The Roles Of Anti-inflammation And Antibacterial Potentiator's Effect In The Protection Of Artesunate For Sepsis Model Mice And Its Molecular Mechanisms

Objective:Sepsis is systemic inflammatory response syndrome (SIRS) caused by infection. This condition may result in septic shock, multiple organ dysfunction syndrome (MODS) and ultimately death. The mortality is as high as to 50~80%. There have been few effective therapeutic method.At present, sepsis is found to be triggered by the presence of invasive bacteria and bacterial components, such as bacterial genomic DNA (bDNA) and lipopolysaccharide (LPS [endotoxin]), etc. Although antibacterial agents could inhibit bacterial growth and treat infectious disease, there are reports that many antibacterial agents, especiallyβ-lactam antibiotics, could induce LPS release from bacterial membrance during killing bacteria and enhance infection. Therefore, it is very important to search drugs to treat bacterail sepsis. In our previous study, we have confirmed that artemisinin (ART) could protect mice against lethal heat-killed E. coli challenge and synergize with antibiotics to protect animals against lethal live E. coli challenge. However, ART is poorly soluble in oil and water and can only be administered orally, which is disadvantageous for treating critically ill patients.There are four derivates of ART; they are dihydroartemisinine (DHA), artemether(AM), arteether and artesunate(AS). The derivates of ART could be administered by intravescular and intramuscular injection, which is suitabl for critically ill patients. Based on their similar chemical structure, we suppose four derivates of ART possibly play role to treat sepsis as well as ART.With these considerations in mind, we undertook the current study to find most effective derivate by screening the effects of their inhibitons on inflammatory cytokines release induced by bacteria and bacterial component. And then, the protection of this derivate on different sepsis model mice and its possible molecular mechanisms will be investigated in orde to search for effective anti-sepsis drug and broaden indications.Methods:1. Screening of inhibitory effect of DHA, AS and AM on TNF-αand IL-6 releases from mice primary peritoneal macrophages and macrophage cell line RAW264.7 cells stimulated by different stimulators using ELISA method.2. The protective effect of AS on bacterial sepsis model mice. Sepsis mice model challenged with heat killed E.coli, live E.coli and CLP model were established. The protective effects of AS were observed.3. The molecular mechanisms of AS on sepsis model mice(1) Mechanism of AS'anti-inflammatory effect The ability of AS to neutralize LPS in vitro was assayed using the LAL test. The direct binding ability of AS to CpG ODN or LPS/lipid A was observed using affinity biosensor technology.Cell-surface binding and accumulation of 6-FAM CpG ODN in RAW264.7 cell lines treated with AS were observed using flow cytometry. TLR4 and TLR9 mRNA expression down-regulated by AS was tested using RT-PCR method; TLR9 expression at protein level within the cells was observed using immunofluorescence assay. Inhibition of AS on NF-κB activation was observed using ELISA assay.(2) Mechanisms of AS as antibacterial potentiator①MICs of AS and different antibacterial agents on E.coli ATCC 35218 and clinical separated strains were observed using micropore dilution, and effects of AS with antibiotics on above strains were observed,too.②Synergistic effect of AS with antibacterial agents on E.coli ATCC35218 and clinical separated strains were observed using dynamic growth curve assay.③Accumulation of daunorubicin within bacteria treated with AS was using confocal scanning microscopy and fluorospectrophotometry.effect of AS on bacterial membrance permeability was observed using transmission electron microscope.Results:1. Among three derivates of ART, AS produced most marked inhibition on inflammatory cytokines release. 2. The protective effect of AS on different sepsis model mice.①AS could delay the death time and decrease the mortality of sepsis mice challenged with heat-killed E.coli. This protection was associated with reductions in serum TNF-αand measurable endotoxin levels.②The administration of AS together with gentamycin or a complex of ampicillin and sulbactam decreased sepsis mice mortality challenged by lethal live E.coli. The administration of AS together with a complex of ampicillin and sulbactam decreased CLP sepsis model mice mortality.3. The molecular mechanisms of AS on sepsis model mice(1) Mechanism of AS'anti-inflammatory effect①AS could not directly bind to LPS or CpG ODN. AS could not neutralize LPS in vitro. AS could not change CpG ODN-binding to the cell-surface of RAW264.7 cells but could promote CpG ODN's accumulation within.③AS down-regulated TLR4 mRNA and TLR9 mRNA expressions up-regulated by LPS, CpG ODN or heat-killed E. coli. AS also down-regulated TLR9 at protein level, too.④AS inhibited heat-killed E. coli-induced NF-κB activation.(2) Mechanisms of AS as antibacterial potentiator①AS had no antibacterial effect, but AS could produce synergistic effect if it with antibacterial agents to E.coli.②AS could increase accumulation of daunomycin within E.coli ATCC35218 in a dose-dependent and time-dependent manners.③AS could destroy the integrity of E.coli cell membrance. The mechanism of AS as antibacterial potentiator was tightly related to increased accumulation of antibacterial agents within bacteria.Conclusions:①Among three derivates of ART, AS produced most marked inhibition on inflammatory cytokines release.②AS could protect sepsis mice challenged with heat-killed E.coli. The administration of AS together with antibacterial agents could produce synergistic protection for sepsis model mice. ③AS-mediated anti-inflammatory effect was associated with a reduction of TNF-αand IL-6 releases via a decrease in TLR4 and TLR9 expressions and NF-κB activation.④Antibacterial potentiator's effect of AS was related to increased drug accumulation within bacteria.⑤Based on anti-inflammatory effect and antibacterial potentiator's effect of AS, and its protection for sepsis model mice, it is significant to further investigate AS.