Artesunate Enhance The Ability Of Mouse Peritoneal Macrophages Endocytosing And Elimination LPS And Escherichia Coli And Its Mechnisms

Sepsis is systemic inflammatory response syndrome (SIRS) with an infectious origin,leading to septic shock, multiple organ dysfunction syndromes (MODS) and even death.However, there are few effective therapeutic methods.Lipopolysaccharide (LPS/endotoxin) is the component of the cell wall ofGram-negative bacteria. LPS is regarded as the major cause of sepsis and septic shock.Mononuclear phagocyte system activated by LPS can release a large of pro-inflammatorymediators such as TNF-α and IL-6through NF-κB and MAPK pathways, and then triggersepsis. Therefore, it is very important to search for drugs to treat LPS-triggered spesis.LPS can be recognized br Toll-like receptors4(TLR4) and scavenger receptors (SRs)and then initiates internalization and degradation in mononuclear phagocyte system.However, there is no evidence indicates that mononuclear phagocyte system canphagocytose and degradate bacteria.Artesunate (AS), a water-soluble hemisuccinate derivative of dihydroartemisinin, iswidely used in antimalaria and antitumor treatment. Previously, we have already observedthat AS could protect mice against a lethal challenge with heatkilled E.coli. This protectionwas associated with reductions in measurable endotoxin levels. However, AS could not bindto LPS directly in vivo. Therefore, the effect was speculated to be associtated withenhancing the ability of mononuclear phagocyte system endocytosing LPS. In theexperiments, the effects and possible mechanism of AS on internalization and eliminatingof of LPS and Escherichia coli (E.coli.) was investigated.ObjectTo investigate the effects of AS on mouse peritoneal macrophages internalizing andeliminating LPS and phagocytosing E.coli.Methods 1. Effects of AS on LPS and E.coli internalization and degradation of mouseperitoneal macrophagesa) The isolation, cultivation and identification of mouse peritoneal macrophagesb) Observe the effects of AS, monodansylcadaverine (MDC) and chloroquine (CQ) onLPS internalization of mouse peritoneal macrophages by confocal laser scaningmicroscopy.c) Observe the effects of AS on E.coli internalization of mouse peritoneal macrophagesby confocal laser scaning microscopy and bacterial clone forming counting test.2. Mechnisms of AS enhance the ability of mouse peritoneal macrophagesendocytosing and eliminating LPS and E.coli.a) Utilizing ELISA method measure the effects of AS, MDC and CQ on mouseperitoneal macrophages TNF-α and IL-6secretion.b) Utilizing RT-PCR method examin the effects of AS on mouse peritonealmacrophages TLR4, SRs and AOAH mRNA expression.c) Utilizing FCM method measure the effects of AS on mouse peritoneal macrophagescell surface TLR4, SRs expression.Results1. Effects of AS on LPS and E.coli internalization and degradation of mouseperitoneal macrophagesa) Succeeded in isolating mouse peritoneal macrophages, and the cell purity and vtalityare appropriate for our experiments.b) AS could increase LPS internalization of mouse peritoneal macrophages, whileMDC and chloroquine could inhibit LPS but artesunate still could increase LPSinternalization of macrophages treated with MDC and CQ.c) AS could increase E.coli internalization of mouse peritoneal macrophages.2. Mechnisms of AS enhance the ability of mouse peritoneal macrophagesendocytosing and eliminating LPS and E.colia) AS, MDC and CQ could decrease TNF-α and IL-6secretion of mouse peritonealmacrophages. AS combined with MDC or CQ could decrease TNF-α、IL-6secretion ofmouse peritoneal macrophages further more. b) AS had effects on mouse peritoneal macrophages TLR4, SRs and AOAH mRNAexpression.c) AS had no effects on mouse peritoneal macrophages cell surface TLR4, SRsexpression.Conclusion1. AS could increase LPS internalization into mouse peritoneal macrophages, whileMDC and chloroquine could inhibit LPS but AS still could increase LPS internalization ofmacrophages treated with MDC and CQ.2. AS could increase E.coli internalization into mouse peritoneal macrophages.3. Mechnisms of AS enhance the ability of mouse peritoneal macrophagesendocytosing and eliminating LPS and E.coli were associated with influence on MyD88dependent and independent signaling pathways, and TLR4, SRs mRNA expression, but notclathrin function and endosome maturation.