Effects of 5,14-HEDGE, a 20-HETE mimetic, on lipopolysaccharide-induced changes in MyD88/TAK1/IKKß/IκB-α/NF-κB pathway and circulating miR-150, miR-223, and miR-297 levels in a rat model of septic shock.

OBJECTIVES: We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (5,14-HEDGE), which mimics the effects of endogenously produced 20-HETE, prevents vascular hyporeactivity, hypotension, tachycardia, inflammation, and mortality in a rodent model of septic shock. The present study was performed to determine whether decreased renal and cardiovascular expression and activity of myeloid differentiation factor 88 (MyD88)/transforming growth factor-activated kinase 1 (TAK1)/inhibitor of κB (IκB) kinase ß (IKKß)/IκB-α/nuclear factor-κB (NF-κB) pathway and reduced circulating microRNA (miR)-150, miR-223, and miR-297 expression levels participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, and inflammation in response to systemic administration of lipopolysaccharide (LPS). METHODS: Conscious male Wistar rats received saline (4 ml/kg) or LPS (10 mg/kg) at time 0. Blood pressure and heart rate were measured using a tail-cuff device. Separate groups of LPS-treated rats were given 5,14-HEDGE (30 mg/kg) 1 h after injection of saline or LPS. The rats were killed 4 h after LPS challenge and blood, kidney, heart, thoracic aorta, and superior mesenteric artery were collected for measurement of the protein expression. RESULTS: LPS-induced fall in blood pressure and rise in heart rate were associated with increased MyD88 expression and phosphorylation of TAK1 and IκB-α in cytosolic fractions of the tissues. LPS also caused an increase in both unphosphorylated and phosphorylated NF-κB p65 proteins in the cytosolic and nuclear fractions as well as nuclear translocation of NF-κB p65. In addition, serum miR-150, miR-223, and miR-297 expression levels were increased in LPS-treated rats. These effects of LPS were prevented by 5,14-HEDGE. CONCLUSIONS: These results suggest that downregulation of MyD88/TAK1/IKKß/IκB-α/NF-κB pathway as well as decreased circulating miR-150, miR-223, and miR-297 expression levels participate in the protective effect of 5,14-HEDGE against hypotension, tachycardia, and inflammation in the rat model of septic shock.

Contribution of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway to ischemia/reperfusion-induced oxidative/nitrosative stress and inflammation leading to distant and target organ injury in rats.

The small G protein RhoA and its downstream effector Rho-kinase play an important role in various physiopathological processes including ischemia/reperfusion (I/R) injury. Reactive oxygen and nitrogen species produced by iNOS and NADPH oxidase are important mediators of inflammation and organ injury following an initial localized I/R event. The aim of this study was to determine whether RhoA/Rho-kinase signaling pathway increases the expression and activity of MEK1, ERK1/2, iNOS, gp91(phox), and p47(phox), and peroxynitrite formation which result in oxidative/nitrosative stress and inflammation leading to hindlimb I/R-induced injury in kidney as a distant organ and gastrocnemius muscle as a target organ. I/R-induced distant and target organ injury was performed by using the rat hindlimb tourniquet model. I/R caused an increase in the expression and/or activity of RhoA, MEK1, ERK1/2, iNOS, gp91(phox), p47(phox), and 3-nitrotyrosine and nitrotyrosine levels in the tissues. Although Rho-kinase activity was increased by I/R in the kidney, its activity was decreased in the muscle. Serum and tissue MDA levels and MPO activity were increased following I/R. I/R also caused an increase in SOD and catalase activities associated with decreased GSH levels in the tissues. Y-27632, a selective Rho-kinase inhibitor, (100µg/kg, i.p.; 1h before reperfusion) prevented the I/R-induced changes except Rho-kinase activity in the muscle. These results suggest that activation of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway associated with oxidative/nitrosative stress and inflammation contributes to hindlimb I/R-induced distant organ injury in rats. It also seems that hindlimb I/R induces target organ injury via upregulation of RhoA/MEK1/ERK1/2/iNOS pathway associated with decreased Rho-kinase activity.

Contribution of MEK1/ERK1/2/iNOS pathway to oxidative stress and decreased caspase-3 activity in endotoxemic rats.

Oxidative stress and apoptosis are the states that can contribute to the pathogenesis of sepsis. In this study we aimed to investigate whether mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2)/inducible nitric oxide synthase (iNOS) pathway plays a role in oxidative stress and apoptosis in endotoxemic rats. Systemic total antioxidant, SOD, GPx, and GR activities as markers of oxidative stress, and tissue caspase-3 enzyme activity as a marker of apoptosis were measured in sera and thoracic aortae of male Wistar rats sacrificed 4 h after being treated with saline (vehicle) or lipopolysaccharide (LPS) (10 mg/kg, i.p.). A decrease in total antioxidant activity and caspase-3, SOD, GPx, and GR enzyme activities was occured by LPS. These changes caused by LPS were prevented when a selective iNOS inhibitor, 1,3-PBIT (10 mg/kg, i.p.) or a selective inhibitor of ERK1/2 phosphorylation by MEK1, U0126 (5 mg/kg, i.p.) were given 1 h after administration of LPS. Our results suggest that decreased activity of MEK1/ERK1/2/iNOS pathway prevents oxidative stress by increasing systemic antioxidant enzyme activities and restores decreased caspase-3 activity in thoracic aorta in endotoxemic rat.

Increased production of nitric oxide mediates selective organ-specific effects of endotoxin on oxidative stress.

Endotoxemic shock is a systemic inflammatory response that is associated with increased nitric oxide (NO) production by inducible NO synthase (iNOS) which contributes to hypotension, vascular hyporeactivity, and multiple organ failure. Oxidative stress (OS) is a major contributing factor to high morbidity and mortality in endotoxemic shock. We have previously demonstrated that endotoxin-induced fall in blood pressure is associated with an increase in nitrite levels in serum, kidney, heart, thoracic aorta (TA), and superior mesenteric artery (SMA), a decrease in malondialdehyde (MDA) levels in the kidney, heart, TA, and SMA, and an increase in myeloperoxidase (MPO) activity in the heart and TA, but a decrease in the kidney and SMA of rats. In this study, we further investigated whether increased production of iNOS-derived NO contributes to endotoxin induced changes in the biomarkers of OS in the liver, lungs, brain, spleen, and femoral artery (FA) of rats. Endotoxin-induced increase in nitrite production was associated with a decrease in reduced glutathione levels in the liver, lungs, brain, spleen, and FA. MPO activity was increased by endotoxin in the lungs, spleen, and FA, but decreased in the liver and brain. MDA levels were increased by endotoxin in the lungs, brain, spleen, and FA, but were decreased in the liver. Activities of superoxide dismutase and catalase were decreased in the liver and spleen, but were increased in the lungs, brain, and FA. These effects of endotoxin were prevented by a selective iNOS inhibitor, phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide. These data suggest that iNOS-derived NO mediates selective organ-specific effects of endotoxin on OS.