Surfactin C inhibits platelet aggregation

This study was designed to investigate the effect of surfactin C, which is derived from Bacillus subtilis, on platelet aggregation and homotypic leucocyte aggregation. Surfactin C strongly and dose-dependently inhibited platelet aggregation, which was stimulated both by thrombin (0.1 U mL(-1)), a potent agonist that activates the G protein-coupled protease receptor, and by collagen (5 microg mL(-1)), a potent ligand that activates alpha(IIb)beta(3) with IC50 values (concentration inhibiting platelet aggregation by 50%) of 10.9 and 17.0 microM, respectively. Moreover, surfactin C significantly suppressed the intracellular Ca(2+) mobilization in thrombin-activated platelets. Surfactin C, however, did not affect various integrin-mediated U937 cell aggregation, implying that the anti-platelet activity of surfactin C was not due to its detergent effect but by its action on the downstream signalling pathway. Therefore, the results suggest that surfactin C may have a beneficial therapeutic effect on aberrant platelet aggregation-mediated cardiovascular diseases.     

NF‐κB activation mediates LPS‐or zymosan‐induced hypotension and inflammation reversed by BAY61‐3606, a selective Syk inhibitor,in rat models of septic and non‐septic shock

We have previously demonstrated that the activation of the spleen tyrosine kinase (Syk)/inhibitory‐κB (IκB)‐α/nuclear factor‐κB (NF‐κB) p65 signalling pathway contributes to hypotension and inflammatory response in a rat models of zymosan (ZYM)‐induced non‐septic shock. The purpose of this study was to further examine the possible mechanism underlying the effect of inhibition of Syk by BAY61‐3606 via NF‐κB activity at the level of nuclear translocation regarding the production of vasodilator and proinflammatory mediators in lipopolysaccharide (LPS) (septic)‐ and ZYM (non‐septic)‐induced shock. Administration of LPS (10 mg/kg, ip) or ZYM (500 mg/kg, ip) to male Wistar rats decreased mean arterial pressure and increased heart rate that was associated with an increase in the activities of cyclooxygenase and nitric oxide synthase, tumour necrosis factor‐α, and interleukin‐8 levels, and NF‐κB activation and nuclear translocation in sera and/or cardiovascular and renal tissues. BAY61‐3606 (3 mg/kg, ip), the selective Syk inhibitor, given 1 hour after LPS‐ or ZYM injection reversed all the above‐mentioned effects. These results suggest that Syk contributes to the LPS‐ or ZYM‐induced hypotension and inflammation associated with transactivation of NF‐κB in septic and non‐septic shock.     

重组人生长激素对败血症休克大鼠微循环功能障碍的影响

目的观察重组人生长激素(rhGH)对败血症休克大鼠微循环功能障碍的影响。方法ipE.coli复制大鼠败血症休克模型。采用微循环观测系统及激光多普勒观测正常对照组(C组)、败血症休克组(S组)及rhGH治疗组(T组)第1、3天的肠道微循环、耳廓血流量等指标的变化情况。结果S组大鼠肠道微循环血流明显减慢甚至淤滞,血管明显扩张,血管数、管袢数及功能血管数明显减少,尤以第1天时最为明显;T组大鼠肠道微循环血液流速、血管数及功能血管数均明显高于S组,与C组比较无显著性差异。S组大鼠耳廓血流量在各时点均显著低于C组、T组水平,但T组与C组比较无明显差异。结论rhGH可明显改善败血症休克大鼠的微循环功能障碍。     

感染性休克对大鼠肠上皮水通道蛋白8表达的影响

目的 通过建立大鼠感染性休克模型,探讨其对肠上皮水通道蛋白8（AQP8）表达的影响及与肠组织水肿的关系。方法 32只健康SD大鼠随机分成正常对照组（C组）、感染性休克2h、8h和24h组。采用静脉注射大肠杆菌脂多糖（LPs）制作感染性休克模型。Western blot检测肠上皮AQP8蛋白的表达,同时测定肠组织含水量,并做相关分析。结果 感染性休克组各时间点肠组织含水量均明显升高,与C组比较差异有统计学意义（P〈0.05）,且以休克后8h时最高（P〈0.05）。C组AQP8呈现低密度带;感染性休克后2h、8h、24h时AQP8密度带明显增强,活性增加（P〈0.05）;休克后8 h时点AQP8密度带更高,蛋白表达更强（P〈0.05）。相关分析表明,肠道组织的含水量与AQP8蛋白表达呈正相关。结论 感染性休克使AQP8表达增加,与感染性休克后肠组织水肿的形成有关。     

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.     

脓毒症大鼠肝脏细胞TLR4和TNF-α表达及细胞凋亡研究

目的 观察脓毒症时期大鼠肝脏Toll样受体4(toll-like receptor 4,tLR4)及肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)表达及肝脏细胞凋亡情况.方法 (1)建立动物模型及分组:72只SPF级SD大鼠随机分为正常对照组、脓毒症组及脓毒性休克组,脓毒症组腹腔注射10 mg/kg内毒素(lipopolysaccharide,LPS)、脓毒性休克组腹腔注射12 mg/kg内毒素建立大鼠模型;正常对照组腹腔注射12 mg/kg生理盐水.(2)各组大鼠在注射后1h、6h、24 h处死,每时点每组各8只,采集肝脏组织标本;标本制作石蜡切片,并设计原位杂交探针序列,进行mRNA原位杂交检测;TUNEL(原位末端1转移酶标记技术)法测定细胞凋亡;结果进行定量分析,并做数据统计.结果 (1) mRNA原位杂交检测TLR4在脓毒症及脓毒症休克组大鼠肝脏表达在LPS攻击后1h、6h、24 h随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组均显著高于对照组,脓毒性休克组TLR4表达显著高于脓毒症组(P＜0.05).(2)TNF-α在LPS攻击大鼠后1h、6h、24 h随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组均显著高于对照组,脓毒性休克组均显著高于脓毒症组(P＜0.05).(3)脓毒症及脓毒性休克大鼠肝脏组织细胞凋亡情况随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组显著高于对照组,脓毒性休克组细胞凋亡显著高于脓毒症组(P＜0.05).结论 (1)TLR4及TNF-α在肝脏的高表达,尤其在脓毒性休克大鼠肝脏的高表达,进一步证实由于LPS导致机体通过TLR4介导产生TNF-α等炎症因子的过度释放,产生炎症因子的瀑布反应,引起重要脏器组织细胞凋亡,对机体造成严重影响.(2)通过阻断TLR4的表达,可能抑制脓毒症大鼠炎症因子的大量释放,对减轻机体各重要脏器的损害可能有益.     

Inhibition of lipopolysaccharide activity by a bacterial cyclic lipopeptide surfactin

Compounds that inactivate lipopolysaccharide (LPS) activity have the potential of being new anti-inflammatory agents. Therefore, we searched among microbial secondary metabolites for compounds that inhibited LPS-stimulated adhesion between human umbilical vein endothelial cells (HUVEC) and HL-60 cells. By this screening, we found a cyclic lipopeptide surfactin from the culture broth of Bacillus sp. BML752-121F2 to be inhibitory. The addition of the surfactin prior to the LPS stimulation decreased HL-60 cell-HUVEC adhesion without showing any cytotoxicity. We confirmed that surfactin inhibited LPS-induced expression of ICAM-1 and VCAM-1 in HUVEC. It also inhibited the cellular adhesion induced by lipid A, the active component of LPS; but it did not inhibit TNF-alpha or IL-1 beta-induced cell adhesion. Then, surfactin was shown to suppress the interaction of lipid A with LPS-binding protein (LBP) that mediates the transport of LPS to its receptors. Finally, surface plasmon resonance (SPR) analysis revealed the surfactin to interact reversibly with lipid A. Thus, this Bacillus surfactin was shown to be an inhibitor of LPS-induced signal transduction, directly interacting with LPS.     

Bartonella quintana lipopolysaccharide effects on leukocytes,CXC chemokines and apoptosis: a study on the human whole blood and a rat model

Bartonella quintana, an emerging gram-negative pathogen, may cause trench fever, endocarditis, cerebral abscess and bacillary angiomatosis usually with the absence of septic shock in humans. B. quintana lipopolysaccharide (LPS), a deep rough endotoxin with strong reactivity in the limulus amebocyte lysate (LAL)-assay, was studied in human whole blood and in a rat model. A significant (P<0.05) increase of interleukin-8 (IL-8) concentration, comparable to the level induced by enterobacterial LPS, was stimulated in the human whole blood by B. quintana LPS. Isolated human neutrophils delayed their apoptotic behavior in the presence of B. quintana LPS. In the rat, B. quintana LPS induced a significant (P<0.001) increase in white blood cell count, both 30 and 60 min after intravenous injection. Such leukocytosis was inhibited by pretreatment with prazosin, an alpha-adrenergic antagonist. B. quintana LPS did not significantly change heart rate (HR), hematocrit (HCT) and platelet count in the above reported in vivo model, and regarding mean blood pressure (MAP) only a very early (5 min after LPS) and mild (yet significant) hypotension was observed. In contrast, a long-lasting decrease of MAP was found in Salmonella minnesota R595 LPS-treated animals. Blood TNFalpha levels did not change significantly from the baseline in rats injected with either saline or with B. quintana LPS, on the contrary S. minnesota R595 LPS-injected animals showed substantial increase of TNFalpha levels up to 2924 pg/ml at 60 min after LPS injection. B. quintana LPS as well as Salmonella LPS-injected rats exhibited an increase of the blood levels of GRO/CINC-1, particularly at 240 min after LPS administration. Apical part of rat gut villi showed several TUNEL-positive cells in tissue sections from B. quintana LPS-treated animals. Taken together, our data demonstrates that B. quintana LPS is able to selectively stimulate some inflammatory mediators. B. quintana LPS-induced leukocytosis appears mediated by an alpha-adrenergic receptor. The delayed apoptotic process of leukocytes and the chemokine increase may explain the apoptotic cells found in the rat gut and the inflammatory reactions in some human Bartonella diseases. This peculiar inflammatory pattern induced by B. quintana LPS, may partially account for the lack of severe septic shock, observed in human B. quintana infections.     

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.     

LIPOCORTIN-1 PRESERVES MYOCARDIAL RESPONSIVENESS TO β-ADRENERGIC STIMULATION IN RAT PAPILLARY MUSCLE

1. During septic shock, myocardial contractile dysfunction is accompanied by the release of cytokines and enhanced production of nitric oxide, and the contractile dysfunction is prevented by glucocorticoids. 2. Myocardial dysfunction was induced in vitro by incubation of rat papillary muscle for 15 h with endotoxin (lipopolysaccharide, LPS) and interferon-gamma (IFN-gamma). 3. Both baseline contractile function and inotropic responsiveness to isoprenaline were markedly reduced by the combination of LPS plus IFN-gamma. 4. Lipocortin-1 (LC-1) is induced by glucocorticoids, and LC-1(2-26), its N-terminal fragment, protected the papillary muscle inotropic responsiveness to isoprenaline, but did not affect the decline in baseline contractile function induced by LPS plus IFN-gamma. 5. The mechanisms of this protective action need to be explored further, but LC-1 may prove to be a novel cardioprotective agent for the management of septic shock.     

Radiation and primary immune response to lipopolysaccharide: lymphocyte distribution and function

INTRODUCTION: Lipopolysaccharide (LPS) is a major cause of septic shock and death due to infection with Gram-negative bacteria. The purpose of this study was to quantify the effects of whole-body irradiation on lymphocyte populations during response to challenge with LPS. MATERIALS AND METHODS: C57BL/6 mice (n = 10/group) were irradiated whole-body with 3 gray (Gy) gamma-rays in a single fraction at 0.8 Gy/min. LPS (E. coli serotype 0111:B4) at 1 mg/kg was injected intraperitoneally 10 days later and mice were euthanized at 60 min and days 1, 7, and 14 post-inoculation for analyses. RESULTS: Significant interactions between radiation and LPS were noted in circulating and splenic lymphocyte subpopulations, including T-, B-, and NK-cells, particularly at the early time points. There were significant interactions on circulating, but not splenic, CD62L+ T-cell populations. However, there were no interactions on CD62L+ B-cells. Finally, there were significant interactions in both early and late blastogenic responses. CONCLUSION: The data support that response to infection with Gram-negative bacteria may be significantly compromised by exposure to ionizing radiation.     

Structure-Bioactivity Relationships of Bacterial Endotoxins

Abstract

Endotoxins, chemically lipopolysaccharides (LPS), are major components of the cell envelope of Gram-negative bacteria which are an important contributing factor to septic shock, in general, and Gram-negative septic shock, in particular. The structure of the lipid moiety of LPS, lipid A, has been intensively investigated in Escherichia coli and Salmonella spp. and based on the structural data, synthetic compounds prepared. In general, these compounds exhibit identical endotoxic activities compared with bacterial lipid A and LPS, thus confirming that lipid A is the endotoxic center of LPS. Analysis of lipid A from various Gram-negative bacterial species showed that lipid A is a family of (phospho)glycolipid molecules that are closely related in general architecture, but whose fine structure varies. The various structures of selected lipid As of different bacterial species are reviewed in this article and the influence of their structure on endotoxicity is discussed. The resulting Deductions on structure-bioactivity relationships from the latter studies is supported and extended by investigations on the bioactivity (endotoxicity) of synthetic lipid A analogs and partial structures. In particular, endotoxicity is not dependent on one lipid A constituent, i.e., a toxophore group, but it is a unique molecular structure, a peculiar surpramolecular conformation, which allows optimal expression of endotoxicity activity. Furthermore, LPS is not a direct toxin, rather endotoxic shock is a syndrome that results from the host's own response to LPS whereby there is systemic release of endogenous substances (cytokines) that control the cascade of events leading to shock. Considerable advances have been made in understanding the specific interaction of endotoxin with serum proteins and monocyte/macrophage-bound recognition molecules. Since this is the central event of endotoxin activity leading to endotoxic shock, therapeutic approaches have been proposed blocking this event.     

The search for molecular determinants of LPS inhibition by proteins and peptides

Lipo-poly-saccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis. The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E. coli K-12 (Ferguson et al., Science 1999, 282, 2215). This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins. Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect. The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means. The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.     

Thymosin beta(4) reduces lethality and down-regulates inflammatory mediators in endotoxin-induced septic shock

Thymosin beta(4) (Tbeta(4)), a highly conserved peptide with immunomodulatory properties, is the major actin-sequestering peptide in mammalian cells. Recent studies have established that Tbeta(4) can accelerate wound healing in full thickness skin wounds and following burn injuries to the cornea. In the eye studies, the accelerated healing due to Tbeta(4) was accompanied by a significant reduction in polymorphonuclear leukocyte (PMN) infiltration and a several-fold decrease in interleukin-1beta (p< or =0.015) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF1alpha, p< or =0.05). Given the recognized role of proinflammatory cytokines in septic shock and of extracellular F- and G-actin in the pathophysiology of multiple organ dysfunction, we have investigated the role of Tbeta(4) in sepsis. We report that an LD(50) dose of LPS (24 mg/kg) in rats resulted in a significant reduction of Tbeta(4) levels in the blood. Furthermore, administration of 100 microg of Tbeta(4) immediately following and at 2 and 4 h after an LD(50) dose of LPS (60 mg/kg) in mice significantly reduced mortality rates (p< or =0.024) and lowered blood levels of a number of inflammatory cytokines, eicosanoids, and other molecules that are highly elevated following endotoxin administration. In studies in human subjects given low doses of endotoxin (4 ng/kg LPS) and in patients with septic shock, we have also observed significant decreases in blood levels of Tbeta(4). The rapid disappearance of Tbeta(4) in the blood following LPS administration or during septic shock suggests that Tbeta(4) may be involved in early events leading to activation of the inflammatory cascade and ultimately the clinical sequelae of sepsis. The results of this study indicate that Tbeta(4) may have utility in the clinic in the treatment of septic shock and in syndromes associated with actin toxicities.     

Evodia rutaecarpa protects against circulation failure and organ dysfunction in endotoxaemic rats through modulating nitric oxide release

Using a rat model of septic shock we studied the effects of Evodia rutaecarpa, a Chinese herbal medicine with antimicrobial and anti-inflammatory activity, on haemodynamic parameters, biochemical markers of organ function and nitric oxide (NO) production. Anaesthetized rats challenged with a high dosage of endotoxin (Escherichia coli lipopolysaccharide; LPS; 50 mg kg(-1), i.v.) for 6 h showed a severe decrease in mean arterial pressure. This was accompanied by delayed bradycardia, vascular hyporeactivity to phenylephrine and increase in plasma levels of lactate dehydrogenase, aspartate aminotransferase, bilirubin and creatinine, as well as NOx (NO2- plus NO3-). Pretreatment with ethanol extract of E. rutaecarpa (25, 50 and 100 mg kg(-1), i.v.), 1 h before LPS, dose-dependently prevented the circulation failure, vascular hyporeactivity to phenylephrine, prevented liver dysfunction and reduced the NOx over-production in plasma in endotoxaemic rats. A selective inducible NO-synthase (iNOS) inhibitor, aminoguanidine (15 mg kg(-1), i.v.), also effectively ameliorated the above pathophysiological phenomenon associated with endotoxaemia so that the normal condition was approached. Endotoxaemia for 6 h resulted in a significant increase in iNOS activity in the liver homogenate, which was attenuated significantly by E. rutaecarpa pretreatment. In summary, E. rutaecarpa, at the dosages used, exerted these beneficial effects probably through inhibition of iNOS activity and subsequent modulation of the release of NO. These significant results may offer E. rutaecarpa as a candidate for the treatment of this model of endotoxaemia.     

Protective action of amlodipine on cardiac negative inotropism induced by lipopolysaccharide in rats

Amlodipine has been shown to prevent decrease in vascular responsiveness induced by injection of Salmonella typhosa lipopolysaccharide (LPS); however, there is no study reporting if this protection by amlodipine extends to ventricular contractility. Therefore, we have investigated in rat isolated right ventricle strips contracted by electrical stimulation (1 and 3 Hz and subsequently 1 Hz) whether pre-treatment with amlodipine (15 mg/kg orally for 1 week) precludes the decrease in ventricular contractility related to the induction of nitric oxide synthase stimulated by LPS injection (4 mg/kg intraperitoneally). The induction of septic shock was confirmed in isolated aortic rings from LPS-injected rats by verifying that the contractile response to 1 microM noradrenaline had been (i) decreased after LPS injection and (ii) markedly potentiated by the addition of 10 microM l-arginine. The injection of saline to untreated and amlodipine-treated rats produced a non-significant effect on right ventricular contractility during 180 min. at 3 Hz; the recovery of the contractile response was improved when the stimulation frequency was subsequently returned to 1 Hz after 30 min. In contrast, injection of LPS to untreated and amlodipine-treated rats (amlodipine + LPS) produced a decrease in right ventricular contractility during 180 min. at 3 Hz, an effect that was more pronounced in LPS than in amlodipine-treated rats. These ex vivo results obtained after LPS injection suggest that amlodipine may have inhibited, at least in part, the induction of nitric oxide synthase with a resulting preclusion of the cardiovascular failure produced by septic shock.     

Selective versus non-selective suppression of nitric oxide synthase on regional hemodynamics in rats with or without LPS-induced endotoxemia

The late phase of severe septic shock is associated with reduced cardiac output (CO) and activation of the inducible isoform of nitric oxide synthase (NOS). This study examined the effects of 1400 W (N-3-aminomethyl-benzyl-acetamidine), a new selective inhibitor of inducible NOS (iNOS), relative to those of N(G)-nitro-L-arginine (L-NNA, non-selective inhibitor of NOS) and the vehicle, on mean arterial pressure (MAP), CO, total peripheral resistance (TPR) and tissue blood flow (BF) in thiobutabarbital-anesthetized rats with lipopolysaccharide (LPS, 10 mg/kg, i.v.) induced endotoxemia. At 2.5 as well as 4 h after injection of LPS, MAP, CO, and BF of the stomach, skeletal muscle and skin were decreased, but TPR was increased, BF to the heart and kidneys were also decreased at 4 h after injection of LPS. Treatment of endotoxemic rats with 1400 W (3 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h after endotoxin challenge prevented the late phase fall in MAP without exacerbating the decreases in CO and tissue BF. In contrast, treatment with L-NNA (8 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h did not prevent the decline in MAP in the LPS-treated rats. Furthermore, CO drastically decreased, TPR markedly increased, and BF to the heart, brain, intestine and skeletal muscle were decreased at 4 h relative to the readings in saline- or 1400 W-treated endotoxemic rats. Therefore, selective inhibition of iNOS by 1400 W restores MAP without compromising CO, but non-selective inhibition of NOS is detrimental at the late stage of septic shock.     

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.     

Protective effects of early administration of alpha-lipoic acid against lipopolysaccharide-induced plasma lipid peroxidation

Lipopolysaccharide (LPS), called endotoxin, is a major component of Gram-negative bacteria cell wall. LPS stimulates the synthesis and release of several metabolites from mammalian phagocytes which leads to fulminant systemic inflammation (endotoxic shock). Among LPS-induced metabolites, reactive oxygen species are considered to play crucial role in the pathogenesis of endotoxic shock via oxidative stress generation. In this study, the effect of early administration of antioxidant alpha-lipoic acid (LA) on plasma lipid peroxidation and total antioxidant blood capacity was evaluated in endotoxic shock in rats. Lipid peroxidation was measured as plasma thiobarbituric acid reactive substances (TBARS) levels, while total blood antioxidant capacity was assessed as ferric reducing ability of plasma (FRAP). The endotoxic shock was induced by administration of LPS (Escherichia coli 026:B6, 30 mg/kg, iv) in anesthetized rats. Then, 30 min later, animals were treated intravenously (iv) with LA at 60 mg/kg. After 5 h observation animals were killed and blood from heart was taken for TBARS and FRAP measurements. LPS injected to saline-pretreated animals resulted in development of oxidative stress indicated by significant increases in plasma TBARS and significant decrease in total antioxidant capacity of plasma. Conversely, LA injected to saline pretreated animals caused an increase in FRAP values and the decrease in TBARS levels. The administration of LA 0.5 h after LPS challenge resulted in an increase in FRAP values and decrease in plasma lipid peroxidation as compared to LPS group. Moreover, the levels of TBARS and FRAP in LPS + LA group were similar to those observed in LA group. In conclusion, our present study demonstrates that early treatment with LA significantly protects against endotoxin-induced oxidative stress in rats.     

盐酸戊乙奎醚对大鼠急性肺损伤支气管肺泡灌洗液TNF—α和IL-6水平的影响

目的探讨盐酸戊乙奎醚（PHC）预处理对大鼠急性肺损伤支气管肺泡灌洗液TNF—α和IL-6水平的影响。方法48例SD大鼠,随机分4组：C组：尾静脉注射生理盐水1ml／kg;M组：尾静脉注射大肠杆菌脂多糖（LPS）5mg／kg;P1组：尾静脉注射大肠杆菌LPS5mg／kg＋PHC0．25mg／kg;P2组：尾静脉注射大肠杆菌LPS5mg／kg＋PHC0．75mg／kg。每组12只。观察与C组和M组比较,支气管肺泡灌洗液TNF—α和IL-6结果。结果TNF-α：与C组比较,3组升高（P〈0．01）;与M组比较,P1和P2组降低（P〈0．01）;与P1组比较,P2组降低（P〈0．05）。IL-6：与C组比较,3组升高（P〈0．01）;与M组比较,P1和P2组降低（P〈0．01）;与P1组比较,P2组降低（P〈0．05）。结论PHC预处理能有效减低脓毒症大鼠肺组织血浆TNF—α和IL-6水平,并且在一定剂量范围内呈剂量效应关系。