Therapeutic effects of TAK-242, a novel selective Toll-like receptor 4 signal transduction inhibitor, in mouse endotoxin shock model

Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule that selectively inhibits Toll-like receptor 4-mediated signaling, inhibits various kinds of inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, IL-10, macrophage inhibitory protein (MIP)-2 and prostaglandin E2 from lipopolysaccharide (LPS)-stimulated macrophages. The effects of TAK-242 were evaluated in a mouse model of endotoxin shock. Intravenous administration of TAK-242 to mice 1 h before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-alpha, IL-1beta, IL-6, IL-10, MIP-2, and NO metabolites. TAK-242 protected mice from LPS-induced lethality in a similar dose-dependent manner, and rescued 100% of mice at a dose of 1 mg/kg. Interestingly, TAK-242 worked quickly, and showed beneficial effects even when administered after LPS challenge. Even though increases in serum levels of IL-6 and hypothermia were already evident 2 h after LPS challenge, TAK-242 administration inhibited further increase in IL-6 levels and decrease in body temperature. LPS-induced increases in serum levels of organ dysfunction markers, such as alanine aminotransferase, total bilirubin, and blood urea nitrogen, were also significantly suppressed by post-treatment as well as pre-treatment. Furthermore, administration of 3 mg/kg TAK-242 significantly increased survival of mice, even when given 4 h after LPS challenge. These results suggest that TAK-242 protects mice against LPS-induced lethality by inhibiting production of multiple cytokines and NO. TAK-242 has a quick onset of action and provides significant benefits by post-treatment, suggesting that it may be a promising drug candidate for the treatment of sepsis.     

Responses of exposure to cigarette smoke at three dosage levels on soleus muscle fibers in Wistar-Kyoto and spontaneously hypertensive rats

Overproduction of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) is importantly involved in the pathogenesis of endotoxemia and atherosclerosis. Calcium antagonists are commonly used as cardiovascular drugs and have a beneficial effect on prolonging survival in various models of endotoxin shock. The present study was to investigate the effect of a calcium antagonist amlodipine on nitrite, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) formation and iNOS induction both in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and in a rat model of endotoxemia. Incubation with amlodipine (0.1 - 10 microM) for 24 h resulted in a significant and dose-dependent attenuation in medium nitrite, TNF-alpha and IL-1beta formation as well as iNOS protein expression in LPS/IFN-gamma-treated RASMC. In addition, amlodipine inhibited leucigenin-induced superoxide formation in RASMC. In the rat endotoxic model, the serum nitrite/nitrate, TNF-alpha and IL-1beta levels as well as iNOS protein expression of lungs were also suppressed by administration of amlodipine (50 microg/kg, i.v.). These results suggest that amlodipine may exert vascular beneficial effects by suppressing pro-inflammatory cytokines and free radical generation as well as iNOS induction in smooth muscle cells during activation of inflammatory mechanism.     

The lazaroid, U-74389G, inhibits inducible nitric oxide synthase activity, reverses vascular failure and protects against endotoxin shock

The aim of our study was to investigate the effect of the 21-aminosteroid U-74389G [21- < 4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl-pregna-1,4,9,(11) triene-3,20-dione(z)-2-butenedionate] on the l-arginine-nitric oxide (NO) pathway in a rat model of endotoxin shock. Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg/kg of Salmonella Enteritidis lipopolysaccharide (LPS). Rats were treated with U-74389G (7.5, 15 and 30 mg/kg i.v.) or vehicle (1 ml/kg i.v.) 5 min after endotoxin challenge. Lipopolysaccharide administration reduced survival rate (0%, 72 h after endotoxin administration) decreased mean arterial blood pressure, enhanced plasma concentration of bilirubin and alanine aminotransferase and increased plasma nitrite concentrations. Lipopolysaccharide injection also increased the activity of inducible NO synthase in the liver and in the aorta. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (1 nM-10 microM). In addition lipopolysaccharide (50 microg/ml for 4 h) in vitro stimulation significantly increased nitrite production in peritoneal macrophages harvested from normal rats. Treatment with U-74389G (15 and 30 mg/kg i.v., 5 min after endotoxin challenge) significantly protected against lipopolysaccharide-induced lethality (90% survival rate 24 h and 80% 72 h after lipopolysaccharide injection, respectively, following the highest dose of the drug), reduced hypotension, ameliorated liver function, decreased plasma nitrite levels, restored the hyporeactivity of aortic rings to their control values and inhibited the activity of inducible NO synthase in the liver and in the aorta. Finally, U-74389G in vitro (12.5, 25 and 50 microM) significantly inhibited nitrite production in endotoxin stimulated peritoneal macrophages. The data suggest that U-74389G may exert beneficial effects in an experimental model of septic shock by inhibiting the activity of the inducible NO synthase.     

A synthetic peptide derived from bactericidal/permeability-increasing protein neutralizes endotoxin in vitro and in vivo

Lipopolysaccharide (LPS [endotoxin]), a structural component of gram-negative bacteria, is implicated in the pathogenesis of septic shock. Lipid A is an evolutionarily conserved region of LPS that has been identified as the toxic component of LPS. Therapeutic strategies for the treatment of septic shock in humans are currently focused on neutralization of LPS. Here, the anti-endotoxin activity of BNEP, a synthetic peptide derived from the human bactericidal/permeability-increasing protein (BPI; aa 148-161) was investigated in vitro and in experimental animal endotoxemia models in vivo. The ability of BNEP to bind LPS from Escherichia coli O55:B5 and lipid A from Salmonella Re 595 was tested using an affinity sensor assay, and its ability to neutralize LPS was tested using a sensitive Limulus amebocyte lysate (LAL) assay. Polymyxin B (PMB) was used as the positive control in the in vitro experiments and in mouse experiments. We found that BNEP and PMB bound LPS with a similar affinity (Kd values of 25.4 and 25.8 nM, respectively). In contrast, BNEP bound lipid A with a slightly lower affinity than that of PMB (Kd values of 8 and 5.6 nM, respectively). The exact capacity of BNEP binding to LPS was approximately 0.53 microg peptide per 1 ng of LPS, as shown by affinity sensor assay. The LAL test showed that 256 microg of BNEP almost completely neutralized 2 ng LPS. In vivo, mice were randomized, intravenously injected with BNEP (0.5-10 mg/kg) or 1 mg/kg PMB, and then lethally challenged with 20 mg/kg LPS. We found that 5 mg/kg BNEP significantly protected mice from LPS challenge. In an endotoxemia rat model, animals were co-treated with 5 or 10 mg/kg BNEP and 10 mg/kg LPS via cardiac catheter. BNEP treatment resulted in significant reduction of tumor necrosis factor alpha (TNF-alpha) and IL-6, compared with LPS-only control animals. In addition, 10 mg/kg BNEP-treated animals showed a significant decrease in plasma endotoxin levels in comparison to animals treated with LPS alone. These results provide evidence that BNEP effectively neutralizes LPS in vitro and in vivo, and could protect animals from the lethal effects of LPS via decreasing plasma endotoxin and proinflammatory cytokines. Our work suggests that this peptide is worthy of further investigation as a possible novel treatment for septic shock.     

Nitric oxide-releasing flurbiprofen reduces formation of proinflammatory hydrogen sulfide in lipopolysaccharide-treated rat

The biosynthesis of both nitric oxide (NO) and hydrogen sulfide (H2S) is increased in lipopolysaccharide (LPS)-injected mice and rats but their interaction in these models is not known. In this study we examined the effect of the NO donor, nitroflurbiprofen (and the parent molecule flurbiprofen) on NO and H2S metabolism in tissues from LPS-pretreated rats. Administration of LPS (10 mg kg(-1), i.p.; 6 h) resulted in an increase (P<0.05) in plasma TNF-alpha, IL-1beta and nitrate/nitrite (NO(x)) concentrations, liver H2S synthesis (from added cysteine), CSE mRNA, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO) activity (marker for neutrophil infiltration) and nuclear factor-kappa B (NF-kappaB) activation. Nitroflurbiprofen (3-30 mg kg(-1), i.p.) administration resulted in a dose-dependent inhibition of the LPS-mediated increase in plasma TNF-alpha, IL-1beta and NO(x) concentration, liver H2S synthesis (55.00+/-0.95 nmole mg protein(-1), c.f. 62.38+/-0.47 nmole mg protein(-1), n = 5, P<0.05), CSE mRNA, iNOS, MPO activity and NF-kappaB activation. Flurbiprofen (21 mg kg(-1), i.p.) was without effect. These results show for the first time that nitroflurbiprofen downregulates the biosynthesis of proinflammatory H2S and suggest that such an effect may contribute to the augmented anti-inflammatory activity of this compound. These data also highlight the existence of 'crosstalk' between NO and H2S in this model of endotoxic shock.     

Tetramethylpyrazine prevents inducible NO synthase expression and improves survival in rodent models of endotoxic shock

This study is to investigate the possible mechanism of beneficial effects of tetramethylpyrazine (TMP) on endotoxic shock which we showed in our preliminary study (Liao et al. 1998; Proc Natl Sci Counc Repub China B 22:46-54). Here, we have confirmed the beneficial effects of TMP on the hypotension, vascular hyporeactivity to noradrenaline (NA), release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). In addition, we further examined the expression of inducible NO synthase in the lung and in the aorta from these rats and evaluated the effect of TMP on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg(-1), i.v.) resulted in an acute fall followed by a substantial fall in MAP within 4 h and an increase in HR. In contrast, animals pretreated with TMP (10 mg kg(-1), i.p.; at 30 min prior to LPS) maintained a significantly higher MAP but the tachycardia was further enhanced at 1-2 h when compared to rats given only LPS (LPS rats). The pressor effect of NA (1 microg kg(-1), i.v.) was also significantly reduced after the treatment of rats with LPS. Similarly, the thoracic aorta obtained from rats at 4 h after LPS showed a significant reduction in the contractile responses elicited by NA (1 microM). Pretreatment of LPS rats with TMP partially, but significantly, prevented this LPS-induced hyporeactivity to NA in vivo and ex vivo. The injection of LPS resulted in a bell-shaped change in plasma TNF-alpha level which reached a maximum at 1 h, whereas the effect of LPS on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increment of both TNF-alpha and nitrate levels was significantly reduced in LPS rats pretreated with TMP. Endotoxaemia for 4 h caused a significantly increased protein expression of iNOS in the lung and the aorta. In LPS rats pretreated with TMP, iNOS protein expression in lung and aorta homogenates was attenuated by 75+/-3% and 57+/-6%, respectively. In addition, the lack of evidence of pressor effect of TMP on rats with endotoxaemia for 4 h suggested that TMP inhibits the induction of iNOS rather than directly inhibiting NOS activity. Treatment of conscious ICR mice with a high dose of endotoxin (60 mg kg(-1), i.p.) resulted in a survival rate of only 15% at 36 h (n=20). However, therapeutic application of TMP (10 mg kg(-1), i.p.; at 0, 6, 15 and 24 h after LPS) increased the 36 h survival rate to 55% (n=20). Thus, TMP inhibits the expression of iNOS and mitigates the delayed circulatory failure caused by endotoxic shock in the rat. In addition, TMP also improves survival in a murine model of severe endotoxaemia.     

脂多糖刺激体外大鼠小胶质细胞产生细胞肽和一氧化氮

AIM: To study the characterization of interleukin (IL)-1, IL-2, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production in microglia stimulated with lipopolysaccharides (LPS). METHODS: Primary cultured neonatal rat microglia were incubated with LPS (0-10 mg.L-1) for 0-72 h. The supernatants and lysates were collected. IL-1, IL-2, and TNF-alpha were assayed by mouse thymocyte proliferation, mouse spleen cell proliferation, and 1929 cytotoxity, respectively. NO was assayed by Griess reaction. RESULTS: Extracellular IL-1, TNF-alpha, and NO production reached peak levels at LPS 1 mg.L-1. Intracellular IL-1 production reached its peak level at LPS 100 micrograms.L-1. Intracellular TNF-alpha level was very low. IL-1, TNF-alpha, and NO activities were detected at 1, 4, and 8 h, after the cells were stimulated with LPS. IL-1 got to its peak value at 8 h, TNF-alpha, and NO reached the highest levels at 24 h. However, IL-2 activity was not detected after the microglia were stimulated with LPS 0-10 mg.L-1 during the incubation period. CONCLUSION: Rat microglia stimulated with LPS in vitro produced proinflammatory cytokines and NO.     

Influence of enalapril, quinapril and losartan on lipopolysaccharide (LPS)-induced serum concentrations of TNF-alpha, IL-1 beta, IL-6 in spontaneously hypertensive rats (SHR)

Immunopharmacological studies of drugs used in cardiovascular diseases provide new data concerning their modulating effect on the levels of proinflammatory cytokines, chemokines and adhesion molecules. Therefore, we have made an attempt to find out whether enalapril, quinapril and losartan (drugs used in the treatment of arterial hypertension) are able to modulate lipopolysaccharide (LPS)-induced proinflammatory cytokine serum concentrations (tumor necrosis factor alpha - TNF-alpha, interleukin-1 beta _ IL-1 beta, interleukin-6 - IL-6) in spontaneously hypertensive rats (SHR). The animals were divided into four groups as follows: SHR + M (control rats receiving 1% solution of methylcellulose), SHR + E (rats receiving enalapril - 10 mg/kg), SHR + Q (rats receiving quinapril - 10 mg/kg) and SHR + L(rats receiving losartan - 20 mg/kg). 1% solution of methylcellulose and hypotensive drugs were administered by a gavage for 21 days. Arterial blood pressure was measured in conscious rats, using the tail-cuff method. Twenty four hours after the last administration of enalapril, quinapril, losartan or 1% solution of methylcellulose, the rats received a single dose of LPS (ip; 0.1 mg/kg). After 2 h, the rats were anesthetized with ether and the blood samples were collected by heart puncture. Serum TNF-alpha, IL-1 beta and IL-6 concentrations were measured with enzyme-linked immunosorbent assay kits. Additionally, total cholesterol and high density lipoprotein (HDL) cholesterol were evaluated. Enalapril, quinapril and losartan significantly decreased LPS-stimulated TNF-alpha and IL-1 beta level after 21 days. Three-week administration of quinapril lowered IL-6 serum concentration after LPS stimulation. Enalapril and losartan did not affect the IL-6 level. The results were accompanied by a statistically significant decrease in systolic, diastolic and mean blood pressure. Hypotensive drugs also showed no effect on lipid level. The latest data indicate additional properties of hypotensive drugs. However, further studies are necessary to elucidate precisely the role of proinflammatory cytokines in arterial hypertension.     

Cloricromene, a coumarine derivative, protects against lethal endotoxin shock in rats.

Endotoxin shock was induced in male rats by an intravenous (i.v.) injection of Salmonella enteriditis lipopolysaccharide (LPS; 20 mg/kg i.v.). Survival rate, macrophage and serum tumor necrosis factor (TNF-alpha), mean arterial blood pressure (MAP) and white blood cell count were then evaluated. Furthermore the in vitro effect of cloricromene on peritoneal macrophage phagocytosis and TNF-alpha release by primed peritoneal macrophages was investigated. LPS administration caused animal death (0% survival 24 h after endotoxin challenge), hypotension, marked leukopenia and increased the levels of TNF-alpha in both serum and macrophage supernatants. Cloricromene administration (0.5, 1 and 2 mg/kg i.v. 15 min after endotoxin) protected against LPS-induced lethality (100% survival rate 24 h after endotoxin challenge), reverted LPS-induced hypotension and leukopenia, and decreased TNF-alpha in both serum and macrophage supernatants. Finally, cloricromene, added in vitro to peritoneal macrophages collected from endotoxin-treated rats increased macrophage phagocytosis and reduced TNF-alpha formation by activated mononuclear phagocytes. Our data suggest that cloricromene increases survival rate in endotoxin shock through an inhibition of TNF-alpha production.     

Amplified proinflammatory cytokine expression and toxicity in mice coexposed to lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol).

A single oral exposure to the trichothecene vomitoxin (VT) has been previously shown in the mouse to increase splenic mRNA levels for several cytokines in as little as 2 h. Since one underlying mechanism for these effects likely involves superinduction of transiently expressed cytokine genes, VT may also potentially amplify cytokine responses to inflammatory stimuli. To test this possibility, the effects of oral VT exposure on tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta expression were measured in mice that were intraperitoneally injected with lipopolysaccharide (LPS), a prototypic inflammatory agent. As anticipated, VT alone at 1, 5, and 25 mg/kg body weight increased splenic mRNA expression of all three cytokines after 3 h in a dose-response fashion. LPS injection at 1 and 5 mg/kg body weight also induced proinflammatory cytokine mRNA expression. There was a synergistic increase in TNF-alpha splenic mRNA levels in mice treated with both VT and LPS as compared to mice treated with either toxin alone, whereas the effects were additive for IL-6 and IL-1beta mRNA expression. When relative mRNA levels were examined over a 12-h period in mice given LPS (1 mg/kg) and/or VT (5 mg/kg), significant enhancement was observed up to 6, 12, and 3 h for TNF-alpha, IL-6, and IL-1beta, respectively. When plasma cytokine concentrations were measured, TNF-alpha was found to peak at 1 h and was significantly increased at 1, 3, and 6 h if mice were given LPS and VT, whereas LPS or VT alone caused much smaller increases in plasma TNF-alpha Plasma IL-6 peaked at 3 h in LPS, VT, and LPS/VT groups, with the combined toxin group exhibiting additive effects. Plasma IL-1beta was not detectable. The potential for VT and LPS to enhance toxicity was examined in a subsequent study. Mortality was not observed up to 72 h in mice exposed to a single oral dose of VT at 25 mg/kg body weight or to an intraperitoneal dose of LPS at 1 or 5 mg/kg body weight; however, all mice receiving VT and either LPS dose became moribund in less than 40 h. The principal histologic lesions in the moribund mice treated with VT and LPS were marked cell death and loss in thymus, Peyer's patches, spleen, and bone marrow. In all of these lymphoid tissues, treatment-induced cell death had characteristic histologic features of apoptosis causing lymphoid atrophy. These results suggest that LPS exposure may markedly increase the toxicity of trichothecenes and that the immune system was a primary target of these interactive effects.     

Lipopolysaccharide-induced down-regulation of organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is independent of tumor necrosis factor-alpha, Interleukin-1beta, interleukin-6, or inducible nitric oxide synthase.

Organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is expressed almost exclusively in liver, where it mediates uptake of a variety of compounds, including bile acids, as well as other endo- and xenobiotics, across hepatic sinusoidal membranes in a Na+-independent manner. Lipopolysaccharide (LPS) has been shown to decrease Oatp4 mRNA levels in a dose- and time-dependent manner in Toll-like receptor 4 (TLR4)-normal (C3H/OuJ) mice, but not in TLR4-mutant (C3H/HeJ) mice. Moreover, after LPS administration, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) are markedly lower in TLR4-mutant mice than in TLR4-normal mice. Thus, TLR4 is considered an upstream mediator of LPS-induced decrease in mouse Oatp4 mRNA. LPS is thought to alter liver gene expression through LPS-induced cytokines or nitric oxide (NO). TNF receptor p55 (TNFRp55) and type I IL-1 receptor (IL-1RI) mediate the biological functions of TNF-alpha and IL-1beta, respectively. Therefore, to determine whether endogenous cytokines or NO are mediators of LPS-induced down-regulation of Oatp4, Oatp4 mRNA levels were determined in mice deficient in the TNFRp55, IL-1RI, IL-6, or inducible nitric oxide synthase (iNOS) after LPS administration. Mice homozygous for a targeted deletion of genes for TNFRp55, IL-1RI, IL-6, or iNOS exhibited similar decreases in Oatp4 mRNA levels as wild-type mice after LPS administration. Moreover, in mouse hepatoma cells, treatment with TNF-alpha, IL-1beta, or IL-6 individually or in combination did not suppress activity of mouse Oatp4 promoter (-4.8 kb to +30). Therefore, LPS-induced down-regulation of Oatp4 appears to be independent of TNF-alpha, IL-1beta, IL-6, or iNOS.     

Effect of a kinin B2 receptor antagonist on LPS- and cytokine-induced neutrophil migration in rats

1 This study examines the involvement of kinins in neutrophil migration into rat subcutaneous air pouches triggered by lipopolysaccharide (LPS), as well as the putative roles played by kinin B(1) and B(2) receptors, tumour necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and selectins in this response. 2 LPS (5 ng to 10 micro g cavity(-1)) injected into the 6-day-old pouch induced a dose- and time-dependent neutrophil migration which peaked between 4 and 6 h, and was maximal following the dose of 100 ng cavity(-1) (saline: 0.46+/-0.1; LPS: 43+/-3.70 x 10(6) cells cavity(-1) at 6 h). 3 Bradykinin (BK) (600 nmol) injected into the pouch of saline-treated rats induced only modest neutrophil migration (0.73+/-0.16 x 10(6) cells cavity(-1)). A more robust response to BK (3.2+/-0.25 x 10(6) cells cavity(-1)) was seen in animals pretreated with captopril, but this was still smaller than the responses to IL-1beta or TNF-alpha (15 pmol: 23+/-2.2 x 10(6) and 75 pmol: 29.5+/-2 x 10(6) cells cavity(-1), respectively). Nevertheless, the B(1) agonist des-Arg(9)-BK (600 nmol) failed to induce neutrophil migration. 4 HOE-140 (1 and 2 mg kg(-1)), a B(2) receptor antagonist, reduced LPS-induced neutrophil migration. HOE-140 also reduced the neutrophil migration induced by BK, but had no effect on the migration promoted by IL-1beta or TNF-alpha. des-Arg(9)-[Leu(8)]-BK, B(1) receptor antagonist was ineffective in changing neutrophil migration caused by any of these stimuli. 5 Neutrophil migration induced by LPS or BK was reduced by interleukin-1 receptor antagonist (IL-1ra) (1 mg kg(-1)), sheep anti-rat TNF serum (anti-TNF serum) (0.3 ml cavity(-1)), and the nonspecific selectin inhibitor fucoidin (10 mg kg(-1)). 6 TNF-alpha levels in the pouch fluid were increased by LPS or BK injection, peaking at 0.5-1 h and gradually declining thereafter up to 6 h. IL-1beta levels increased steadily throughout the 6 h period. HOE-140 markedly inhibited the rise in IL-1beta and TNF-alpha levels in pouch fluid triggered by both stimuli. 7 These results indicate that BK participates importantly in selectin-dependent neutrophil migration into the air pouch triggered by LPS in the rat, by stimulating B(2) receptors coupled to synthesis/release of TNF-alpha and IL-1beta.     

Effects of phycocyanin extract on tumor necrosis factor-alpha and nitrite levels in serum of mice treated with endotoxin

Phycocyanin is a biliprotein which exerts antioxidative and anti-inflammatory effects in various in vivo and in vitro experimental models. In this study phycocyanin effects on tumor necrosis factor-alpha (TNF alpha) and nitrite levels in serum of mice treated with lipopolysaccharide (LPS) was examined. TNF alpha was measured by cytotoxicity on L-929 cells and nitrite by the Griess reaction, after reduction of all nitrates to nitrites by nitrate reductase, 1 h after LPS injection (0.5 mg/kg i.p.) there was a significant increase in TNF alpha levels in mouse serum. Phycocyanin (50-300 mg/kg p.o.), administered 1 h before LPS, reduced dose-dependently the TNF alpha concentration in serum. After 18 h, LPS (30 mg/kg i.p.) also induced a substantial increase in serum nitrite levels, which were reduced dose-dependently by phycocyanin pretreatment (100-300 mg/kg p.o.). The results indicate that phycocyanin exerts inhibitory effects on TNF alpha and NO production which might be ascribed to the antioxidative properties of the biliprotein.     

The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha.

1. Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha). These cytokines depress cardiac mechanical function by a mechanism which is not well defined. 2. Bacterial endotoxin or cytokines cause the expression of Ca(2+)-independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL-1 beta plus TNF-alpha in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation. 3. Rat isolated working hearts perfused with IL-1 beta plus TNF-alpha showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time-matched control hearts. The depressant action of IL-1 beta plus TNF-alpha was first apparent after 1 h of perfusion; no early (15 min) cardiac depressant actions were seen. 4. The competitive inhibitor of Ca(2+)-dependent and Ca(2+)-independent NO synthases, NG-nitro-L-arginine methyl ester (L-NAME, 3 microM) when given concurrently with IL-1 beta plus TNF-alpha prevented the loss in contractile function such that these hearts after 2 h of perfusion had similar function to time-matched controls. L-NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL-1 beta plus TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-dose propofol ameliorates haemorrhagic shock-induced organ damage in conscious rats

1. Propofol is an anti-inflammatory agent commonly used for general anaesthesia and sedation in intensive care unit patients. Haemorrhagic shock (HS) followed by resuscitation can induce the production of several inflammatory mediators, such as tumour necrosis factor (TNF)-a and interleukin (IL)-10, leading to multiple organ dysfunction and death. 2. In the present study, we investigated the effects of treatment with high-dose (10 mg/kg per h) and low-dose (1 mg/kg per h) propofol after HS on the physiopathology and cytokine levels in conscious rats. 3. The experiments were designed to induce HS by withdrawing 40% of total blood volume from a femoral artery catheter (6 mL/100 g bodyweight) over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 48 h after the start of blood withdrawal. Levels of biochemical parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), were measured 30 min before and 0, 1, 3, 6, 9, 12, 18, 24 and 48 h after the end of the 30 min blood withdrawal period. Serum cytokine levels, including TNF-a and IL-10, were measured at 1, 6, 12, 24 and 48 h after HS. The kidneys, liver, lungs and small intestine were removed for pathological assessment 48 h after HS. 4. In the present study, HS significantly increased blood GOT, GPT, BUN, LDH, CPK, TNF-a and IL-10 levels in conscious rats. Post-treatment with high-dose propofol accentuated systemic hypotension, increased serum TNF-a and IL-10 levels and increased the severity of organ damage after HS. In contrast, post-treatment with low-dose propofol did not affect MAP, but did suppress increased serum levels of the inflammatory cytokines and improved the survival rate after HS, thus protecting rats against HS-induced organ damage. 5. In conclusion, post-treatment with low-dose propofol ameliorated HS-induced markers of organ injury, suppressed the release of TNF-a and IL-10 and protected against HS-induced liver, kidney, lung and small intestine damage in conscious rats. These findings suggest the need to investigate whether low-dose propofol may be beneficial for patients with HS-induced organ damage.     

Inflammatory mediator production in swine following endotoxin challenge with or without co-administration of dexamethasone

The inflammatory response in swine challenged with lipopolysaccharide (LPS) has only been partially characterized. As swine are increasingly used in biomedical research, it is important to determine if they respond to endotoxin challenge in a manner similar to other model systems. Accordingly, 24 Poland China x Landrace barrows were treated with saline, LPS, dexamethasone, or LPS and dexamethasone, with six animals in each treatment group.The kinetics of TNFalpha, IL-1beta, IL-6, IL-8, IL-10, nitric oxide (nitrate/nitrite), and neopterin production in swine plasma were examined at 1, 3, 6, 9, and 24 h after acute LPS challenge. Lipopolysaccharide increased plasma TNFalpha levels, which peaked 1 h post-challenge. Dexamethasone decreased LPS-induced TNFalpha by approximately 60%. Plasma IL-6 levels peaked 3 h post-LPS challenge, returning to basal levels by 9 h. Swine given both LPS and dexamethasone had minimal IL-6 levels. Control and dexamethasone-only treated animals never exhibited systemic TNFalpha or IL-6 levels. Lipopolysaccharide increased plasma IL-10 1 h after challenge. Dexamethasone did not alter plasma IL-10 levels in LPS-challenged swine. Interleukin-1beta was constitutively present in plasma and was not altered by any combination of treatments. Plasma IL-8 was not observed in any treatment group. Plasma nitrate/nitrite levels were maximal 24 h post-challenge. Dexamethasone treatment prevented increases in plasma nitrate/nitrite levels in LPS-treated animals. Lipopolysaccharide induced levels of neopterin; dexamethasone served to further increase plasma neopterin levels in LPS-challenged animals. The discordant regulation of inflammatory mediators suggests that the immunological responses by swine to LPS are distinct from the responses seen in rodent and human studies.     

Lipopolysaccharide neutralization by the antibacterial peptide CM4

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. Binding of LPS to the CD14+ murine macrophage cell line RAW264.7 results in pro-inflammatory cytokine secretion. In extreme cases, it leads to septic shock in vivo. Therefore, the pursuit for molecules with antiendotoxin properties is urgent. In this study, we investigated the efficacy of antibacterial peptide CM4 in binding Escherichia coli LPS in vitro. CM4 avidly bound to E. coli LPS, as proven by the limulus amoebocyte lysate assay. Furthermore, the killing activity of CM4 against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Flow cytometric analysis revealed that CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, the inhibition of peptide to LPS-dependent cytokine induction was analyzed. CM4 suppressed LPS-induced TNF-alpha and IL-6 mRNA expression and blocked release of TNF-alpha and NO following LPS challenge in RAW264.7 cells. Together these observations indicate that antibacterial peptide CM4 probably exerts protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells.     

Discovery of novel and potent small-molecule inhibitors of NO and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(N-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate

To develop a new therapeutic agent for sepsis, screening of the Takeda chemical library was carried out using mouse macrophages stimulated with lipopolysaccharide (LPS) to identify a new class of small-molecule inhibitors of inflammatory mediator production. The lead compound 5a was discovered, from which a series of novel cyclohexene derivatives I bearing a sulfamoyl and ester group were designed, synthesized and tested for their inhibitory activity against nitric oxide (NO) production. Derivatives I were synthesized by the coupling of sulfonyl chlorides and anilines with concomitant double bond migration in the presence of triethylamine, and phenyl ring substitution and modification of the ester and cyclohexene moieties were carried out. Among the compounds synthesized, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [(R)-(+)-5n, TAK-242] was found to exhibit the most potent suppressive activity for the production of not only NO but also inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) induced by LPS-stimulated mouse macrophages with IC50 values of 1.8, 1.9 and 1.3 nM, respectively. It shows marked beneficial effects in vivo also. Intravenous administration of (R)-(+)-5n at doses of 0.1 mg/kg or more suppressed the production of NO and various cytokines [TNF-alpha, IL-6 and IL-1beta] in the mouse endotoxin shock model. Furthermore, it protected mice from death dose-dependently and all mice survived at a dose of 3 mg/kg. The minimum effective dose to protect mice from lethality in this model was 0.3 mg/kg, which was consistent with those for inhibitory effects on the production of NO and cytokines. Compound (R)-(+)-5n is currently undergoing clinical trials for the treatment of sepsis.     

Role of p38 MAP kinase in LPS-induced airway inflammation in the rat

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED(50)=15.8 mg kg(-1)) plasma levels of TNF-alpha in rats challenged with LPS (1.5 mg kg(-1), i.p.). Aerosolized LPS induced a peak in TNF-alpha levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL-1beta levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100 mg kg(-1)), had no effect on peak TNF-alpha levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg(-1)) significantly reduced both TNF-alpha levels and neutrophilic inflammation. However, the BAL fluid from rats pre-treated with either compound significantly inhibited TNF-alpha release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100 mg kg(-1)) produced dose-related inhibition of BAL IL-1beta levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4 h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL-1beta and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.     

Increase in gastric intramucosal hydrogen ion concentration following endotoxin challenge in the rat and the actions of nitric oxide synthase inhibitors

1. Cardiovascular events and outcome in septic shock may be predicted by monitoring the fall in intramural pH (pHi), as an index of splanchnic perfusion and mucosal ischaemia. In the present study, a small animal model for monitoring the changes of gastric pHi or intramucosal [H+] following challenge with the endotoxin lipopolysaccharide (LPS) was developed in the rat. The role of nitric oxide (NO) in these events in this model was evaluated using the non-selective NO synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA). 2. The pHi and intramucosal [H+] were evaluated in omeprazole-pretreated rats (30 mg/kg, i.p.) using the Henderson equation after estimating the PCO2 and the bicarbonate concentration in gastric wall. To measure gastric wall PCO2, the oesophagus was intubated and the pylorus ligated. The PCO2 was measured by a blood gas analyser in 2 mL saline instilled for 30 min in the gastric lumen to equilibrate with the gastric wall. The pHi was measured under basal conditions and 3 and 5 h after LPS (3 mg/kg) administration. Separate groups received treatment with L-NMMA (25-50 mg/kg) or L-NAME concomitantly or 2.5 h after administration of LPS. 3. Intravenous administration of Escherichia coli LPS provoked a significant fall in gastric pHi from 7.37 to 7.18 (median values; n =10-19) determined after 5 h. In groups treated concurrently with LPS and L-NAME (5 mg/kg; n = 19), there was a similar increase in intramucosal [H+] as that induced by LPS alone (n = 15) in those animals that survived. In contrast, L-NAME (5 mg/kg; n = 12), given 2.5 h after LPS challenge, at a time at which inducible NOS is known to be significantly expressed, prevented the increase in intramucosal [H+] at 3 and 5 h after LPS challenge. Similarly, L-NMMA (25-50 mg/kg; n = 23), given 2.5 h after LPS challenge, dose-dependently inhibited the increase in intramucosal [H+] at 3 and 5 h. 4. In conclusion, these findings indicate that this rat model could be useful in exploring the pathophysiology of acute endotoxin shock. Delayed administration of L-NAME and L-NMMA abolished the increase in gastric intramucosal [H+], supporting the involvement of excess NO in the tissue dysfunction associated with endotoxin shock. This suggests the potential value of this small animal model in evaluating the therapeutic activity of novel agents for use in septic shock.