Li Bin, Zhang Rong, Li Jun, Zhang Lezhi, Ding Guofu, Luo Ping, He Shengqi, Dong Yan, Jiang Weiwei, Lu Yongling, Cao Hongwei, Zheng Jiang, Zhou Hong
Department of Pharmacology, College of Pharmaceutical, Third Military Medical University, Chongqing 400038, China.
Int Immunopharmacol. 2008 Mar;8(3):379-89. doi: 10.1016/j.intimp.2007.10.024. Epub 2007 Dec 7.
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.
细菌DNA（bDNA）和脂多糖（LPS）是单核细胞和巨噬细胞等免疫细胞的强效激活剂，可导致全身炎症反应综合征（SIRS）和脓毒症。不幸的是，许多基于实验性炎症拮抗剂的疗法在脓毒症试验中均告失败，目前临床使用的辅助疗法仅有一种，如活化蛋白C。青蒿琥酯（AS）是双氢青蒿素的水溶性衍生物，最近已证明它可通过抑制肿瘤坏死因子-α（TNF-α）mRNA表达来保护细胞免受LPS诱导的人脐静脉内皮细胞（HUVEC）激活和损伤。在本研究中，用热灭活的大肠杆菌在动物模型中诱导脓毒症。我们观察到AS能以剂量依赖性方式保护小鼠免受热灭活大肠杆菌的致命攻击。这种保护作用与血清TNF-α和可测量的内毒素水平降低有关。此外，用AS处理小鼠腹膜巨噬细胞可强烈抑制CpG寡脱氧核苷酸（CpG ODN）、LPS或热灭活大肠杆菌诱导的TNF-α和IL-6释放，且呈剂量依赖性。使用亲和传感器技术的实验表明，AS不能直接结合CpG ODN或LPS。此外，AS在体外不能中和LPS。此外，流式细胞术显示，AS不会改变CpG ODN与细胞表面的结合，但可促进CpG ODN在RAW264.7细胞内的积累。此外，AS降低了LPS、CpG ODN或热灭活大肠杆菌刺激的TLR4和TLR9 mRNA表达，并抑制热灭活大肠杆菌诱导的NF-κB激活。总之，我们的结果表明，AS介导的对热灭活大肠杆菌致命攻击的保护作用与促炎细胞因子释放和内毒素水平降低有关，其机制涉及TLR4、TLR9 mRNA表达降低和NF-κB激活。
