Features of Metabolite Changes in Disease Evolution in Cholecystolithiasis.

BACKGROUND: Cholecystolithiasis is defined as a disease caused by complex and changeable factors. Advanced age, female sex, and a hypercaloric diet rich in carbohydrates and poor in fiber, together with obesity and genetic factors, are the main factors that may predispose people to choledocholithiasis. However, serum biomarkers for the rapid diagnosis of choledocholithiasis remain unclear. AIMS: This study was designed to explore the pathogenesis of cholecystolithiasis and identify the possible metabolic and lipidomic biomarkers for the diagnosis of the disease. METHODS: Using UHPLC-MS/MS and GC-MS, we detected the serum of 28 cholecystolithiasis patients and 19 controls. Statistical analysis of multiple variables included Principal Component Analysis (PCA). Visualization of differential metabolites was performed using volcano plots. The screened differential metabolites were further analyzed using clustering heatmaps. The quality of the model was assessed using random forests. RESULTS: In this study, dramatically altered lipid homeostasis was detected in cholecystolithiasis group. In addition, the levels of short-chain fatty acids and amino acids were noticeably changed in patients with cholecystolithiasis. They detected higher levels of FFA.18.1, FFA.20.1, LPC16.0, and LPC20.1, but lower levels of 1-Methyl-L-histidine and 4-Hydroxyproline. In addition, glycine and L-Tyrosine were higher in choledocholithiasis group. Analyses of metabolic serum in affected patients have the potential to develop an integrated metabolite-based biomarker model that can facilitate the early diagnosis and treatment of the disease. CONCLUSION: Our results highlight the value of integrating lipid, amino acid, and short-chain fatty acid to explore the pathophysiology of cholecystolithiasis disease, and consequently, improve clinical decision-making.

[Screening and molecular identification of endophytic fungi promoting accumulation of flavonoids in callus of Scutellaria baicalensis].

To screen and identify the endophytic fungal strains that could promote the accumulation of flavonoids in the callus of Scutellaria baicalensis. Seventeen endophytic fungal strains from S. baicalensis were used to prepare mycelium elicitors and fermentation broth elicitors. Their effects on flavonoid accumulation in S. baicalensis callus were then determined. The results showed that the fermentation broth elicitors of two strains(CL79, CL105) promoted the accumulation of flavonoids. The fermentation broth elicitor of CL79 significantly promoted accumulation of baicalin, wogonoside, baicalein, and wogonin, with the maximum levels increased by 37.8%, 40.4%, 44.7%, and 42.2%(vs. blank), respectively. Similarly, the fermentation broth elicitor of CL105 significantly promoted the accumulation of baicalin, wogonoside, baicalein, and wogonin, with the maximum levels increased by 78.1%, 140.9%, 275.6%, and 208.5%(vs. blank), respectively. CL79 was identified as Alternaria alternata, and CL105 as Fusarium solani. The fermentation broth elicitors of A. alternata CL79 and F. solani CL105 were able to promote the flavonoid accumulation in the callus of S. baicalensis, which enriched the resources of endophytic fungi and provided candidate strains for the development of microbial fertili-zers for improving the quality of S. baicalensis.

The cultivable endophytic fungal community of Scutellaria baicalensis: diversity and relevance to flavonoid production by the host.

Scutellaria baicalensis (SB), a traditional Chinese medicinal plant, is widely used because of its important pharmacological activities. However, the endophytic fungi that promote flavonoid accumulation in SB remain unclear. Therefore, we analyzed the endophytic fungal community of SB and screened the endophytic fungi that might promote flavonoid synthesis in SB. ITS1/ITS4Blast was used to identify the endophytic fungi in SB. In total, 687 strains were identified in 57 genera. The dominant genus in the leaves and stems was Alternaria and that in the roots was Fusarium. Alternaria was the dominant genus in SB collected from all sites and in wild and cultivated SB. Alpha diversity indexes indicated more abundant endophytic fungi in samples from Chengde, the genuine producing area of SB, than in those from other sites. Beta diversity index analysis indicated that SB plants with closer geographical relationships showed more similar endophytic fungal community profiles. Spearman correlation analysis revealed that baicalin, wogonoside, wogonin, and oroxylin A contents were significantly correlated with the relative abundance of Alternaria. Overall, the results indicate the importance of geographical factors in influencing the endophytic fungal community of SB and suggest that the presence of Alternaria spp. might contribute to flavonoid synthesis in SB.

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

OBJECTIVE: To determine the mechanism responsible for ghrelin's 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 ß-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 ghrelin's 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.

Ghrelin attenuates brain injury after traumatic brain injury and uncontrolled hemorrhagic shock in rats.

Traumatic brain injury (TBI) and hemorrhagic shock often occur concomitantly due to multiple injuries. Gastrointestinal dysfunction occurs frequently in patients with TBI. However, whether alterations in the gastrointestinal system are involved in modulating neuronal damage and recovery after TBI is largely neglected. Ghrelin is a "gut-brain" hormone with multiple functions including antiinflammation and antiapoptosis. The purpose of this study was to determine whether ghrelin attenuates brain injury in a rat model of TBI and uncontrolled hemorrhage (UH). To study this, brain injury was induced by dropping a 450-g 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. At 45 min after TBI/UH, ghrelin (4, 8 or 16 nmol/rat) or 1 mL normal saline (vehicle) was intravenously administered. Brain levels of TNF-α and IL-6, and cleaved PARP-1 levels in the cortex were measured at 4 h after TBI/UH. Beam balance test, forelimb placing test and hindlimb placing test were used to assess sensorimotor and reflex function. In additional groups of animals, ghrelin (16 nmol/rat) or vehicle was subcutaneously (s.c.) administered daily for 10 d after TBI/UH. The animals were monitored for 28 d to record body weight changes, neurological severity scale and survival. Our results showed that ghrelin downregulated brain levels of TNF-α and IL-6, reduced cortical levels of cleaved PARP-1, improved sensorimotor and reflex functions, and decreased mortality after TBI/UH. Thus, ghrelin has a great potential to be further developed as an effective resuscitation approach for the trauma victims with brain injury and severe blood loss.

Vasoactive hormone adrenomedullin and its binding protein: anti-inflammatory effects by up-regulating peroxisome proliferator-activated receptor-gamma.

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-gamma (PPAR-gamma) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-gamma expression in vivo and in vitro and was associated with increased TNF-alpha production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-gamma levels in both models, resulting in TNF-alpha suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-alpha production in the absence of PPAR-gamma. 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 TauNuF-alpha release, it did not alter PPAR-gamma 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-gamma 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-gamma.

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-microL Alzet mini-pump for 15 hours. At 20 hours after CLP, plasma and peritoneal fluid levels of TNF-alpha 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-alpha and IL-6 levels in sepsis. In contrast, ghrelin did not inhibit TNF-alpha and IL-6 release from LPS-stimulated Kupffer cells or peritoneal macrophages. However, vagotomy, but not sham vagotomy, prevented ghrelin's down-regulatory effect on TNF-alpha 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.

Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia-reperfusion.

BACKGROUND: Recent studies have shown that adrenomedullin (AM) and AM-binding protein-1 (AMBP-1) possess anti-inflammatory properties in sepsis. We hypothesized that administration of AM/AMBP-1 after gut ischemia-reperfusion (I/R) downregulates inflammatory cytokines and attenuates tissue injury. METHODS: Male Sprague-Dawley rats (275-325 g) were used. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery (SMA) for 90 minutes. Upon release of the SMA clamp, the animals were treated by AM (12 microg per kilogram of body weight) and AMBP-1 (40 microg per kilogram of body weight) in combination, or vehicle (1 mL 0.9% NaCl) over 30 minutes via a femoral vein catheter. The animals undergoing sham operation or ischemia for 90 minutes only, did not receive AM/AMBP-1 treatment. At 60 minutes after the completion of the treatment (ie, 90 minutes after reperfusion), blood samples were collected. Plasma AM and AMBP-1 were measured by radioimmunoassay and Western blot analysis, respectively. Serum levels of TNF-alpha, interleukin (IL)-1beta, IL-6, IL-10, transaminases (ie, alanine aminotransaminase, aspartate aminotransaminase), lactate, and creatinine were determined with the use of enzyme-linked immunosorbent assay and other standard methods. In additional groups of animals, the 10-day survival rate was recorded after gut I/R. RESULTS: Ischemia alone was sufficient to downregulate both AM and AMBP-1. Unlike AMBP-1 that remained decreased, AM levels increased significantly after reperfusion. I/R but not ischemia alone significantly increased serum levels of inflammatory cytokines. Moreover, I/R-induced tissue injury was evidenced by increased levels of transaminases, lactate, and creatinine. Administration of AM/AMBP-1 after ischemia, however, markedly reduced cytokine levels, attenuated tissue injury, and improved survival. CONCLUSIONS: AM/AMBP-1 may be a novel treatment to attenuate the reperfusion injury after gut ischemia.

Suppression of hepatocyte CYP1A2 expression by Kupffer cells via AhR pathway: the central role of proinflammatory cytokines.

The hepatic cytochrome P-450 (CYP) enzyme system provides a major aspect of liver function, yet alterations of CYP in sepsis remain largely unknown. Although we have recently shown that CYP1A2, one of the major isoforms of CYP in rats, is downregulated in sepsis, the underlying mechanism and possible therapeutic approaches warrant further investigation. The aim of this study was to determine whether Kupffer cells (KCs) play any role in suppressing CYP1A2 in the hepatocytes (HCs) and if so, how to modulate CYP1A2 expression in sepsis. To study this, primary KCs and HCs were cultured separately or together with or without transwells. Cells and supernatant samples were collected after various stimulations. Additionally, polymicrobial sepsis was induced in rats by cecal ligation and puncture (CLP) with or without curcumin pretreatment. Liver samples were harvested 20 h post-CLP. The results show that lipopolysaccharide (LPS) did not suppress CYP1A2 in HC or HC/KC coculture with transwells. However, LPS downregulated CYP1A2, aryl hydrocarbon receptor (AhR, a nuclear receptor) and AhR nuclear translocator (Arnt) in coculture without transwells. Anti-TNF-alpha and anti-IL-1beta antibodies attenuated this downregulation. Moreover, elevated hepatic levels of TNF-alpha and IL-1beta post-CLP were decreased by curcumin pre-treatment. This reduction was associated with increased expression of AhR and CYP1A2. These results indicate that KCs-derived proinflammatory cytokines may play an important role in downregulating CYP1A2 in sepsis. The reduction of AhR/Arnt may be the underlying mechanism for such downregulation. Inhibition of proinflammatory cytokines by curcumin may provide a novel approach to modulate the hepatic CYP function in sepsis.

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

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)-gamma, 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 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/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)-alpha 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-alpha and PPAR-gamma 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-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma 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-gamma.

Ghrelin improves tissue perfusion in severe sepsis via downregulation of endothelin-1.

OBJECTIVES: Severe sepsis is associated with increased total peripheral resistance (TPR) and decreased organ blood flow, in which endothelin-1 (ET-1) plays an important role. Plasma levels of ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor and a potent vasodilatory peptide, are significantly reduced in sepsis. Ghrelin downregulation heralds the hypodynamic response in severe sepsis. Therefore, we hypothesized that the administration of exogenous ghrelin improves organ blood flow by downregulation of ET-1 under such conditions. METHODS: Male adult Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 h post-CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed mini-pump over 15 h. At 20 h post-CLP (i.e., severe sepsis), cardiac output (CO), stroke volume (SV), TPR, and organ blood flow were measured using (141)Ce-microspheres. Plasma ET-1 levels and preproET-1 gene expression in the liver, small intestine, and kidneys were measured by ELISA and RT-PCR, respectively. The direct effect of ghrelin on ET-1 production was studied using cultured human umbilical vein endothelial cells (HUVECs) treated with tumor necrosis factor-alpha (TNF-alpha). RESULTS: Ghrelin administration reduced TPR, increased CO, SV, and organ blood flow, downregulated preproET-1 gene expression, and decreased plasma levels of ET-1 in sepsis. Ghrelin inhibited TNF-alpha-induced ET-1 release from HUVECs in a dose-dependent manner. Moreover, ghrelin inhibited TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVECs. CONCLUSIONS: The improvement of tissue perfusion by ghrelin in severe sepsis appears to be mediated by downregulation of ET-1 involving a NF-kappaB-dependent pathway.

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 Ringer's 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-alpha, 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-alpha 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.

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 microg/kg of body weight) in combination with AMBP-1 (40 microg/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.

A novel approach to maintaining cardiovascular stability after hemorrhagic shock: beneficial effects of adrenomedullin and its binding protein.

BACKGROUND: Vascular responsiveness to adrenomedullin (AM), a recently discovered vasodilator peptide, is depressed after hemorrhage and resuscitation. Downregulation of AM binding protein-1 (ie, AMBP-1) appears to be responsible for this hyporesponsiveness. Therefore, we hypothesize that administration of AM/AMBP-1 improves cardiovascular responses after hemorrhagic shock and resuscitation. METHODS: Male rats were bled to and maintained at a mean blood pressure of 40 mm Hg for 90 minutes. The animals were then resuscitated with 4 times the volume of shed blood with Ringer's lactate over 60 minutes. At 15 minutes after the beginning of resuscitation in hemorrhaged animals, AM alone, AMBP-1 alone, AM in combination with AMBP-1, or vehicle (phosphate-buffered saline solution) was administered intravenously over 45 minutes. At 4-hour postresuscitation, in vivo left ventricular contractility parameters, maximal rates of ventricular pressure increase (+dP/dt max ) and decrease (-dP/dt max ), were determined. Cardiac output and organ blood flow were measured with the use of radioactive microspheres. In an additional group of animals, cardiac tumor necrosis factor-alpha (TNF-alpha) levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Four hours after resuscitation, +dP/dt max , -dP/dt max , cardiac output, and organ blood flow in the liver, small intestine, and kidneys were decreased while treatment with AM/AMBP-1 increased these parameters ( P < .05). Moreover, cardiac TNF-alpha levels were elevated at 4 hours after hemorrhage and resuscitation, while AM/AMBP-1 treatment reduced them to sham levels ( P < .05). CONCLUSIONS: Administration of AM/AMBP-1 appears to be a useful approach for restoring and maintaining cardiovascular stability after severe hemorrhagic shock and crystalloid resuscitation.

Delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis.

OBJECTIVE: We have recently shown that administration of human inter-alpha inhibitor proteins (IalphaIp) very early after the onset of sepsis maintains cardiovascular stability and reduced mortality. However, it remains unknown whether injection of IalphaIp at later time points of sepsis has any beneficial effects. We therefore hypothesized that IalphaIp and its active component bikunin are reduced in sepsis and that the delayed administration of IalphaIp also improves survival rate. DESIGN: : Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: : Male adult Sprague-Dawley rats. INTERVENTIONS: Rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). MEASUREMENTS AND MAIN RESULTS: : Bikunin gene expression in the liver was measured by reverse transcription polymerase chain reaction. Plasma concentrations of IalphaIp were determined by Western blot at 5 and 20 hrs after CLP. IalphaIp clearance was assessed by injecting radioactive IalphaIp at 12 hrs post-CLP, and the half-life was determined. In addition, IalphaIp (30 mg/kg of body weight) or vehicle was administered at 1, 5, or 10 hrs (single treatment) or at both 10 and 20 hrs (double treatment) post-CLP. The necrotic cecum was excised at 20 hrs post-CLP, and 10-day survival was recorded. The results indicate that bikunin gene expression decreased significantly at 20 hrs post-CLP. Moreover, IalphaIp concentrations decreased significantly at 5 and 20 hrs post-CLP, and its half-life increased from 5.6 +/- 0.3 hrs to 11.8 +/- 2.7 hrs (p <.05), suggesting down-regulation of IalphaIp in sepsis despite the decreased clearance. Administration of IalphaIp at 1 hr post-CLP improved the survival rate from 50% to 92% (p <.05), whereas there was no significant improvement when IalphaIp was administrated at 5 or 10 hrs post-CLP. However, double injection of IalphaIp at 10 and 20 hrs post-CLP (i.e., severe sepsis) increased the survival rate from 44% to 81% (p <.05). CONCLUSION: Since delayed but repeated administration of human IalphaIp improves survival after CLP, this compound appears to be a useful agent for the treatment of severe sepsis.

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.

Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis.

Ghrelin, a newly identified endogenous ligand for growth hormone secretagogue receptor 1a (GHSR-1a, i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide. Although sepsis is characterized by an early, hyperdynamic phase, it remains unknown whether ghrelin or GHSR-1a plays a role in the cardiovascular response to sepsis. To determine this, polymicrobial sepsis was induced by cecal ligation and puncture in male adult rats. At 5 h (i.e., early sepsis) or 20 h (i.e., late sepsis) after cecal ligation and puncture, blood and tissue samples were collected. Ghrelin levels and ghrelin and GHSR-1a mRNA expression were assessed by RIA and RT-PCR, respectively. In addition, GHSR-1a protein levels in aorta, heart, and small intestine were determined by Western blotting. The vascular response to ghrelin was determined by using an isolated gut preparation. A primary rat aortic smooth muscle cell culture was used to determine the effects of LPS on GHSR-1a expression. The results indicate that although ghrelin levels decreased at early and late sepsis, its receptor was markedly elevated in early sepsis. Moreover, ghrelin-induced relaxation in resistance blood vessels of the isolated small intestine increased significantly during early sepsis but was not altered in late sepsis. Furthermore, GHSR-1a expression in smooth muscle cells was significantly increased at mRNA and protein levels with stimulation by LPS at 10 ng/ml. These results demonstrate that GHSR-1a expression is upregulated and vascular sensitivity to ghrelin stimulation is increased in the hyperdynamic phase of sepsis.

Ghrelin clearance is reduced at the late stage of polymicrobial sepsis.

The cardiovascular response to sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Ghrelin, a newly-identified endogenous ligand for growth hormone secretagogue receptor (i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide in addition to its effects on growth hormone release and energy homeostasis. We have shown that ghrelin (via its receptor) may play an important role in regulating cardiovascular responses in the progression of polymicrobial sepsis. However, it remains unknown whether the clearance of this peptide is altered in sepsis. To determine this, male adult rats were injected with 125I-ghrelin through the jugular vein at 5 or 20 h after cecal ligation and puncture (CLP, i.e., sepsis model) or sham operation. The blood sample was collected every 2 min for 30 min for determining half-life (t1/2). Tissue samples (i.e., kidneys, liver, brain, heart, lungs, spleen, stomach, small intestine, large intestine, skin and muscle) were then harvested. The radioactivities of samples were counted. The results indicate that 125I-ghrelin's t1/2 and its distribution were not significantly altered in early sepsis (5 h after CLP). However, the t1/2 increased significantly in late sepsis (20 h after CLP). Tissue distribution of 125I-ghrelin was far greater in the kidneys than in any other tissues tested in both sham and septic animals. Moreover, the kidneys and liver had significantly less radioactive uptake at 20 h after CLP, but the radioactivity in blood was much higher at the same time point. There were no significant changes in 125I-ghrelin distribution in other organs at the late stage of sepsis. These results indicate that the kidneys are the primary site of ghrelin clearance, which is significantly diminished in late sepsis. In addition, the liver also plays a role in the clearance of ghrelin, which was also reduced in late sepsis. The decreased clearance of ghrelin by the kidneys and liver may be due to renal and hepatic dysfunctions under such conditions.