An anti-sepsis monomer, 2',5,6',7-tetrahydroxyflavanonol (THF), identified from Scutellaria baicalensis Georgi neutralizes lipopolysaccharide in vitro and in vivo

Lipopolysaccharide (LPS) is a known trigger in the pathogenesis of sepsis, lipid A being the toxic component. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on the neutralization of LPS. In order to obtain the components from traditional Chinese herbs that can neutralize the endotoxin, aqueous extractions of twelve herbs were tested using affinity biosensor technology. From twelve herbs, Scutellaria baicalensis Georgi (Huang Qin) found to possess high lipid A-binding abilities, and was selected in subsequent experiments. After subjected to macroporous adsorptive resins and HPLC, we obtained 2',5,6',7-tetrahydroxyflavanonol (THF) from S. baicalensis Georgi under the direction of neutralization of LPS and reducing proinflammatory cytokines. In vitro, THF directly bound to LPS and neutralized its activity. THF not only down-regulated TNF-alpha mRNA expression but also decreased TNF-alpha and IL-6 release from RAW264.7 cells induced by LPS in a dose-dependent manner. THF-mediated inhibition on proinflammatory cytokine release is probably associated with downregulation of LPS-induced TLR4 mRNA augmentation. In vivo, THF could significantly protect mice against a lethal challenge with heat-killed E. coli 35218 (E. coli 35218) in a dose-dependent manner, and decreased the plasma LPS level in endotoxemia mice. These findings provide compelling evidence that THF may be an important potential drug for sepsis treatment. Considering the inhibitory effects of THF on LPS-induced cytokine release are unlikely due to its nonspecific cellular toxicity, THF should be considered as a safe putative candidate for development as a drug for sepsis treatment.     

Identification of a new anti-LPS agent,geniposide, from Gardenia jasminoides Ellis,and its ability of direct binding and neutralization of lipopolysaccharide in vitro and in vivo

Lipopolysaccharide (LPS/endotoxin) is a key pathogen recognition molecule for sepsis. Currently, one of the therapeutic approaches for severe sepsis is focusing on the neutralization of LPS, and clinical trials have shown a lot of traditional Chinese herbs possess anti-sepsis function. Herein, to elucidate the bioactive components of traditional Chinese herbs that can neutralize LPS, the lipid A-binding abilities of sixty herbs were tested using affinity biosensor technology. The aqueous extract of Gardenia jasminoides Ellis, traditionally used to treat inflammation in Asian countries for centuries, was further investigated. Subsequently, a monomer, identified as geniposide, was isolated. In vitro, geniposide was found to directly bind LPS and neutralize LPS. It dose-dependently inhibited cytokines release from RAW264.7 cells induced by LPS without affecting the cell viability, and inhibited TNF-α mRNA expression up-regulated by LPS. However, geniposide did not decrease TNF-α release induced by CpG DNA, Poly I:C or IL-1β. Significantly, geniposide dose-dependently down-regulated TLR4 mRNA expression up-regulated by LPS, and suppressed the phosphorylations of p38 MAKP induced by LPS but not by IL-1β. In vivo, geniposide (40 mg/kg) could significantly protect mice challenge with lethal heat-killed E. coli, and dose-dependently decreased the level of serum endotoxin which was tightly associated with the cytokine levels in endotoxemia mice. In summary, we successfully isolated geniposide from G. jasminoides Ellis. Geniposide directly bound LPS and neutralized LPS in vitro, and significantly protected sepsis model mice. Therefore, geniposide could be as a useful lead compound for anti-sepsis drug development.     

Dual targets guided screening and isolation of Kukoamine B as a novel natural anti-sepsis agent from traditional Chinese herb Cortex lycii

Treating sepsis remains challenging at present. Bacterial lipopolysaccharide (LPS) and bacterial DNA/CpG DNA are important pathogenic molecules and drug targets for sepsis. It is thus a promising strategy to treat sepsis by discovering agents that neutralize LPS and CpG DNA simultaneously. In this study, we present evidences of the biosensor based screening and isolation of active anti-sepsis fractions and monomers from traditional Chinese herbs using dual targets (LPS and CpG DNA) guided drug discovery strategy. Firstly, LPS or CpG DNA was immobilized on surfaces of cuvettes in the biosensor to establish a screening platform. Then, Cortex lycii with both highest affinities was selected out from one hundred and fourteen traditional Chinese herbs. In subsequent experiments, chromatography was utilized and coupled with the biosensor to purify fractions with a higher affinity for LPS and CpG DNA. In line with affinity assay, these fractions were shown to neutralize LPS and CpG DNA and inhibit their activity in vitro and in vivo. Lastly, the contributing monomer Kukoamine B (KB) was purified. KB neutralized LPS and CpG DNA in vitro. It inhibited TLR4, TLR9 and MyD88 mRNA expressions up-regulated by LPS and CpG DNA, and also attenuated the LPS and CpG DNA elicited nuclear translocation of NF-κB p65 protein in RAW264.7 cells. It also protected mice from lethal challenge of heat-killed E. coli, a mixture of LPS and CpG DNA. In conclusion, we presented a dual target guided discovery of a novel anti-sepsis agent KB from traditional Chinese herbs via combination of biosensor technology and chromatography methods.     

以lipid A为靶点应用生物传感器技术筛选抗炎中药

目的应用生物传感器跟踪检测技术从传统抗炎中药中筛选具有拮抗内毒素作用的中药，并对其进行活性物质含量排序。方法将lipid A包被于生物传感器的非衍生板，建立靶点，测定22种去鞣质后的中药水煎液与lipid A的亲和力；选择具有较高亲和力的药物与定量脂多糖（5ng／mL）于37℃混合孵育30min，再测定其与lipidA的亲和力，以评价药物水提液中活性物质的含量。结果22种中药均具有与lipidA较高的亲和力（大于100RU）；侧柏叶、地骨皮、石榴皮等16种中药中与lipidA特异性结合的活性物质含量较高。结论应用生物传感器技术建立的筛选平台快速而客观，筛选出的16种中药均含有能与lipidA发生特异性结合作用的非鞣质类活性物质。     

Increment of plasma soluble CD14 level in carrageenan-primed endotoxin shock model mice

CD14 is membrane-associating or free soluble glycoprotein which recognizes lipopolysaccharide (LPS) and is assumed to be involved in the onset of endotoxin shock. There are some reports suggesting the relationship between increased expression of CD14 in infectious or inflammatory diseases. However, little has been reported concerning the soluble CD14 (sCD14) level, especially in mice. In this study, we measured the plasma level of sCD14, TNF-alpha and IL-6 in the iota-carrageenan (CAR)-primed endotoxin shock model in addition to the D-galactosamine (D-galN)-primed endotoxin shock model mice. It was confirmed that all mice were dead within 12 h after a higher dose of LPS-treatment in both animal models. The level of TNF-alpha, IL-6 and sCD14 significantly increased in the CAR-primed endotoxin shock model mice. However, the D-galN-primed endotoxin shock model mice showed only a slight increment of TNF-alpha and IL-6 level, and sCD14 was below the detectable level. In the examination using several doses of LPS in CAR-primed model mice, IL-6 and sCD14 were increased dependent on the LPS dose, but TNF-alpha remained at an almost equal level at any dose of LPS in this study condition. In conclusion, the production of TNF-alpha, IL-6 and sCD14 was significantly enhanced in the CAR-primed model mice, compared to the D-galN-primed model mice. Therefore, these data indicate the possibility that the sCD14 level did not increase consistently, even under a fatal condition in endotoxin shock. Also, CAR-primed endotoxin shock would be an important experimental model to examine the elevation mechanisms for sCD14 and IL-6 production.     

Targeting CpG DNA to screen and isolate anti-sepsis fraction and monomers from traditional Chinese herbs using affinity biosensor technology

Bacterial DNA/CpG DNA is recognized as a key molecule during the pathogenesis of sepsis. Therefore, preventing CpG DNA from binding to its receptor is considered as the most promising strategy. In the present experiments, Radix et Rhizoma Rhei had the highest CpG DNA-binding ability among the seventy-eight traditional Chinese herbs. After the isolation of silica gel chromatography and high performance liquid chromatography (HPLC) and evaluation with affinity biosensor, the active fraction was confirmed and named Fraction D. It was found that in vitro, Fraction D bound to both CpG DNA and lipid A with high affinity, and strongly inhibited LPS- and CpG DNA-induced TNF-α release from RAW264.7 cells in a dose-dependent manner. Furthermore, Fraction D reduced the expression of TLR9 mRNA up-regulated by CpG DNA. In vivo, Fraction D protected mice challenged with lethal heat-killed E. coli. Using HPLC method, two monomers with high affinity for CpG DNA were isolated and identified as rhein and emodin. Rhein could significantly reduce CpG DNA- and LPS-induced TNF-α release, but emodin only reduced CpG DNA-induced TNF-α release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF-α release, which contributed to the bioactivity of Fraction D. In conclusion, we successfully established the platform to screen anti-CpG DNA components of traditional Chinese herbs using affinity biosensor technology, got active Fraction D from Radix et Rhizoma Rhei and determined rhein and emodin as the main bioactive ingredients in Fraction D.     

G1-4A, an immunomodulatory polysaccharide from Tinospora cordifolia, modulates macrophage responses and protects mice against lipopolysaccharide induced endotoxic shock

Pro-inflammatory cytokines are known to be the mediators of endotoxic shock and several immunomodulatory herbs can modulate the expression of these cytokines. Therefore we have investigated the possibility of using an arabinogalactan polysaccharide, G1-4A, from the stem of Tinospora cordifolia, for protection against endotoxin induced sepsis. There was 100% protection against lipopolysaccharide (LPS) induced mortality in mice pretreated with G1-4A. To elucidate the mechanism of action, its effect on macrophages, the primary source of these pro-inflammatory molecules was evaluated. G1-4A was shown to bind to the murine macrophages leading to their activation and reciprocally inhibited binding of LPS to macrophages. Following treatment with G1-4A, there was a small increase in serum TNF-alpha and IL-1beta levels. However, challenge with LPS elicited significantly reduced levels of TNF-alpha in G1-4A pretreated mice as compared to the controls while the level of soluble TNFR was enhanced. An increase in serum IL-1beta, IL-6, IFN-gamma levels and decrease in that of IL-10 was observed following challenge with LPS in mice pretreated with G1-4A as compared to the controls. In addition, G1-4A also modulated the release of nitric oxide by murine macrophages. Similar phenomenon was observed in a human monocytic cell line, U937. Thus G1-4A appeared to induce tolerance against endotoxic shock by modulation of cytokines and nitric oxide.     

Inhibitory effects of dimethylacetyl-beta-cyclodextrin on lipopolysaccharide-induced macrophage activation and endotoxin shock in mice

The potential use of hydrophilic cyclodextrins (CyDs) as an inhibitor for lipopolysaccharide (LPS) was examined. Of the five CyDs used in this study, dimethylacetyl-beta-cyclodextrin (DMA7-beta-CyD) had greater inhibitory activity than other CyDs against the production of nitric oxide (NO) and various proinflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) in murine macrophages stimulated with two serotypes of LPS and lipid A. The inhibitory effect of DMA7-beta-CyD on NO production was also observed in macrophages stimulated with lipoteichoic acid (LTA), but not peptidoglycan (PGN), polyinosinic-polycytidylic acid (poly I:C) or CpG oligonucleotide (CpG-ODN). Several studies have suggested that the inhibitory effects of DMA7-beta-CyD could be ascribed to the interaction with LPS. Simultaneous administration of DMA7-beta-CyD not only intraperitoneally but also intravenously and intraperitoneal injection of aqueous solution containing LPS and d-galactosamine in murine endotoxin shock model suppressed fatality. Also, DMA7-beta-CyD decreased blood level of TNF-alpha as well as serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in mice. In conclusion, DMA7-beta-CyD may have promise as a new therapeutic agent for endotoxin shock induced by LPS.     

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.     

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-alpha) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-alpha and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-alpha and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-kappaB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-kappaB activation.     

Effects of post-treatment with low-dose propofol on inflammatory responses to lipopolysaccharide-induced shock in conscious rats

1. In the present study, we used a low dose of propofol (5 mg/kg per h) to investigate its effects on the pro-inflammatory cytokines (tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10) and changes in nitric oxide (NO) following lipopolysaccharide (LPS) for a period of 12 h in conscious rats. 2. Experiments were designed to induce endotoxin shock by intravenous injection of Klebsiella pneumoniae LPS (10 mg/kg) in conscious rats. Arterial pressure (AP) and heart rate (HR) were monitored continuously for 12 h after LPS administration. Tumour necrosis factor-alpha, IL-1beta, IL-10 and plasma nitrates/nitrites were determined before and 0.5, 1, 3, 6, 9 and 12 h after LPS administration. A low dose of intravenous propofol (5 mg/kg per h) was administered to investigate the effects on cytokine responses and changes in NO in endotoxin shock. 3. Lipopolysaccharide significantly increased TNF-alpha, IL-1beta, IL-10, nitrites/nitrates and HR, whereas mean AP was decreased. Post-treatment with propofol suppressed the release of TNF-alpha, IL-1beta, IL-10 and NO production after endotoxin shock. 4. Lipopolysaccharide also caused a decrease in the white blood cell count and haematocrit. 5. Post-treatment with propofol slightly, but not significantly, affected the LPS-induced systemic hypotension, tachycardia, leukocytopenia and anaemia. 6. These findings suggest that low-dose propofol may be beneficial to the inflammatory change in sepsis.     

Protective effect of baicalein against endotoxic shock in rats in vivo and in vitro

Dried roots of Scutellaria baicalensis Georgi (Huang qin) are widely used in traditional Chinese medicine. Baicalein is a major bioactive flavonoid component of H. qin that shows a wide range of biological activities, including antioxidant and anti-inflammatory actions. We evaluated therapeutic effects and possible mechanisms of action of baicalein on circulatory failure and vascular dysfunction during sepsis induced by lipopolysaccharide (LPS; 10 mg/kg, i.v.) in anesthetized rats. Treatment of the rats with baicalein (20 mg/kg, i.v.) significantly attenuated the deleterious hemodynamic changes of hypotension and tachycardia caused by LPS and significantly inhibited the elevation of plasma tumor necrosis factor alpha (TNF-alpha). Baicalein also decreased levels of inducible nitric oxide synthase (iNOS) and the overproduction of NO and superoxide anions caused by LPS. It also increased the survival rate of ICR mice (25-30 g) challenged by LPS (60 mg/kg). Moreover, infiltration of neutrophils into the liver and lungs of rats 6h after treatment with LPS was also reduced by baicalein. To investigate the mechanism of action of baicalein on sepsis, RAW 264.7 cells were used as a model. Baicalein inhibited iNOS protein production, and suppressed LPS-induced degradation of IkappaBalpha, the formation of a nuclear factor kappa B (NF-kappaB)-DNA complex and NF-kappaB-dependent reporter gene expression. Thus, the therapeutic effects of baicalein were associated with reductions in TNF-alpha and superoxide anion levels during sepsis. The inhibitory effects of baicalein on iNOS production may be mediated by inhibition of the activation of NF-kappaB. Baicalein may thus prove a potential agent against endotoxemia.     

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.     

A synthetic cyclic peptide derived from Limulus anti-lipopolysaccharide factor neutralizes endotoxin in vitro and in vivo

Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infections. Recently, much attention has been focused on cationic peptides which possess the potential to detoxify LPS. Limulus anti-LPS factor (LALF), a protein found in the horseshoe crab (Limulus polyphemus), has been proved with striking anti-LPS effects. We synthesized a cyclic peptide (CLP-19), and then investigated its bioactivity both in vitro and in vivo. The ability of CLP-19 to neutralize LPS in vitro was tested using a Limulus amebocyte lysate (LAL) assay and the LPS-binding affinity was measured with an affinity biosensor method. The synthetic peptide LALF31-52 (residues 31 to 52 of LALF) was used as the positive control peptide in this study. It was found that CLP-19 exhibited the significant activity to antagonize LPS without observable cytotoxicity effect on mouse macrophages. CLP-19 directly bound to LPS, and neutralized it in a dose-dependent manner in the LAL assay. Moreover, CLP-19 also showed the remarkable ability to protect mice from lethal LPS attack and to inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) release by decreasing serum LPS in vivo. Our work suggests that this peptide is worthy of further investigation as a potential anti-LPS agent in the treatment of septic shock.     

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.     

N-Allylsecoboldine as a novel agent prevents acute renal failure during endotoxemia

Blockades of cytokine and oxygen radicals release are considered to be beneficial in reducing multiple organ injury and increasing the survival rate in sepsis/septic shock. Thus, we examined the protective efficacy of N-allylsecoboldine, a secoaporphine derivative with antioxidant and alpha1-adrenoceptor blocking activities, in rats treated with endotoxin (E. coli lipopolysaccharide, LPS). Pretreatment of LPS-treated rats with N-allylsecoboldine significantly attenuated the late-phase hypotension, hypoglycemia and incremental plasma tumor necrosis factor (TNF)-alpha. Overproduction of plasma nitrate in endotoxemia was not changed but the continuous decrease of urinary nitrate appeared to be partially ameliorated by N-allylsecoboldine. However, N-allylsecoboldine inhibited the inducible nitric oxide synthase (iNOS) protein expression in the renal cortex of endotoxemic rats. N-allylsecoboldine also improved the endotoxemia-induced organ injury as demonstrated from the conspicuous recovery of marker enzymes in the LPS-treated rats. Endotoxemia was associated with renal dysfunctions as indicated by decreases in renal blood flow, urinary potassium excretion, and renal nitrate clearance. However, pretreatment with N-allylsecoboldine showed significant alleviation of these renal dysfunctions. In addition, a lower dose of N-allylsecoboldine ameliorated the mortality of LPS-treated mice. This study demonstrates N-allylsecoboldine's ability to avail against acute renal failure and increase survival rate during endotoxemia. These beneficial effects may be attributed to the inhibition of iNOS expression, TNF-alpha production, and free radical scavenging activities. However, the role of alpha1-adrenoceptor antagonism for N-allylsecoboldine in sepsis remains unclear.     

Vasodilatory and anti-inflammatory effects of the 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) via a nitric oxide-cGMP pathway

Vasorelaxant and anti-inflammatory effects of a 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG) isolated from the root barks of Paeonia suffruticosa and possible mechanisms responsible were investigated. PGG induced a concentration-dependent relaxation of the phenylephrine-precontracted rat aorta. This effect disappeared with the removal of functional endothelium. Pretreatment of the aortic tissues with either N(G)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]-oxadiazole-[4,3-alpha]-quinoxalin-1-one (ODQ) inhibited the relaxation induced by PGG. Incubation of human umbilical vein endothelial cells (HUVECs) or carotid arteries isolated from rats with PGG increased the production of cGMP in a dose-dependent manner, but this effect was blocked by pretreatment with L-NAME and ODQ, respectively. PGG treatment attenuated tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappaB (NF-kappaB) p65 translocation in human umbilical vein endothelial cells. In addition, PGG suppressed the expression levels of adhesion molecules including intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha. TNF-alpha-induced monocyte chemoattractant protein-1 (MCP-1) expression was also attenuated by addition of PGG. PGG treatment inhibited cellular adhesion of U937 cells onto human umbilical vein endothelial cells induced by TNF-alpha. Taken together, the present study suggests that PGG dilates vascular smooth muscle and suppresses the vascular inflammatory process via endothelium-dependent nitric oxide (NO)/cGMP signaling.     

Endotoxin induces late increase in the production of pulmonary proinflammatory cytokines in murine lupus-like pristane-primed modelp

Lupus-like syndrome is characterized by multiple organ injuries including lungs and kidneys. Endotoxin induces a transiently intent systemic inflammatory response and indirectly transient acute lung injury in normal condition. However, whether endotoxin may trigger the persistent development of lung injury in chronic, inflammatory lupus-like syndrome compared with normal condition remains unclear. We examined the pulmonary vascular permeability and production of proinflammatory cytokines, such as TNF-alpha, IL-6, IL-10 and IFN-gamma, which play prominent roles in the pathogenesis of lupus-like tissue injury, 6 h and 72 h after i.p. lipopolysaccharide (LPS; endotoxin) injection in pristane-primed chronic inflammation ICR mice characterized by a lupus-like syndrome. These results demonstrated that levels of serum IL-6, IL-10 and IFN-gamma and bronchoalveolar lavage (BAL) IL-6 and IFN-gamma were remarkably increased 6 h in LPS-exposed pristane-primed mice compared with pristane-primed controls, while pulmonary vascular permeability and levels of serum and BAL TNF-alpha were not. And levels of BAL TNF-alpha, IL-6 and IL-10 were significantly enhanced 72 h in LPS-exposed pristane-primed mice compared with pristane-primed controls. Also, LPS significantly induced the increased in vitro production of TNF-alpha, IL-6 and IL-10 by lung cells obtained from LPS-exposed pristane-primed mice compared with LPS-exposed normal mice. Our findings indicate that LPS may trigger persistent progression of lung injury through late overproduction of BAL TNF-alpha, IL-6, and IL-10 in lupus-like chronic inflammation syndrome compared with normal condition.