Hongwei Cao

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

The Antimalarial Artemisinin Synergizes with Antibiotics To Protect against Lethal Live Escherichia coli Challenge by Decreasing Proinflammatory Cytokine Release

ABSTRACT In the present study artemisinin (ART) was found to have potent anti-inflammatory effects in animal models of sepsis induced by CpG-containing oligodeoxy-nucleotides (CpG ODN), lipopolysaccharide (LPS), heat-killed Escherichia coli 35218 or live E. coli. Furthermore, we found that ART protected mice from a lethal challenge by CpG ODN, LPS, or heat-killed E. coli in a dose-dependent manner and that the protection was related to a reduction in serum tumor necrosis factor alpha (TNF-α). More significantly, the administration of ART together with ampicillin or unasyn (a complex of ampicillin and sulbactam) decreased mortality from 100 to 66.7% or 33.3%, respectively, in mice subjected to a lethal live E. coli challenge. Together with the observation that ART alone does not inhibit bacterial growth, this result suggests that ART protection is achieved as a result of its anti-inflammatory activity rather than an antimicrobial effect. In RAW264.7 cells, pretreatment with ART potently inhibited TNF-α and interleukin-6 release induced by CpG ODN, LPS, or heat-killed E. coli in a dose- and time-dependent manner. Experiments utilizing affinity sensor technology revealed no direct binding of ART with CpG ODN or LPS. Flow cytometry further showed that ART did not alter binding of CpG ODN to cell surfaces or the internalization of CpG ODN. In addition, upregulated levels of TLR9 and TLR4 mRNA were not attenuated by ART treatment. ART treatment did, however, block the NF-κB activation induced by CpG ODN, LPS, or heat-killed E. coli. These findings provide compelling evidence that ART may be an important potential drug for sepsis treatment.

Artesunate protects sepsis model mice challenged with Staphylococcus aureus by decreasing TNF-α release via inhibition TLR2 and Nod2 mRNA expressions and transcription factor NF-κB activation

Gram-positive bacteria have become the most common organisms responsible for the development of sepsis. Staphylococcus aureus (S. aureus) is the major gram-positive pathogen in both community-acquired and nosocomial infections. The Mortality associated with nosocomial infections caused by S. aureus may vary but are generally high. In the present study, we found that artesunate (AS) could protect mice against a lethal challenge with heat-killed S. aureus in a dose-dependent manner, and AS in combination with ampicillin sodium-sulbactam sodium (AMPS) could further increase survival of mice challenged with live S. aureus than AMPS alone. This protection was associated with reductions of serum at TNF-alpha level. In in vitro experiments, AS-pretreatment strongly inhibited TNF-alpha release from murine peritoneal macrophage induced by heat-killed S. aureus or peptidoglycan in a dose-dependent manner. AS reduced the Toll like receptor 2 (TLR2) and nucleotide-binding oligomerization domain containing 2 (Nod2) mRNA expressions up-regulated by heat-killed S. aureus and inhibited NF-kappaB activation induced by heat-killed S. aureus. In conclusion, our results demonstrated that AS-mediated protection on septic mice challenged with S. aureus was associated with its reduction on TNF-alpha release via inhibition of TLR2 and Nod2 mRNA expressions and transcription factor NF-kappaB activation.

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 (40mg/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.

Inhibition of clathrin/dynamin-dependent internalization interferes with LPS-mediated TRAM-TRIF-dependent signaling pathway.

Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM-TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM-TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM-TRIF-dependent signaling pathway.

Artesunate in combination with oxacillin protect sepsis model mice challenged with lethal live methicillin-resistant Staphylococcus aureus (MRSA) via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin.

Sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA) has worse outcome because of multiresistance to a large group of antibiotics, which may lead to death from septic shock. In the present study, we firstly found that artesunate in combination with oxacillin was capable of protecting mice challenged with live MRSA WHO-2 (WHO-2) and the protection was related to the reduced TNF-α and IL-6 levels and decreased bacterial load. Based on above results, artesunate was further investigated from two aspects in vitro, anti-inflammation effect and antibacterial enhancement effect on antibiotics. Artesunate not only inhibited TNF-α and IL-6 release but also inhibited mRNA and protein expressions of TLR2 and Nod2, two important receptors, in murine peritoneal macrophages stimulated with heat-killed WHO-2, further demonstrating anti-inflammatory effect of artesunate was related to the inhibition of TLR2- and Nod2-mediated proinflammatory cytokines. Significantly, artesunate enhanced antibacterial activity of oxacillin and ampicillin not levofloxacin against WHO-2 and a clinical MRSA strain; the fractional inhibitory concentration indexes were lower than 0.5. Further, artesunate possessed moderate affinity for penicillin-binding protein 2a (PBP2a) and reduced the mecA mRNA expression up-regulated by oxacillin, suggesting that artesunates enhancement on antibacterial activity of β-lactams was related to the inhibition of PBP2a and down-regulation of mecA mRNA expression. In conclusion, our results demonstrated that artesunate in combination with oxacillin protected mice challenged with lethal live MRSA via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin. Artesunate could be further investigated as a candidate drug for MRSA sepsis.

Ulinastatin is a novel candidate drug for sepsis and secondary acute lung injury, evidence from an optimized CLP rat model

Ulinastatin is a potent multivalent serine protease inhibitor, which was recently found with therapeutic potentials in treating sepsis, and the most life-threatening complication of critically ill population. However, the pharmacological features and possible mechanisms need to be further elucidated in reliable and clinical relevant sepsis models. As known, sepsis induced by surgery of cecal ligation and puncture (CLP) is widely accepted as the gold standard animal model, but the inconsistency of outcomes is the most obvious problem. In the present experiments, we reported an improved rat CLP model with much more consistent outcomes using self-made three edged puncture needles in our lab. Results from this optimized model revealed that ulinastatin improved survivals of CLP rats, attenuated proinflammatory response and prevented systemic disorder and organ dysfunction. Ulinastatin was also found to be effective in ameliorating sepsis-related ALI, a syndrome most frequent and fatal in sepsis. The molecular mechanism investigation showed that ulinastatins protection against ALI was probably related to the down-regulation of NF-κB activity and inhibition of TNF-α, IL-6 and elastase expressions in the lung tissue. In conclusion, based on a successful establishment of optimized rat CLP model ulinastatin is proved to be an effective candidate for sepsis treatment, due to its anti-inflammation and anti-protease activities that ameliorate systemic disorders, prevent organ injuries and thus improve the survival outcomes of sepsis in animals.

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.

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-alpha 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-alpha release, but emodin only reduced CpG DNA-induced TNF-alpha release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF-alpha 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.

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.