TLR4和TLR2激动剂协同诱导SR-A受体表达及其在内毒素耐受中的作用

发布时间：2018-09-17 17:20

【摘要】： 小剂量的内毒素预处理后的单核细胞和巨噬细胞对LPS再次刺激的反应性明显降低,表现为炎症因子的分泌减少;而且能提高小鼠在致死剂量内毒素再次刺激后的存活率,即内毒素耐受现象。自从证实TLR (Toll-like receptor) 4是LPS的信号受体以来,内毒素耐受的研究主要集中于信号受体和TLR4胞内信号分子的变化。基因敲除试验证实SR-A(macrophage scavenger receptor A)能通过调理素非依赖的方式吞噬细菌,从而提高小鼠对病原体攻击的抵抗能力,然而SR-A是否同样参与内毒素目前还不清楚。我们发现LPS能以剂量和时间依赖的方式诱导RAW264.7表达SR-A,这与RAW264.7对LPS的再次刺激后分泌的炎症因子(TNF-α和IL-6)减少之间有着很好的相关性。过量表达或瞬时转染SR-A能抑制LPS刺激RAW264.7分泌炎症因子或活化NF-κB,说明LPS诱导上调的SR-A参与了内毒素耐受。另外,LPS能增强RAW264.7对FITC-LPS的结合和吞噬,但是能被清道夫受体SR配体Fucoidan和抗SR-A抗体2F8特异地抑制,提示RAW264.7结合和吞噬FITC-LPS的SR-A受体特异性。过量表达或瞬时转染SR-A?1-49也同样能抑制LPS刺激RAW264.7分泌炎症因子或活化NF-κB,提示SR-A介导的RAW264.7对FITC-LPS的结合也能一定程度上抑制炎症反应。虽然,TLR4是LPS的信号受体,但是TLR4/MD-2抗体抑制TLR4信号后并不能抑制LPS诱导RAW264.7表达SR-A。然而,p38特异性抑制剂SB203580却能完全抑制LPS诱导RAW264.7表达SR-A。因此,p38,而不是TLR4,依赖的SR-A上调可能通过结合或吞噬LPS参与RAW264.7细胞内毒素耐受的形成。已知sLPS(来源于Sigma的Escherichia coli O111:B4 LPS)能诱导RAW264.7表达SR-A,但是许多商品化LPS,包括我们所用的sLPS,通常含有TLR4和TLR2两种激动剂,有关它们在诱导RAW264.7表达SR-A中的相对作用目前还不清楚。荧光素酶试验证实我们所用的sLPS确实具有活化TLR4和TLR2两种活性,但是重新纯化去除TLR2激动剂或用多粘霉素(PmB)特异中和TLR4激动剂后的sLPS均只能微弱地诱导RAW264.7表达SR-A,但是等量的uLPS(re-extracted sLPS)和Pam3CSK4混合物却能协同地诱导RAW264.7表达SR-A,而对TLR2和TLR4受体的表达没有协同作用。虽然,
[Abstract]:The reactivity of monocytes and macrophages pretreated with small doses of endotoxin to LPS restimulation was significantly decreased, showing that the secretion of inflammatory factors was decreased, and the survival rate of mice was improved after the lethal dose of endotoxin was stimulated again. The phenomenon of endotoxin tolerance. Since it was confirmed that TLR (Toll-like receptor) 4 is the signal receptor of LPS, the study of endotoxin tolerance mainly focuses on the changes of signal receptors and intracellular signal molecules of TLR4. Gene knockout test confirmed that SR-A (macrophage scavenger receptor A) could phagocystify bacteria in a non-dependent manner, thus enhancing the ability of mice to resist pathogen attack. However, it is not clear whether SR-A is involved in endotoxin. We found that LPS can induce the expression of SR-A, in RAW264.7 in a dose-and time-dependent manner, which has a good correlation with the decrease of TNF- 伪 and IL-6 secreted by RAW264.7 after the restimulation of LPS. Overexpression or transient transfection of SR-A could inhibit the secretion of inflammatory factors or activate NF- 魏 B in RAW264.7 stimulated by LPS, suggesting that the up-regulated SR-A induced by LPS was involved in endotoxin tolerance. In addition, lipopolysaccharide could enhance the binding and phagocytosis of RAW264.7 to FITC-LPS, but could be specifically inhibited by scavenger receptor SR ligand Fucoidan and anti-SR-A antibody 2F8, suggesting that RAW264.7 binding and phagocytosis of FITC-LPS SR-A receptor were specific. Overexpression or transient transfection of SR-A?1-49 could also inhibit the secretion of inflammatory factors or activation of NF- 魏 B in RAW264.7 stimulated by LPS, suggesting that SR-A mediated RAW264.7 binding to FITC-LPS could also inhibit the inflammatory response to some extent. Although TLR4 is the signal receptor of LPS, TLR4/MD-2 antibody can not inhibit the expression of SR-A. in RAW264.7 induced by LPS after inhibiting TLR4 signal. However, SB203580, a specific inhibitor of p38, could completely inhibit the expression of SR-A. by RAW264.7 induced by LPS. Therefore, the up-regulation of p38, not TLR4, dependent SR-A, may be involved in the formation of endotoxin tolerance in RAW264.7 cells by binding or phagocytosis of LPS. It is known that sLPS (Escherichia coli O111:B4 LPS derived from Sigma) can induce RAW264.7 to express SR-A,. However, many commercial LPS, including the sLPS, we use, usually contain two agonists, TLR4 and TLR2, and the relative role of these two agonists in inducing RAW264.7 expression SR-A is still unknown. Luciferase tests confirmed that the sLPS we used did have activated TLR4 and TLR2 activities. However, the repurification of sLPS after removal of TLR2 agonist or specific neutralization of TLR4 agonist with polymycin (PmB) could only slightly induce RAW264.7 to express SR-A, but the same amount of uLPS (re-extracted sLPS) and Pam3CSK4 mixture could co-induce RAW264.7 to express SR-A, and to TLR2 and TLR4. There is no synergism in the expression of receptors. although
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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