐2 expression and PGI2 and PGE2 release was detected. Dexamethasone (DEX) inhibited SP‐mediated COX‐2 expression. Mitogen‐activated protein kinases (MAPK) p38 and p42/44 were activated by SP, whereas SB202190 and PD98059, inhibitors of these kinases, blocked COX‐2 expression. 5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone (DFU), an experimental selective COX‐2 inhibitor, blocked SP‐induced PG release. By RT–PCR and Western blot analysis, we demonstrated that NK1 and NK2 but not NK3 receptors are present on HUVEC. Selective NK1 and NK2 agonists, namely [Sar9, Met(O2)11]SP and [β‐Ala8] NKA(4–10), upregulated COX‐2 protein expression and PG production, whereas senktide (Suc–Asp–Phe–MePhe–Gly–Leu–Met–NH2), a selective NK3 agonist, was ineffective in this respect. The NK1 selective antagonist L703,606 ((cis)‐2‐(diphenylmethyl)‐N‐((2‐iodophenyl)‐methyl)‐1‐azabicyclo(2.2.2)octan‐3‐amine) and the NK2 selective antagonist SR 48,968 ((S)‐N‐methyl‐N‐(4‐(4‐acetylamino‐4‐phenylpiperidino)‐2‐(3,4 dichlorophenyl)butyl) benzamide) competitively antagonised SP‐induced effects. The study shows HUVEC to possess functional NK1 and NK2 receptors, which mediate the ability of SP to induce expression of COX‐2 in HUVEC, thus showing a previously‐undetected effect of SP on endothelial cells.

High Sugar Intake and Development of Skeletal Muscle Insulin Resistance and Inflammation in Mice: A Protective Role for PPAR-δ Agonism

Peroxisome Proliferator Activated Receptor (PPAR)-δ agonists may serve for treating metabolic diseases. However, the effects of PPAR-δ agonism within the skeletal muscle, which plays a key role in whole-body glucose metabolism, remain unclear. This study aimed to investigate the signaling pathways activated in the gastrocnemius muscle by chronic administration of the selective PPAR-δ agonist, GW0742 (1 mg/kg/day for 16 weeks), in male C57Bl6/J mice treated for 30 weeks with high-fructose corn syrup (HFCS), the major sweetener in foods and soft-drinks (15% wt/vol in drinking water). Mice fed with the HFCS diet exhibited hyperlipidemia, hyperinsulinemia, hyperleptinemia, and hypoadiponectinemia. In the gastrocnemius muscle, HFCS impaired insulin and AMP-activated protein kinase signaling pathways and reduced GLUT-4 and GLUT-5 expression and membrane translocation. GW0742 administration induced PPAR-δ upregulation and improvement in glucose and lipid metabolism. Diet-induced activation of nuclear factor-κB and expression of inducible-nitric-oxide-synthase and intercellular-adhesion-molecule-1 were attenuated by drug treatment. These effects were accompanied by reduction in the serum concentration of interleukin-6 and increase in muscular expression of fibroblast growth factor-21. Overall, here we show that PPAR-δ activation protects the skeletal muscle against the metabolic abnormalities caused by chronic HFCS exposure by affecting multiple levels of the insulin and inflammatory cascades.

Urinary metabolic fingerprinting of mice with diet-induced metabolic derangements by parallel dual secondary column-dual detection two-dimensional comprehensive gas chromatography

This study investigates the potential of a parallel dual secondary column-dual detection two-dimensional comprehensive GC platform (GC×2GC-MS/FID) for metabolic profiling and fingerprinting of mouse urine. Samples were obtained from a murine model that mimics a typical unhealthy Western diet featuring both high fat and sugar (HFHS) intake, which induces obesity, dyslipidemia, and insulin resistance. Urines collected at different steps of the study were used to obtain pivotal and comparative data on the presence and relative distributions of early markers of metabolic disease. The data elaboration and interpretation work-flow includes an advanced untargeted fingerprinting approach, with peak-region features to locate relevant features to be quantified by external standard calibration. The reliability of untargeted fingerprinting is confirmed by quantitative results on selected relevant features that showed percentages of variations consistent with those observed by comparing raw data quantitative descriptors (2D peak-region volumes and percent of response). Analytes that were up-regulated with % of variation ranging from 30 to 1000, included pyruvic acid, glycerol, fructose, galactose, glucose, lactic acid, mannitol and valine. Down-regulation was evidenced for malonic acid, succinic acid, alanine, glycine, and creatinine. Advanced fingerprinting also is demonstrated for effectively evaluating individual variations during experiments, thus representing a promising tool for personalized intervention studies. In this context, it is interesting to observe that informative features that were not discriminant for the entire population may be relevant for individuals.