细菌脂多糖诱导人单核细胞株对细菌脂蛋白的耐受性及其与肌动蛋白骨架关系

LPS-induced BLP Tolerance and the Role of Actin Cytoskeleton in THP-1 Cell Line

细菌脂多糖(LPS)可诱导宿主对LPS的耐受,但对细菌脂蛋白(BLP)是否存在交叉耐受,目前报道不一。采用人单核细胞株(THP-1),建立小剂量LPS诱导THP-1对LPS耐受的细胞模型;观察细胞肌动蛋白骨架、炎症因子TNF-α、IL-1β、IL-6的浓度及NF-κB的DNA结合活力的变化情况;探讨BLP交叉耐受及细胞骨架在其中的作用。结果显示,THP-1细胞经小剂量(10ng/ml)LPS、大剂量(100ng/ml)LPS或BLP刺激后,细胞形态严重变形,肌动蛋白重组,细胞周边肌动蛋白丝带消失,出现明显的肌动蛋白收缩团块及伪足,细胞核内NF-κB的DNA结合活性显著升高,培养上清液中炎症因子(TNF-α、IL-1β及IL-6)的释放显著增加;而小剂量LPS预刺激12h后,再用大剂量的LPS或BLP刺激6h,上述指标明显改善;采用细胞骨架肌动蛋白聚集破坏剂鬼笔环肽预处理后的THP-1细胞,可取消由小剂量LPS诱导的自身耐受及对BLP的交叉耐受;可见,细菌LPS、BLP(100ng/ml)可诱导THP-1细胞肌动蛋白骨架的改变,激活NF-κB信号通路,诱导炎性细胞因子TNF-α、IL-1、IL-6过度释放,激活宿主炎症细胞的炎症反应;而小剂量LPS预刺激后可诱导出THP-1细胞对LPS的自身耐受和对BLP的交叉耐受;细胞骨架肌动蛋白参与了小剂量LPS诱导THP-1细胞对LPS自身耐受和对BLP交叉耐受的形成。

Abstract It is remain uncertain whether bacterial lipopolysaccharide （LPS）-toleranced monocyte cross-toleranced with bacterial lipoprotein （BLP）. The study was aimed to observe the LPS tolerance and BLP cross-tolerance induced by LPS （10 ng/ml） and the changes of actin, and to explore the role of actin cytoskeleton in LPS-tolerance and BLP cross-tolerance in THP-1. The human monocyte cell line （THP-1） were adopted to establish the LPS-toleranced and BLP cross-toleranced cellular models. Actin skeleton, supernatant concentration of cytokins （TNF-α, IL-1β and IL-6） and NF-κB DNA binding activity was tested. When THP-1 were insulted with LPS （100 ng/ml） or BLP （100 ng/ml）, The cellular morphologic changes were significant with the formation of pseudopod and the actin reorganized to form the actin-band, NF-κB DNA binding activity upregulated in nuclear, and the concentration of cytokins （TNF-α, IL-1β and IL-6） in supernatant increased in THP-1. Pretreatment THP-1 with LPS （10 ng/ ml）, the morphology, cytokines and NF-κB DNA binding activity were improved. Phalladin, a specifical actin cytoskeleton reorganization inhibitor, party abolished the LPS tolerance and BLP cross-tolerance in THP- 1 cell induced by 10 ng/ml LPS. The results suggested that LPS （100 ng/ml） and BLP （100 ng/ml） could trigger the reorganization of actin cytoskeleton, activate NF-κB transcription, and increase the cytokins （TNF-α, IL- 1β and IL-6） releasing, which were closely associated with the activatation of inflammation. LPS （10 ng/ml） pretreatment could induce LPS tolerance and BLP cross-tolerance in THP-1 which was at least partly associated with actin cytoskeleton.