Massimo Collino

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.

PPARγ stimulation promotes mitochondrial biogenesis and prevents glucose deprivation-induced neuronal cell loss

Peroxisome proliferator-activated receptor (PPAR)gamma stimulation provides protection in several models of neurological disorders, but the mechanisms underlying these effects remain to be fully elucidated. Here we have studied whether two PPARgamma agonists, pioglitazone and rosiglitazone, prevent loss of differentiated SH-SY5Y cells transiently exposed to glucose deprivation (GD). Nanomolar drug concentrations prevented GD-induced cell loss in a concentration- and time-dependent manner. These effects were abolished by malonate, a reversible mitochondrial Complex II inhibitor, while significantly potentiated by pyruvate, thus suggesting that they are related to mitochondrial function. During cell pretreatment, PPARgamma agonists promoted biogenesis of functional mitochondria, as indicated by the up-regulation of PPARgamma coactivator (PGC)-1alpha, NRF1, TFAM, cytochrome c oxidase subunit (CO) I and CO IV, and the increased level of mtDNA, while did not significantly change mitochondrial membrane potential. In addition, the analysis of the concentration-response and time-course curves for the protective effects and the up-regulation of mitochondrial biogenesis markers suggests that mitochondrial biogenesis and cell loss prevention are related effects. In conclusion our data indicate that a prolonged PPARgamma stimulation, by repeated administration of nanomolar pioglitazone or rosiglitazone concentrations, decreases GD-induced loss of differentiated SH-SY5Y cells. In addition, they suggest that mitochondrial biogenesis may contribute to these effects.

SREBP-1c in nonalcoholic fatty liver disease induced by Western-type high-fat diet plus fructose in rats.

This study concentrated on the initial events triggering the development of nonalcoholic fatty liver disease induced by a high-fat plus fructose (HF-F) diet and on the possibility of delaying nonalcoholic fatty liver disease progression by adding dehydroepiandrosterone (DHEA) to the diet. Sterol regulatory element binding protein-1c (SREBP-1c) activation plays a crucial role in the progression of nonalcoholic fatty liver disease induced by an HF-F diet. This study investigated the protective effects of DHEA, a compound of physiological origin with multitargeted antioxidant properties, against the induction of SREBP-1c and on liver insulin resistance in rats fed an HF-F diet, which mimics a typical unhealthy Western diet. An HF-F diet, fortified or not with DHEA (0.01%, w/w), was administered for 15 weeks to male Wistar rats. After HF-F the liver showed unbalanced oxidative status, fatty infiltration, hepatic insulin resistance, and inflammation. The addition of DHEA to the diet reduced both activation of oxidative-stress-dependent pathways and expression of SREBP-1c and partially restored the expression of liver X-activated receptor-alpha and insulin receptor substrate-2 genes. DHEA supplementation of the HF-F diet reduced de novo lipogenesis and delayed progression of nonalcoholic fatty liver disease, demonstrating a relationship between oxidative stress and nonalcoholic fatty liver disease via SREBP-1c.

Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation

Background and purpose:  Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator‐activated receptor (PPAR)γ agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARγ are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)‐3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARγ activation in a non‐genetic insulin‐resistant animal model.

Insulin Reduces Cerebral Ischemia/Reperfusion Injury in the Hippocampus of Diabetic Rats: A Role for Glycogen Synthase Kinase-3β

OBJECTIVE—There is evidence that insulin reduces brain injury evoked by ischemia/reperfusion (I/R). However, the molecular mechanisms underlying the protective effects of insulin remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3β (GSK-3β). Here, we investigate the role of GSK-3β inhibition on I/R-induced cerebral injury in a rat model of insulinopenic diabetes. RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were subjected to 30-min occlusion of common carotid arteries followed by 1 or 24 h of reperfusion. Insulin (2–12 IU/kg i.v.) or the selective GSK-3β inhibitor TDZD-8 (0.2–3 mg/kg i.v.) was administered during reperfusion. RESULTS—Insulin or TDZD-8 dramatically reduced infarct volume and levels of S100B protein, a marker of cerebral injury. Both drugs induced phosphorylation of the Ser9 residue, thereby inactivating GSK-3β in the rat hippocampus. Insulin, but not TDZD-8, lowered blood glucose. The hippocampi of the drug-treated animals displayed reduced oxidative stress at 1 h of reperfusion as shown by the decreased generation of reactive oxygen species and lipid peroxidation. I/R-induced activation of nuclear factor-κB was attenuated by both drug treatments. At 24 h of reperfusion, TDZD-8 and insulin significantly reduced plasma levels of tumor necrosis factor-α; neutrophil infiltration, measured as myeloperoxidase activity and intercellular-adhesion-molecule-1 expression; and cyclooxygenase-2 and inducible-NO-synthase expression. CONCLUSIONS—Acute administration of insulin or TDZD-8 reduced cerebral I/R injury in diabetic rats. We propose that the inhibitory effect on the activity of GSK-3β contributes to the protective effect of insulin independently of any effects on blood glucose.

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.

Review: PPARs as new therapeutic targets for the treatment of cerebral ischemia/reperfusion injury

Stroke is a leading cause of death and long-term disability in industrialized countries. Despite advances in understanding its pathophysiology, little progress has been made in the treatment of stroke. The currently available therapies have proven to be highly unsatisfactory (except thrombolysis) and attempts are being made to identify and characterize signaling proteins which could be exploited to design novel therapeutic modalities. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that control lipid and glucose metabolism. PPARs regulate gene expression by binding with the retinoid X receptor (RXR) as a heterodimeric partner to specific DNA sequences, termed PPAR response elements. In addition, PPARs may modulate gene transcription also by directly interfering with other transcription factor pathways in a DNA-binding independent manner. To date, three different PPAR isoforms, designated α, β/δ, and γ, have been identified. Recently, they have been found to play an important role for the pathogenesis of various disorders of the central nervous system and accumulating data suggest that PPARs may serve as potential targets for treating ischemic stroke. Activation of all PPAR isoforms, but especially of PPARγ, was shown to prevent post-ischemic inflammation and neuronal damage in several in vitro and in vivo models, negatively regulating the expression of genes induced by ischemia/ reperfusion (I/R). This paper reviews the evidence and recent developments relating to the potential therapeutic effects of PPAR-agonists in the treatment of cerebral I/R injury.

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.

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.