Department of Emergency, General Hospital of PLA, Beijing 100853, Peoples Republic of China.
Cell Biol Int. 2012 May 1;36(5):475-81. doi: 10.1042/CBI20110535.
Bacterial endotoxins may lead to vascular endothelial cell injury. Our study explored the role of TLR4 (Toll-like receptor 4) and STIM1 (stromal interaction molecule 1) in bacterial endotoxin-induced calcium overload and inflammatory reactions in HUVECs (human umbilical vein endothelial cells). It showed that under LPS (lipopolysaccharide) stimulation, LBP (LPS-binding protein) mRNA levels peaked at 24 h, TLR4 levels at 12 h and NF-κB (nuclear factor κB) levels at 6 h (all P<0.01). LBP levels increased gradually and peaked at 24 h of LPS treatment. TLR4 protein levels increased significantly at 1 h and peaked at 12 h. NF-κB protein levels markedly increased at 1 h and peaked at 6 h. Knockdown of STIM1 alone, TLR4 alone or both STIM1 and TLR4 together, markedly abolished LPS-induced increase in calcium influx into cells (P<0.05, P<0.01 and P<0.01 respectively). LBP-TLR4 and STIM-NF-κB interactions were detected without LPS treatment, enhanced by LPS stimulation, and markedly reduced by knocking down TLR4 and STIM respectively. Both the NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate) and TLR4 knockdown could block LPS induction of NF-κB, STIM, TNFα (tumour necrosis factor α) and IL-6 (interleukin 6). The data indicate LPS-LBP may activate TLR4 signalling and downstream transcription factor NF-κB, which further can activate STIM1 and eventually lead to calcium influx and injury of HUVECs. Inhibition of TLR4 effectively reverses LPS induction of inflammatory mediator generation and extracellular calcium influx mediated by STIM1.
细菌内毒素可能导致血管内皮细胞损伤。本研究探讨了 TLR4（Toll 样受体 4）和 STIM1（基质相互作用分子 1）在细菌内毒素诱导的 HUVECs（人脐静脉内皮细胞）钙超载和炎症反应中的作用。结果表明，在 LPS（脂多糖）刺激下，LBP（LPS 结合蛋白）mRNA 水平在 24 h 时达到峰值，TLR4 水平在 12 h 时达到峰值，NF-κB（核因子 κB）水平在 6 h 时达到峰值（均 P<0.01）。LBP 水平逐渐升高，在 LPS 处理 24 h 时达到峰值。TLR4 蛋白水平在 1 h 时显著升高，在 12 h 时达到峰值。NF-κB 蛋白水平在 1 h 时明显升高，在 6 h 时达到峰值。单独敲低 STIM1、TLR4 或同时敲低 STIM1 和 TLR4，均可显著抑制 LPS 诱导的细胞内钙内流增加（P<0.05、P<0.01 和 P<0.01）。在没有 LPS 处理的情况下检测到 LBP-TLR4 和 STIM-NF-κB 相互作用，LPS 刺激增强了这种相互作用，而分别敲低 TLR4 和 STIM 则显著减少了这种相互作用。NF-κB 抑制剂 PDTC（吡咯烷二硫代氨基甲酸盐）和 TLR4 敲低均可阻断 LPS 诱导的 NF-κB、STIM、TNFα（肿瘤坏死因子 α）和 IL-6（白细胞介素 6）的产生。这些数据表明 LPS-LBP 可能激活 TLR4 信号通路和下游转录因子 NF-κB，进而激活 STIM1，最终导致钙内流和 HUVECs 损伤。抑制 TLR4 可有效逆转 LPS 诱导的 STIM1 介导的炎症介质生成和细胞外钙内流。
