The Antimalarial Drug Artesunate Attenuates Cardiac Injury in A Rodent Model of Myocardial Infarction.

Ischemic heart disease remains the leading cause of morbidity and mortality in the Western world. Artesunate is the WHO-recommended drug of choice for complicated malaria (with organ failure). The administration of high doses of artesunate is safe in healthy volunteers (up to 8 mg/kg i.v.) and patients with severe malaria (2.4 mg/kg i.v.). We investigated the effects of artesunate (1 mg/kg) or its active metabolite dihydroartemisinin (DHA; 0.1 mg/kg) in a model of transient myocardial ischemia/reperfusion (I/R) and evaluated the mechanism of action of the observed cardioprotective effects of artesunate and DHA. We report here for the first time that the administration of artesunate at the onset of reperfusion attenuates the myocardial injury associated with I/R. The observed beneficial effects of artesunate are associated with activation of the PI3K/Akt/ERK 1/2 (RISK) pathway, activation of endothelial nitric oxide synthase, inhibition of glycogen synthase kinase-3ß, inhibition of nuclear factor kappa B, and activation of the STAT3 (SAFE) pathway. In conclusion, as artesunate has an excellent safety profile, the above data should stimulate clinical trials in patients with acute coronary syndromes.

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1ß. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

A nitric oxide-donor furoxan moiety improves the efficacy of edaravone against early renal dysfunction and injury evoked by ischemia/reperfusion.

Edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, EDV) is a free-radical scavenger reduces organ ischemic injury. Here we investigated whether the protective effects of EDV in renal ischemia/reperfusion (I/R) injury may be enhanced by an EDV derivative bearing a nitric oxide- (NO-) donor furoxan moiety (NO-EDV). Male Wistar rats were subjected to renal ischemia (45 minutes), followed by reperfusion (6 hours). Administration of either EDV (1.2-6-30 µmol/kg, i.v.) or NO-EDV (0.3-1.2-6 µmol/kg, i.v.) dose-dependently attenuated markers of renal dysfunction (serum urea and creatinine, creatinine clearance, urine flow, urinary N-acetyl-ß-D-glucosaminidase, and neutrophil gelatinase-associated lipocalin/lipocalin-2). NO-EDV exerted protective effects in the dose-range 1.2-6 µmol/kg, while a higher dose (30 µmol/kg) was needed to obtain protection by EDV. Both EDV and NO-EDV modulated tissue markers of oxidative stress and lipid peroxidation. NO-EDV, but not EDV, activated endothelial NO synthase (NOS) and blunted I/R-induced upregulation of inducible NOS, secondary to modulation of Akt and NF-κB activation, respectively. Besides NO-EDV administration inhibited I/R-induced IL-1ß, IL-18, IL-6, and TNF-α overproduction. Overall, these findings demonstrate that the NO-donor moiety contributes to the protection against early renal I/R injury and suggest that NO-donor EDV codrugs are worthy of additional study as innovative pharmacological tools.

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-ß-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)-1ß, 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.

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.

Advanced glycation end products promote hepatosteatosis by interfering with SCAP-SREBP pathway in fructose-drinking mice.

Clinical studies have linked the increased consumption of fructose to the development of obesity, dyslipidemia, and impaired glucose tolerance, and a role in hepatosteatosis development is presumed. Fructose can undergo a nonenzymatic reaction from which advanced glycation end products (AGEs) are derived, leading to the formation of dysfunctional, fructosylated proteins; however, the in vivo formation of AGEs from fructose is still less known than that from glucose. In the present study C57Bl/6J mice received 15% (wt/vol) fructose (FRT) or 15% (wt/vol) glucose (GLC) in water to drink for 30 wk, resembling human habit to consume sugary drinks. At the end of the protocol both FRT- and GLC-drinking mice had increased fasting glycemia, glucose intolerance, altered plasma lipid profile, and marked hepatosteatosis. FRT mice had higher hepatic triglycerides deposition than GLC, paralleled by a greater increased expression and activity of the sterol regulatory element-binding protein 1 (SREBP1), the transcription factor responsible for the de novo lipogenesis, and of its activating protein SCAP. LC-MS analysis showed a different pattern of AGE production in liver tissue between FRT and GLC mice, with larger amount of carboxymethyl lysine (CML) generated by fructose. Double immunofluorescence and coimmunoprecipitation analysis revealed an interaction between CML and SCAP that could lead to prolonged activation of SREBP1. Overall, the high levels of CML and activation of SCAP/SREBP pathway associated to high fructose exposure here reported may suggest a key role of this signaling pathway in mediating fructose-induced lipogenesis.

Inhibition of IκB kinase reduces the multiple organ dysfunction caused by sepsis in the mouse.

Nuclear factor κB (NF-κB) plays a pivotal role in sepsis. Activation of NF-κB is initiated by the signal-induced ubiquitylation and subsequent degradation of inhibitors of kappa B (IκBs) primarily via activation of the IκB kinase (IKK). This study was designed to investigate the effects of IKK inhibition on sepsis-associated multiple organ dysfunction and/or injury (MOD) and to elucidate underlying signaling mechanisms in two different in vivo models: male C57BL/6 mice were subjected to either bacterial cell wall components [lipopolysaccharide and peptidoglycan (LPS/PepG)] or underwent cecal ligation and puncture (CLP) to induce sepsis-associated MOD. At 1 hour after LPS/PepG or CLP, mice were treated with the IKK inhibitor IKK 16 (1 mg/kg body weight). At 24 hours, parameters of organ dysfunction and/or injury were assessed in both models. Mice developed a significant impairment in systolic contractility (echocardiography), and significant increases in serum creatinine, serum alanine aminotransferase and lung myeloperoxidase activity, thus indicating cardiac dysfunction, renal dysfunction, hepatocellular injury and lung inflammation, respectively. Treatment with IKK 16 attenuated the impairment in systolic contractility, renal dysfunction, hepatocellular injury and lung inflammation in LPS/PepG-induced MOD and in polymicrobial sepsis. Compared with mice that were injected with LPS/PepG or underwent CLP, immunoblot analyses of heart and liver tissues from mice that were injected with LPS/PepG or underwent CLP and were also treated with IKK 16 revealed: (1) significant attenuation of the increased phosphorylation of IκBα; (2) significant attenuation of the increased nuclear translocation of the NF-κB subunit p65; (3) significant attenuation of the increase in inducible nitric oxide synthase (iNOS) expression; and (4) a significant increase in the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Here, we report for the first time that delayed IKK inhibition reduces MOD in experimental sepsis. We suggest that this protective effect is (at least in part) attributable to inhibition of inflammation through NF-κB, the subsequent decrease in iNOS expression and the activation of the Akt-eNOS survival pathway.

Erythropoietin attenuates acute kidney dysfunction in murine experimental sepsis by activation of the ß-common receptor.

The ß-common receptor (ßcR) plays a pivotal role in the nonhematopoietic tissue-protective effects of erythropoietin (EPO). Here we determined whether EPO reduces the acute kidney injury (AKI) caused by sepsis and whether this effect is mediated by the ßcR. In young (2 months old) C57BL/6 wild-type and ßcR knockout mice, lipopolysaccharide caused a significant increase in serum urea and creatinine, hence AKI. This AKI was not associated with any overt morphological alterations in the kidney and was attenuated by EPO given 1 h after lipopolysaccharide in wild-type but not in ßcR knockout mice. In the kidneys of endotoxemic wild-type mice, EPO enhanced the phosphorylation of Akt, glycogen synthase kinase-3ß, and endothelial nitric oxide synthase, and inhibited the activation of nuclear factor-κB. All these effects of EPO were lost in ßcR knockout mice. Since sepsis is more severe in older animals or patients, we tested whether EPO was renoprotective in 8-month-old wild-type and ßcR knockout mice that underwent cecal ligation and puncture. These older mice developed AKI at 24 h, which was attenuated by EPO treatment 1 h post cecal ligation and puncture in wild-type mice but not in ßcR knockout mice. Thus, activation of the ßcR by EPO is essential for the observed reduction in AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO.

Erythropoietin attenuates cardiac dysfunction in experimental sepsis in mice via activation of the ß-common receptor.

There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the ß-common receptor ('tissue-protective receptor'), which is pharmacologically distinct from the 'classical' erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the ß-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the ß-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and ß-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16-18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a ß-common-receptor-dependent fashion. Activation by erythropoietin of the ß-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3ß, nuclear factor-κB and interleukin-1ß). All the above pleiotropic effects of erythropoietin were lost in ß-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the ß-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation.

Reversal of the deleterious effects of chronic dietary HFCS-55 intake by PPAR-δ agonism correlates with impaired NLRP3 inflammasome activation.

Although high-fructose corn syrup (HFCS-55) is the major sweetener in foods and soft-drinks, its potential role in the pathophysiology of diabetes and obesity ("diabesity") remains unclear. Peroxisome-proliferator activated receptor (PPAR)-δ agonists have never been tested in models of sugar-induced metabolic abnormalities. This study was designed to evaluate (i) the metabolic and renal consequences of HFCS-55 administration (15% wt/vol in drinking water) for 30 weeks on male C57Bl6/J mice and (ii) the effects of the selective PPAR-δ agonist GW0742 (1 mg/kg/day for 16 weeks) in this condition. HFCS-55 caused (i) hyperlipidemia, (ii) insulin resistance, and (iii) renal injury/inflammation. In the liver, HFCS-55 enhanced the expression of fructokinase resulting in hyperuricemia and caused abnormalities in known insulin-driven signaling events. In the kidney, HFCS-55 enhanced the expression of the NLRP3 (nucleotide-binding domain and leucine-rich-repeat-protein 3) inflammasome complex, resulting in caspase-1 activation and interleukin-1ß production. All of the above effects of HFCS-55 were attenuated by the specific PPAR-δ agonist GW0742. Thus, we demonstrate for the first time that the specific PPAR-δ agonist GW0742 attenuates the metabolic abnormalities and the renal dysfunction/inflammation caused by chronic HFCS-55 exposure by preventing upregulation of fructokinase (liver) and activation of the NLRP3 inflammasome (kidney).

Pharmacological preconditioning with erythropoietin attenuates the organ injury and dysfunction induced in a rat model of hemorrhagic shock.

Pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue-protective effects against 'ischemia-reperfusion'-type injuries. This protection might be mediated by mobilization of bone marrow endothelial progenitor cells (EPCs), which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, for example, prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). Recombinant human EPO (1000 IU/kg/day i.p.) was administered to rats for 3 days. Rats were subjected to HS on day 4 (pre-treatment protocol). Mean arterial pressure was reduced to 35 ± 5 mmHg for 90 minutes, followed by resuscitation with 20 ml/kg Ringer's lactate for 10 minutes and 50% of the shed blood for 50 minutes. Rats were sacrificed 4 hours after the onset of resuscitation. EPC (CD34(+)/flk-1(+) cell) mobilization was measured following the 3-day pre-treatment with EPO and was significantly increased compared with rats pre-treated with phosphate-buffered saline. EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic and neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3ß (GSK-3ß; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing with EPO prior to injury might protect against the organ injury and dysfunction induced by HS, by a mechanism that might involve mobilization of CD34(+)/flk-1(+) cells, resulting in the activation of the Akt-eNOS survival pathway and inhibition of activation of GSK-3ß and NF-κB.

Delayed administration of pyroglutamate helix B surface peptide (pHBSP), a novel nonerythropoietic analog of erythropoietin, attenuates acute kidney injury.

In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion-associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the ß-common receptor (termed "tissue-protective receptor"). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 µg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3ß (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.