Xiaoxuan Cui

Ghrelin Down-regulates Proinflammatory Cytokines in Sepsis Through Activation of the Vagus Nerve

Objective:To test the hypothesis that administration of ghrelin attenuates inflammatory responses in sepsis through vagal nerve stimulation. Summary Background Data:Ghrelin has been demonstrated to possess multiple functions, including stimulation of the vagus nerve. Our recent study has shown that plasma levels of ghrelin were significantly reduced in sepsis; and ghrelin administration improved organ perfusion and function. However, it remained unknown whether ghrelin also decreases proinflammatory cytokines in sepsis and, if so, whether the down-regulatory effect of ghrelin is mediated by activation of the vagus nerve. Methods:Male rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 hours after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed 200-&mgr;L Alzet mini-pump for 15 hours. At 20 hours after CLP, plasma and peritoneal fluid levels of TNF-α and IL-6 were determined. The direct effect of ghrelin on cytokine production was studied using cultured normal rat Kupffer cells or peritoneal macrophages stimulated by lipopolysaccharide (LPS). In additional animals, vagotomy or sham vagotomy was performed in sham and septic animals immediately prior to ghrelin administration and cytokine levels were then measured. Results:Ghrelin significantly reduced TNF-α and IL-6 levels in sepsis. In contrast, ghrelin did not inhibit TNF-α and IL-6 release from LPS-stimulated Kupffer cells or peritoneal macrophages. However, vagotomy, but not sham vagotomy, prevented ghrelins down-regulatory effect on TNF-α and IL-6 production. Conclusions:Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve. Pharmacologic stimulation of the vagus nerve may offer a novel approach of anti-sepsis therapy.

The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-γ*

Objective:Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-γ, an anti-inflammatory nuclear receptor, plays any role. Design:Prospective, controlled, and randomized animal study. Setting:A research institute laboratory. Subjects:Male Sprague-Dawley rats. Interventions:A bolus injection of 0.2 &mgr;mol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 &mgr;mol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP). Measurements and Main Results:Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-α were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-α and PPAR-γ expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-α in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-γ in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-γ antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-α expression and markedly up-regulated PPAR-γ expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin. Conclusions:The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-γ.

Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-γ

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-γ expression in vivo and in vitro and was associated with increased TNF-α production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-γ levels in both models, resulting in TNF-α suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-α production in the absence of PPAR-γ. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced ΤΝF-α release, it did not alter PPAR-γ expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-γ and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-γ.

Adrenomedullin and its binding protein attenuate the proinflammatory response after hemorrhage

Objective:The neuroendocrine response to hemorrhage is to maintain perfusion to the heart and brain, often at the expense of other organ systems. Systemic inflammation and tissue injury are important components of pathophysiologic consequences of hemorrhage. We have recently shown that administration of adrenomedullin (AM, a potent vasodilator peptide) and adrenomedullin binding protein-1 (AMBP-1) prevented the transition from the hyperdynamic to the hypodynamic stage in the progression of sepsis. However, the effect of AM/AMBP-1 on the inflammatory response after hemorrhage remains unknown. We therefore hypothesized that administration of AM/AMBP-1 during fluid resuscitation in hemorrhaged animals (i.e., posttreatment) attenuates tissue injury and the proinflammatory response. Design:Prospective, controlled, and randomized animal study. Setting:A research institute laboratory. Subjects:Male adult rats. Interventions:Rats were bled, and then a mean arterial pressure was maintained at 40 mm Hg for 90 mins. They were then resuscitated by infusion of four times the volume of shed blood using Ringer’s lactate solution for 60 mins. Measurements and Main Results:Fifteen minutes after the beginning of resuscitation, AM (12 &mgr;g/kg of body weight) in combination with AMBP-1 (40 &mgr;g/kg of body weight) was administered via a femoral venous catheter for 45 mins. Blood samples were collected 4 hrs postresuscitation and assayed for levels of liver enzymes (i.e., alanine aminotransferase and aspartate aminotransferase), lactate, creatinine, proinflammatory cytokines tumor necrosis factor and high mobility group box 1, and anti-inflammatory cytokine interleukin-10. The results indicate that levels of alanine aminotransferase, aspartate aminotransferase, creatinine, lactate, tumor necrosis factor, and high mobility group box 1 markedly elevated after hemorrhage and resuscitation, and AM/AMBP-1 treatment significantly attenuated these increases. In contrast, the serum concentration of anti-inflammatory cytokine interleukin-10 was increased by the treatment of AM/AMBP-1. Moreover, AM/AMBP-1 treatment significantly improved the survival rate from 35% in vehicle-treated animals to 73% in AM/AMBP-1-treated animals in a low-volume resuscitation model of hemorrhage. Conclusion:The combined administration of AM and AMBP-1 effectively suppresses hemorrhage-elicited organ injury and reduces hemorrhage-induced mortality, partly through down-regulation of proinflammatory cytokines (tumor necrosis factor and high mobility group box 1) and up-regulation of the anti-inflammatory cytokine interleukin-10.

Mechanisms Responsible for Vascular Hyporesponsiveness to Adrenomedullin after Hemorrhage: The Central Role of Adrenomedullin Binding Protein-1

Objective:Irreversible hypovolemia remains a major clinical problem. Preliminary studies indicate that administration of adrenomedullin and adrenomedullin binding protein-1 in combination (AM/AMBP-1) after hemorrhage, improves cardiovascular function despite the increased levels of AM. Our aim was to determine whether vascular responsiveness to AM is reduced after hemorrhage and, if so, to elucidate the possible mechanism responsible for such hyporesponsiveness. Methods:Male rats were bled to and maintained at a mean arterial pressure of 40 mm Hg for 90 minutes. The animals were then resuscitated with 4 times the volume of shed blood with lactated Ringers solution over 60 minutes. At 1.5 hours postresuscitation, vascular responses to AM and AMBP-1, plasma levels of AM and AMBP-1, AMBP-1 and AM receptor gene expression were measured. In additional animals, AM and AMBP-1 were administered intravenously at 15 minutes after resuscitation over 45 minutes. Serum levels of liver enzymes, lactate, creatinine, TNF-α, IL-6, and IL-10 were measured at 1.5 hours postresuscitation. Results:AM-induced vascular relaxation decreased significantly after hemorrhage and resuscitation, which was markedly improved by AMBP-1. However, AM receptor gene expression did not change under such conditions. Hemorrhage-induced AM hyporesponsiveness was accompanied by the decreased expression and release of AMBP-1. Moreover, AM/AMBP-1 treatment down-regulated TNF-α and IL-6, up-regulated IL-10, and attenuated organ injury. Conclusions:The decreased AMBP-1 levels rather than alterations in AM receptors are responsible for producing AM hyporesponsiveness after hemorrhage. Thus, administration of AMBP-1 in combination with AM can be useful to reduce organ injury after severe hypovolemia.

Ghrelin protects rats against traumatic brain injury and hemorrhagic shock through upregulation of UCP2.

Objective:To determine the mechanism responsible for ghrelins neuroprotective effects after traumatic brain injury (TBI) and hemorrhagic shock. Background:Ghrelin, a gastrointestinal hormone, has been demonstrated to possess multiple functions, including upregulation of uncoupling protein 2 (UCP2) and stimulation of the vagus nerve. Recent evidence has indicated that ghrelin is neuroprotective. We, therefore, hypothesized that ghrelin protects rats against TBI and hemorrhagic shock through upregulation of UCP2, involving stimulation of the vagus nerve. Methods:Brain injury was induced by dropping a 450 g of weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a midline laparotomy was performed, and both lumbar veins were isolated and severed at the junction with the vena cava. The abdomen was kept open for 20 minutes. At 45 minutes after TBI and uncontrolled hemorrhage (UH), ghrelin (4, 8, or 16 nmol/rat) or 1 mL of normal saline (vehicle) was intravenously administered. The Neurological Severity Scale (NSS), morphological alterations and &bgr;-amyloid precursor protein expression in the brain, systemic organ injury markers (ie, alanine aminotransferase, aspartate aminotransferase, and lactate), and UCP2 expression in the cortex were measured. To determine whether the protective effect of ghrelin is mediated through upregulation of UCP2, genipin, a specific UCP2 antagonist, was administered intravenously before the injection of ghrelin in animals with TBI and UH. The role of the vagus nerve was assessed by performing vagotomy immediately before ghrelin administration. Results:Ghrelin attenuated brain injury and facilitated functional recovery after TBI and UH. Ghrelin increased UCP2 expression in the cortex, and administration of genipin abolished ghrelins protection after TBI and UH. Furthermore, vagotomy prevented the beneficial effects of ghrelin and eliminated ghrelin-induced UCP2 upregulation after TBI and UH. Conclusions:The protective effects of ghrelin after TBI and UH seem to be related to upregulation of UCP2 expression in the brain and requiring the intact vagus nerve.

Differential expression of cytochrome P450 isoforms in the lungs of septic animals

Objective:Sepsis is characterized by an early, hyperdynamic phase and a late, hypodynamic phase. Although studies have shown that cytochrome P450 (CYP) plays an important role in the regulation of vascular reactivity, alterations of vascular CYP isoforms in sepsis remain unknown. Since CYP2C11 and CYP2J4 convert arachidonic acid to vasodilative epoxyeicosatrienoic acids, and CYP4A3 metabolizes arachidonic acid to both epoxyeicosatrienoic acids and vasoconstrictive 19,20-hydroxyeicosatetraenoic acid, the aim of this study was to examine the expression of these isoforms in sepsis and their association with hemodynamic changes. Design:Prospective, controlled, and randomized animal study. Setting:An institute research laboratory. Subjects:Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). Interventions:At 5 hrs (early sepsis) or 20 hrs (late sepsis) after cecal ligation and puncture, blood vessel-rich tissues (i.e., lungs) were harvested. The expression of CYP isoforms at both messenger RNA and protein levels was determined by reverse transcription polymerase chain reaction and Western blot analysis (CYP2C11), respectively. Hemodynamic variables were measured by radioactive microspheres. Main Results:The results indicate that the gene expression of CYP2C11 and CYP2J4 was significantly down-regulated at 20 hrs after cecal ligation and puncture, whereas the expression of CYP4A3 was markedly up-regulated at 5 hrs. The protein concentrations of CYP2C11 also decreased significantly at 20 hrs after cecal ligation and puncture. Although total peripheral resistance markedly increased, mean arterial pressure did not change significantly at 20 hrs after the onset of sepsis. In contrast, cardiac output and pulmonary perfusion markedly decreased in late sepsis. Conclusions:Since the up-regulated CYP4A3 is associated with the early, hyperdynamic phase of sepsis and the down-regulated CYP2C11 and CYP2J4 are associated with the late, hypodynamic phase, vascular CYP isoforms that metabolize arachidonic acid may be involved in regulating the cardiovascular response during the progression of sepsis.