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.     

Kukoamine B, a novel dual inhibitor of LPS and CpG DNA, is a potential candidate for sepsis treatment

BACKGROUND AND PURPOSE

Lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) are important pathogenic molecules for the induction of sepsis, and thus are drug targets for sepsis treatment. The present drugs for treating sepsis act only against either LPS or CpG DNA. Hence, they are not particularly efficient at combating sepsis as the latter two molecules usually cooperate during sepsis. In this study, a natural alkaloid compound kukoamine B (KB) is presented as a potent dual inhibitor for both LPS and CpG DNA.

EXPERIMENTAL APPROACH

The affinities of KB for LPS and CpG DNA were assessed using biosensor technology. Direct interaction of KB with LPS and CpG DNA were evaluated using neutralization assays. Selective inhibitory activities of KB on pro-inflammatory signal transduction and cytokine expression induced by LPS and CpG DNA were analysed by cellular assays. Protective effects of KB in a sepsis model in mice were elucidated by determining survival and circulatory LPS and tumour necrosis factor-alpha (TNF-α) concentrations.

KEY RESULTS

KB had high affinities for LPS and CpG DNA. It neutralized LPS and CpG DNA and prevented them from interacting with mouse macrophages. KB selectively inhibited LPS- and CpG DNA-induced signal transduction and expression of pro-inflammatory mediators without interfering with signal pathways or cell viability in macrophages. KB protected mice challenged with heat-killed Escherichia coli, and reduced the circulatory levels of LPS and TNF-α.

CONCLUSIONS AND IMPLICATIONS

This is the first report of a novel dual inhibitor of LPS and CpG DNA. KB is worthy of further investigation as a potential candidate to treat sepsis.     

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.     

The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology

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 were tested using affinity biosensor technology. From amongst 42 herbs, eight were found to possess lipid A-binding abilities. Radix Paeoniae Rubras had the highest lipid A-binding ability; therefore an aqueous extraction from this plant was investigated further. After preparation using standard methods, including silica gel chromatography and HPLC, we obtained 1, 2, 3, 4, 6-beta-d-pentagalloylglucose (PGG), with lipid A-binding ability. It was found that in vitro, PGG directly bound to lipid A, with a Kd of 32 microM, and that it neutralized the endotoxin both in the Limulus Amebocyte Lysate (LAL) assay and in a TNF-alpha release experiment, in a dose-dependent manner. In in vivo experiments, PGG was found to protect mice from a lethal challenge by LPS, and significantly decreased the plasma endotoxin level both in endotoxemic mice and rats, the reduction of the endotoxin level in rats being tightly associated with the TNF-alpha level. In conclusion, we demonstrate the effectiveness of affinity biosensor technology in discovering useful agents amongst traditional Chinese herbs and using this approach we found a new anti-endotoxin agent.     

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.     

Chloroquine protects mice from challenge with CpG ODN and LPS by decreasing proinflammatory cytokine release

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. To date, no effective anti-sepsis drugs have been developed for clinical use. Chloroquine (CQ), a diprotic weak base traditionally used for treating malaria, was recently shown to decrease cytokine release from macrophages induced by LPS and CpG oligonucleotide (CpG ODN). In the present study, Escherichia coli DNA (EC DNA), CpG ODN and LPS were used to induce SIRS/sepsis in animal models. We found that 30 mg/kg of CQ could protect mice from lethal challenge by CpG ODN and LPS, and 25 mg/kg of CQ could decrease serum TNF-alpha and IL-6 in rats injected with sublethal doses of CpG ODN and LPS. In addition, treatment of murine macrophage ANA-1 cells with 2 mM CQ potently inhibited the release of TNF-alpha, IL-6 and IL-12 induced by CpG ODN and LPS. In human peripheral blood mononuclear cells (hPBMC), 100-200 microM CQ almost completely abrogated release of both TNF-alpha and IL-6 induced by CpG ODN and LPS, whereas IL-6 release induced by EC DNA was not significantly affected by 50 microM CQ. Furthermore, CQ reduced the expression of TLR9 and TLR4 mRNA and the activation of NFkappaB and AP-1 stimulated by CpG ODN and LPS in ANA-1 cells. Flow cytometry and confocal microscopy revealed that CQ increased the accumulation of CpG ODN within ANA-1 cells without influence on its uptake, suggesting that the delayed degradation of CpG ODN was associated with the reduction of proinflammatory cytokine release from the cells. Our results demonstrated that CQ-mediated protection of lethal challenge by CpG ODN and LPS was associated with the reduction of proinflammatory cytokine release.     

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.     

应用生物传感器技术筛选拮抗细菌基因组DNA中药的研究

目的:从传统抗炎中药中筛选具有拮抗细菌基因组DNA(Cytidine -phosphate -guanosine DNA,CpG DNA)活性的中药并对其进行活性物质含量的排序。方法:将CpG DNA包被于生物传感器的生物素样品池以建立靶点,测定114种中药水煎液去鞣质后与CpG DNA的亲和力;选择亲和力较高的35种药物与定量CpG DNA (16.5μg.mL-1)于37℃混合孵育30min ,再测定其与CpG DNA的亲和力,以评价药物水提液中活性物质的含量。结果:114种中药中,半枝莲、薄荷、侧柏叶等35种中药与CpGDNA具有较高亲和力( >100RU) ;侧柏叶、地骨皮、大黄等19种中药与CpG DNA特异性结合的活性物质含量较高。结论:应用生物传感器跟踪检测技术筛选具有结合CpG DNA作用的中药具有可行性,筛选出的19种中药均含有非鞣质类能与CpG DNA发生特异性结合作用的活性物质。     

生物传感器技术在筛选赤芍中抗内毒素有效成分的应用研究

目的 :应用生物传感器技术优选赤芍中抗内毒素有效成分的提取方法。方法 :将内毒素包被于生物传感器的疏水样品池中 ,测定赤芍3种方法提取物与内毒素的亲合力及其与内毒素混合孵育后内毒素亲合力的变化。结果 :水浸法提取物与内毒素具有较高的亲合力 ,以1∶40稀释后能够中和78 1 %的内毒素。结论 :赤芍水浸法能够提取到更多的抗内毒素有效成分 ,测定结果与鲎试剂法一致 ;与传统的鲎试剂法比较 ,生物传感器技术具有快速、高效、直观、无干扰等优点。     

Using Chinese natural products for diabetes mellitus drug discovery and development

This paper provides a review of natural Chinese drug products, including phytochemic compounds, medicinal herbs and multi-component herbal formulae, that have been reported to possess hypoglycemic activity with mechanisms for antidiabetic action. Along with a great number of combination formulae, ～ 187 different Chinese medicinal herbs are clinically applied to treat diabetes mellitus and its complications in China, most of which have achieved reasonably good clinical outcomes. These valuable data and practical experience provide a promising opportunity for the discovery and development of drug candidates with good therapeutic efficacy and low toxicity. The concept of treating complex, multifactorial metabolic diseases, such as diabetes, using multi-component therapeutics, including single-herb formulae and combination herbal formulae, shall be regarded as a concerted pharmacologic intervention of multiple compounds interacting with multiple targets and possessing interdependent activities that are required for a synergistic or optimal effect. The conventional approach for the discovery and development of antidiabetic drug products from natural products involving a high-throughput, bioactivity guided drug screening of single compounds obtained from thousands of herbs has proven to be a costly and non-productive effort. Hence, an alternative way of developing new drug candidates, as suggested in this review, is to reduce and simplify a well-established combination herbal formula, along with the pharmacologic evaluation of a small group of phytochemic compounds, which are therapeutically effective as the original formula and have known chemical structures, compositions and mechanisms of action that are similar to chemical drugs.     

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

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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。     

A high expression EGFR/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Rhizoma Polygoni Cuspidati

The epidermal growth factor receptors (EGFRs) in some tumor cells are significant targets for drug discovery. In this work, we have developed an EGFR cell membrane chromatography and online high performance liquid chromatography/mass spectrometry system for screening active component from Rhizoma Polygoni Cuspidati. As a result, resveratrol from Rhizoma Polygoni Cuspidati was found to be the active component acting on EGFR like gefitinib. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. The EGFR/CMC-online-HPLC/MS system demonstrated fast and effective characteristics for screening leading compounds from traditional Chinese medicine.     

巴马汀抗脓毒症潜在靶点的筛选与鉴定

目的 研究巴马汀的分子特性,筛选并鉴定巴马汀抗脓毒症的潜在靶点。方法 通过中药系统药理学数据库和分析平台（TCMSP）分析巴马汀的药理参数和分子特性;通过TCMSP和DRAR-CPI软件筛选巴马汀抗脓毒症的潜在靶点;进一步通过分子对接软件鉴定巴马汀抗脓毒症的潜在靶点,免疫组化法验证潜在靶点。结果 巴马汀口服生物利用率为64.60%,血脑屏障渗透率为0.37,药物相似度为0.65,具有很好的成药性;通过TCMSP和DRAR-CPI软件共筛选到3个潜在靶点,经分子对接软件鉴定NOS3为巴马汀抗脓毒症的潜在靶点,免疫组化结果表明巴马汀能显著下调脓毒症大鼠NOS3的表达。结论 巴马汀具有很好的成药性,其可能通过结合并激活NOS3保护心肌抑制起到抗脓毒症的作用。     

Technological Advances in High-Throughput Screening

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the possibility of candidate drug. SPR can measure the quantity of a complex formed between two molecules in real-time without the need for fluorescent or radioisotopic labels. SPR is capable of characterizing unmodified biopharmaceuticals, studying the interaction of drug candidates with macromolecular targets, and identifying binding partners during ligand fishing experiments. DNA microarrays can be used in HTS be used to further investigate the expression of biological targets associated with human disease, which then opens new and exciting opportunities for drug discovery. Without doubt, the addition of new technologies will further increase the application of HTS in drug screening and its related fields.     

Therapeutic potential of Toll-like receptor 9 activation

In the decade since the discovery that mouse B cells respond to certain unmethylated CpG dinucleotides in bacterial DNA, a specific receptor for these 'CpG motifs' has been identified, Toll-like receptor 9 (TLR9), and a new approach to immunotherapy has moved into the clinic based on the use of synthetic oligodeoxynucleotides (ODN) as TLR9 agonists. This review highlights the current understanding of the mechanism of action of these CpG ODN, and provides an overview of the preclinical data and early human clinical trial results using these drugs to improve vaccines and treat cancer, infectious disease and allergy/asthma.     

Bacterial DNA does not increase serum corticosterone concentration or prevent increases induced by other stimuli

Bacterial DNA containing unmethylated CpG motifs (CpG DNA) and other microbial molecules such as lipopolysaccharide (LPS) have a broad range of immune stimulatory effects, which may include many shared cell signaling pathways leading to enhanced cytokine production. Some cytokines activate the hypothalamic-pituitary-adrenal (HPA) axis, and their production is downregulated by products of the HPA axis (glucocorticoids). Because such interactions have practical implications in the clinical use of CpG DNA, the present study was done to examine the effects of CpG DNA and LPS on serum corticosterone concentrations. In contrast to LPS, administration of CpG DNA (DNA from Escherichia coli) (30-300 microg) alone did not significantly increase serum corticosterone concentrations 1 or 4 h after administration. Administration of CpG DNA to mice prior to LPS caused a synergistic increase in serum tumor necrosis factor-alpha (TNF-alpha), indicative of an immune stimulatory effect. LPS and TNF-alpha, however, induced similar levels of corticosterone with or without concomitant CpG DNA. Increasing doses of LPS caused peak corticosterone levels similar to those induced by LPS in combination with CpG DNA. Exogenous TNF-alpha administered in vivo induced comparable concentrations of corticosterone with or without CpG DNA. An alternative stressor (restraint) yielded similar levels of corticosterone with or without CpG DNA. These results indicate that CpG DNA does not induce corticosterone release or alter its release by other stimuli, indicating biologically important differences in its immune effect compared to those of LPS, and possibly reduced toxicity.     

Suppression of TRIF-dependent signaling pathway of toll-like receptors by allyl isothiocyanate in RAW 264.7 macrophages

Toll-like receptors (TLR) play a significant role in the induction of innate immune responses that are essential for host defense against invading microbial pathogens. In general, TLRs have two major downstream signaling pathways: myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways. Allyl isothiocyanate (AITC) found in cruciferous vegetables has an effect on treatment of many chronic diseases. However, the exact molecular targets of AITC are still unidentified. Here, it was investigated whether AITC can modulate TLR signaling pathways and what is the molecular target of AITC in TLRs signaling pathways. AITC suppressed the activation of nuclear factor-κB by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly[I:C]), but not by macrophage-activating lipopeptide of 2kDa (MALP-2) or cytosine-phosphate-guanine dinucleotide (CpG DNA). AITC also suppressed the activation of interferon regulatory factor 3 (IRF3) and the expression of interferon inducible protein-10 (IP-10) induced by LPS or poly[I:C]. These results suggest that AITC can modulate TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.     

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.