Sesame oil protects against lead-plus-lipopolysaccharide-induced acute hepatic injury

Lead (Pb) increases lipopolysaccharide (LPS)-induced tumor necrosis factor alpha, which causes liver damage. In this study, we investigated the effect of sesame oil on Pb-plus-LPS (Pb + LPS)-induced acute liver damage in mice. Mice were given sesame oil (8 mL/kg orally) just after Pb acetate (10 mmol/kg i.p.) plus LPS (5 mg/kg i.p.). Aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-alpha, interleukin-1beta, nitric oxide, and inducible nitric oxide synthase levels were examined. Sesame oil significantly decreased serum aspartate aminotransferase and alanine aminotransferase levels in Pb + LPS-stimulated mice. Sesame oil reduced Pb + LPS-induced tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide production in serum and liver tissue. Furthermore, sesame oil decreased inducible nitric oxide synthase expression in leukocytes and liver tissue in Pb + LPS-treated mice. We hypothesize that the inhibition of proinflammatory cytokines and nitric oxide might be involved in sesame oil-associated protection against Pb + LPS-induced acute hepatic injury in mice.     

Metalloendopeptidase inhibition regulates phosphorylation of p38-mitogen-activated protein kinase and nitric oxide synthase in heart after endotoxemia

We tested the hypothesis that metalloendopeptidase inhibition using phosphoramidon during induction of endotoxemia 24 h later would down-regulate the protein expression of myocardial inducible nitric oxide synthase (iNOS) and phosphorylation of p38-mitogen-activated protein kinase (p38-MAPK). Male Sprague-Dawley rats (350-400 g) were randomly divided into sham-treated and LPS-treated groups (Escherichia. coli lipopolysaccharide [LPS] 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle- and phosphoramidon (1 mg/kg bolus)-treated subgroups. Blood and heart samples were collected at 2- and 24-h postendotoxemia/phosphoramidon treatment. LPS at 2 h after its administration produced a significant decrease in mean arterial pressure that was blocked by phosphoramidon treatment. LPS at 2 and 24 h produced a significant elevation in the concentration of left ventricular endothelin-1 (ET-1) both in heart and plasma as compared with control group. This LPS-induced left ventricular ET-1 elevation at 24 h was significantly reduced by phosphoramidon. No significant alterations were observed in the myocardial protein expression of preproET-1, iNOS, and eNOS at 2 h post LPS. In 24-h post treatment groups phosphoramidon upregulated the expression of myocardial preproET-1 protein both in control and endotoxemic rat groups. Also, LPS-induced upregulated protein expression of myocardial-inducible nitric oxide synthase and increased levels of nitric oxide byproducts at 24 h were blocked by phosphoramidon. Phosphoramidon inhibited LPS-induced down-regulated expression of myocardial endothelial nitric oxide synthase and upregulated p38-MAPK phosphorylation. These results indicated that inhibition of metalloendopeptidase during induction of endotoxemia could regulate the phosphorylation of myocardial p38-MAPK and iNOS protein expression at 24-h post endotoxemia. We concluded that inhibition of metalloendopeptidases during early endotoxemia not only decreased the biosynthesis of ET-1 in heart locally but also simultaneously down-regulated myocardial protein expression of iNOS and p38-MAPK phosphorylation in the later stage of endotoxemia.     

Oxidant stress in rat liver after lipopolysaccharide administration: effect of inducible nitric-oxide synthase inhibition

The role of inducible nitric-oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced hepatic oxidant stress was evaluated using the iNOS inhibitor L-iminoethyl-lysine (L-NIL). Male rats were divided into three groups. One group received LPS (Salmonella minnesota) 2 mg/kg i.v. A second group received LPS plus L-NIL (3 mg/kg i.p.) at the time of LPS administration followed by a second dose 3 h later. A third group received saline i.v. At 6 h, blood and liver tissue were collected. Serum nitrate/nitrite (metabolic products of nitric oxide) levels were increased from 5.4 +/- 1.5 nmol/ml in the saline group to 360 +/- 48 nmol/ml in the LPS group (n = 5). Values for the LPS + L-NIL group were significantly reduced to 35 +/- 7 nmol/ml. Tissue malondialdehyde levels were increased from 0.20 +/- 0.02 nmol/mg (n = 4) in the saline group to 0.41 +/- 0.03 nmol/mg (n = 4) in the LPS group. L-NIL significantly reduced the values in the LPS group to 0.29 +/- 0.02 nmol/mg (n = 4). 4-Hydroxynonenal-protein adducts levels were increased 3.6-fold by LPS treatment as compared with saline. L-NIL significantly reversed the levels to 1.6-fold (n = 4). Intracellular GSH levels were decreased from 8.49 +/- 0.64 nmol/mg (n = 4) in the saline group to 5.63 +/- 0.51 nmol/mg in the LPS group (n = 7). L-NIL significantly increased the levels in the LPS group to 7.04 +/- 0.46 nmol/mg (n = 7). These data indicate that LPS-induced nitric oxide generation can result in oxidant stress in the liver, and that inhibitors of iNOS may offer some protection in LPS-induced hepatic toxicity.     

Decreased Antipyrine Clearance following Endotoxin Administration: In Vivo Evidence of the Role of Nitric Oxide

Klebsiella pneumoniae endotoxin has been found to decrease hepatic P450-mediated drug-metabolizing enzyme activity in a time-dependent manner. In this study, we investigated the role of nitric oxide (NO) in the decrease in hepatic drug-metabolizing enzyme activity caused by endotoxin in vivo. We measured in vivo pharmacokinetic parameters of antipyrine in rats treated with endotoxin and/or a selective inhibitor of inducible NO synthase (iNOS), S-methylisothiourea. Intraperitoneal injection of endotoxin (1 mg/kg of body weight) dramatically decreased the systemic clearance of antipyrine, reflecting reduced hepatic drug-metabolizing enzyme activity, and significantly increased the level of nitrite and nitrate (NOx) in the plasma. S-Methylisothiourea (10 mg/kg) reversed this decreasing antipyrine clearance and reduced the level of NOx in plasma. Repeated injections of an NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK-409; 10 mg/kg), at a dose which maintained plasma NOx at the same levels as those caused by endotoxin injection, also decreased the systemic clearance of antipyrine. These findings suggest that the overproduction of NO observed in this animal model is at least partially responsible for the significant reduction in the hepatic drug-metabolizing enzyme activity that may happen in a gram-negative bacterial infection.     

Activated protein C suppresses adrenomedullin and ameliorates lipopolysaccharide-induced hypotension

Activated protein C (APC) is an important modulator of vascular function that has antithrombotic and anti-inflammatory properties. Studies in humans have shown modulation of endotoxin-induced hypotension by recombinant human APC, drotrecogin alfa (activated), however, the mechanism for this effect is unclear. We have found that APC suppresses the induction of the potent vasoactive peptide adrenomedullin (ADM) and could downregulate lipopolysaccharide (LPS)-induced ADM messenger RNA (mRNA) and nitrite levels in cell culture. This effect was dependent on signaling through protease-activated receptor 1. Addition of 1400W, an irreversible inducible nitric oxide synthase (iNOS) inhibitor, inhibited LPS-induced ADM mRNA, suggesting that ADM induction is NO mediated. Furthermore, in a rat model of endotoxemia, APC (100 microg/kg, i.v.) prevented LPS (10 mg/kg, i.v.)-induced hypotension, and suppressed ADM mRNA and protein expression. APC also inhibited iNOS mRNA and protein levels along with reduction in NO by-products (NOx). We also observed a significant reduction in iNOS-positive leukocytes adhering to vascular endothelium after APC treatment. Moreover, we found that APC inhibited the expression of interferon-gamma (IFN-gamma), a potent activator of iNOS. In a human study of LPS-induced hypotension, APC reduced the upregulation of plasma ADM levels, coincident with protection against the hypotensive response. Overall, we demonstrate that APC blocks the induction of ADM, likely mediated by IFN-gamma and iNOS, and suggests a mechanism that may account for ameliorating LPS-induced hypotension. Furthermore, our data provide a new understanding for the role of APC in modulating vascular response to insult.     

Immune dysfunction in endotoxicosis: role of nitric oxide produced by inducible nitric oxide synthase

OBJECTIVE: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS). DESIGN:: Prospective animal study with concurrent controls. SETTING: University research laboratory. SUBJECTS: iNOS knockout mice and wild-type littermates. INTERVENTIONS: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level. CONCLUSIONS: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.     

Cardiac response to nitric oxide synthase inhibition using aminoguanidine in a rat model of endotoxemia

This study evaluates the effect of aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase (iNOS), on the prevention of cardiac depression in acute endotoxemia. Cardiac performance was evaluated after 4 h of exposure to endotoxin. Rats (n = 5) were selected randomly to receive, by intraperitoneal injection, one of four treatments: saline, LPS (lipopolysaccharide, E. coli, 4 mg/kg, AG (aminoguanidine 100 mg/kg), and LPS + AG at various times. AG and saline treatments were administered 30 min before LPS and at 1 and 3 h after LPS injection. Hearts were perfused using the Langendorff isolated perfusion system and a balloon-tipped catheter was placed into the left ventricle to measure left ventricular developed pressure (LVDP). Myocyte contractile function was assessed with electrical field stimulation and video microscopy. Tissue was immunostained for the expression of iNOS and for nitrotyrosine, a byproduct of protein nitration by peroxynitrite. Perfused hearts from LPS-treated rats exhibited a 57% decrease (P < 0.05) in LVDP compared to saline-treated animals. No improvement in ventricular function was observed with the administration of AG. Similarly, cardiac myocytes prepared from LPS-treated animals demonstrated a significant (P < 0.05) reduction in percent and velocity of shortening and this effect was unaltered with the same dose of AG. AG administration significantly reduced serum nitrite/nitrate levels (P < 0.05) in endotoxemic rats to control levels. Localized expression of iNOS in the myocardium was lessened with AG treatment and was not associated with peroxynitrite formation in this model of endotoxemia. The results indicate that AG given in vivo before and after endotoxin (at a concentration sufficient to decrease NO production) did not reduce cardiac depression. We conclude that selective inhibition of iNOS and the reduction of NO production do not prevent cardiac dysfunction at an early stage in an acute model of endotoxemia.     

Oral administration of geranylgeranylacetone improves survival rate in a rat endotoxin shock model: administration timing and heat shock protein 70 induction

The present study was performed to determine whether oral pretreatment with geranylgeranylacetone (GGA) inhibits proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated rats and protects rats against death from LPS-induced endotoxin shock, and whether such protection by GGA is related to heat shock protein (HSP) 70 induction in multiple organs of rats. GGA (200 mg/kg) was given orally to rats. LPS (20 mg/kg) was administered intraperitoneally 4, 8, 16, or 24 h after GGA administration. The survival of rats was monitored over 24 h after LPS administration. GGA treatment at 8 or 16 h before LPS dramatically improved the survival rate of LPS-treated rats. Plasma levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and NO 6 h after LPS administration in these GGA-pretreated rats were less than one-half of those in rats treated with LPS alone. A GGA challenge 8 or 16 h before LPS administration enhanced HSP70 expression in rat organs after LPS. Treatment with GGA 8 h before LPS minimized hepatic and renal damage. Furthermore, the protective effect of GGA on mortality in LPS-treated rats was inhibited with quercetin, known as an HSP70 inhibitor. These results suggest that oral administration of GGA at an optimal time before LPS injection induces and enhances HSP70 expression in several organs, inhibits proinflammatory cytokine and NO production, and prevents organ damage, resulting in an improved survival rate.     

Differential involvement of guanylate cyclase and potassium channels in nitric oxide-induced hyporesponsiveness to phenylephrine in endotoxemic rats.

This study evaluated the involvement of nitric oxide (NO), guanylate cyclase, and potassium channels in the long-lasting vascular hyporesponsiveness to phenylephrine induced by Escherichia coli lipopolysaccharide (LPS) in vitro and in vivo. Experiments in rat aorta rings with endothelium incubated with LPS (10 microg/mL) for 12 h showed that the hyporesponsiveness depends on guanylate cyclase activity and tetraethylammonium-sensitive, but not voltage- or ATP-dependent, potassium channels. Pressor responses to phenylephrine were reduced by 50% in rats injected 8 and 24 h before with LPS (10 mg/kg, intraperitoneally). Pretreatment with NO synthase inhibitors (iNOS; Nomega-nitro-L-arginine methyl ester [L-NAME], 55 micromol/kg or aminoguanidine, 244 micromol/kg, intraperitoneally) fully prevented LPS-induced hyporesponsiveness. When administered just before phenylephrine, L-NAME (11 micromol/kg, intravenously) reversed the hyporesponsiveness in rats injected 8 h, but not in those injected 24 h before with LPS, whereas 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1 (ODQ, 11 micromol/kg, intravenously) reversed the hyporesponsiveness in animals injected 24 h, but not in those injected 8 h before with LPS. Tetraethylammonium (360 micromol/kg, intravenously) reestablished normal responses to phenylephrine in rats injected 8 and 24 h before with LPS. Again, neither voltage- nor ATP-dependent potassium channels appears to be involved. Western blot showed that iNOS expression peaked at 8 h, decreasing to low levels 24 h after LPS injection. Therefore, NO is important in initiating LPS-induced hyporesponsiveness to vasoconstrictors, but not in maintaining it for long periods. Once NO has exerted its effects and even when iNOS expression is minimal, the long-lasting hyporesponsiveness appears to depend on a complex interplay between guanylate cyclase and potassium channel activation.     

Effects of electroacupuncture pretreatment on inflammatory response and acute kidney injury in endotoxaemic rats

This study investigated the effect of electroacupuncture pretreatment on the lipopolysaccharide (LPS)-induced inflammatory response and on acute kidney injury in adult male pathogen-free Wistar rats. Rats received electroacupuncture at the Zusanli (ST36) and Neiguan (PC6) acupoints, or electrical stimulation at sham points, for 30 min before stimulation with either 5 mg/kg LPS intravenously or normal saline. Plasma cytokines, plasma nitrite, renal inducible nitric oxide synthase (iNOS) and nuclear factor κB (NF-κB) activity were assessed 240 min after LPS or normal saline injection. Blood urea nitrogen (BUN), creatinine (Cr) and histopathological score for renal tubular damage were also measured. Electroacupuncture pretreatment significantly decreased LPS-induced plasma tumour necrosis factor-α and interleukin (IL)-1β, increased plasma IL-10, and decreased plasma nitrite, renal iNOS and NF-κB activity. It also significantly decreased LPS-induced BUN, Cr and the renal histopathological score. These findings suggest that electroacupuncture pretreatment at the ST36 and PC6 acupoints attenuated the LPS-induced inflammatory response and mitigated acute kidney injury.     

Comparison of tumor necrosis factor-alpha effect on the expression of iNOS in macrophage and cardiac myocytes

Proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), are elevated during cardiopulmonary bypass (CPB), heart failure, and inflammatory cardiac and systemic diseases. Elevated TNF-alpha has been linked to diminished cardiac function, decreased systemic vascular resistance, as well as renal and pulmonary dysfunction. It is understood that myocardial tissues can express TNF-alpha, which results in the induction of inducible nitric oxide synthase (iNOS) leading to a significant decline in cardiac function and other direct effects. The hypothesis of this study was to determine if TNF-alpha would stimulate iNOS and its product nitric oxide (NO) similarly in immortalized macrophage and cardiac myocytes. Cultured macrophages (RAW 264.7) and cardiac myocytes (HL-1) were placed into two treatment groups and a control. The treatments included: (1) TNF-alpha and lipopolysaccharide (LPS); and (2) LPS, TNF-alpha, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) incubated for 8 h. The macrophage expression of iNOS increased by 365% (p < 0.01) and its product, NO, increased proportionally. The expression of iNOS in the cardiac myocyte did not increase with TNF-alpha and LPS. However, with the addition of IFN-alpha and IL-1beta iNOS increased to 140% of control (p < 0.05). Myocyte cGMP and NO did not increase significantly with TNF-alpha treatment. This study suggests that HL-1 myocyte iNOS cannot be induced by TNF-alpha, unlike macrophage iNOS. Furthermore, the resultant cardiac dysfunction, secondary to proinflammatory cytokines effects, is regulated via diverse pathways.     

葛根素抑制脂多糖诱导RAW264.7巨噬细胞表达诱导型一氧化氮合酶

目的观察葛根素对脂多糖（LPS）诱导小鼠RAW264.7巨噬细胞表达诱导型一氧化氮合酶（iNOS）的影响。方法用脂多糖刺激小鼠巨噬细胞RAW264.7,与不同浓度的葛根素进行培养,采用逆转录多聚酶链式反应（RT-PCR）和免疫蛋白印迹法测定（Western blotting）方法分别检测RAW264.7巨噬细胞iNOS mRNA和蛋白质表达。结果随着葛根素的浓度的增加,RAW264.7细胞iNOS的mRNA及其蛋白质表达逐渐减少(P<0.01)。结论葛根素可以通过调节RAW264.7细胞表达iNOS发挥抗炎的作用。