Xiao Jianguo, Song Qing, Li Tanshi, Sun Rongju
Department of Critical Care Medicine, General Hospital of PLA, Beijing 100853, China (Xiao JG, Song Q); Department of Emergency, General Hospital of PLA, Beijing 100853, China (Li TS, Sun RJ). Corresponding author: Sun Rongju, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Feb;29(2):150-155. doi: 10.3760/cma.j.issn.2095-4352.2017.02.011.
To explore the effect of toll-like receptor 4 (TLR4), myeloid differentiation protein-2 (MD2), and stromal interaction molecular 1 (STIM1) for regulating human vascular endothelial calcium overload injury and inflammatory reaction induced by bacterial endotoxin (LPS).
Human umbilical vein endothelial cells (HUVECs) were cultured in Dulbecco's modification of Eagle's medium (DMEM). (1) The levels of TLR4, MD2 and nuclear factor-κB (NF-κB) were detected by reverse transcriotion-polymerase chain reaction (RT-PCR) before and 0.5, 1, 6, 12, 24 hours after LPS stimulation. (2) Intracellular calcium peak level was detected by confocal following probe fluo-3 AM loading in HUVEC cells induced with LPS and transfected by psiSTIM or psiTLR. (3) MD2, STIM1 or NF-κB protein level was detected by immunoprecipitation (IP) and immuno-blotting in HUVEC cells which were transfected by TLR4 inhibited expression (psiTLR) for 12 hours and followed by LPS stimulation for 6 hours. (4) HUVEC cells were randomly divided into 6 groups: control group, LPS group, PDTC 0.1 mg/L group, PDTC 1 mg/L group, psiTLR 1 h group and psiTLR 12 h group. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme linked immunosorbent assay (ELISA) in supernatant. The mRNA levels of STIM1 and NF-κB were detected by RT-PCR.
(1) The mRNA levels of TLR4, MD2, and NF-κB gradually increased after LPS induction and peaked at 6 hours (2: 23.52±2.88, 17.43±3.43, 18.13±2.99, respectively), which were statistically significant before the stimulation with LPS (2: 7.02±2.81, 5.19±3.22, 8.11±1.42, all P < 0.05). (2) Extracellular calcium influx in LPS group was increased significantly higher than control group (nmol/L: 108.13±22.33 vs. 41.57±13.19, P < 0.01). Extracellular calcium influx in psiSTIM+LPS group (nmol/L: 62.61±14.12 vs. 108.13±22.33, P < 0.05) and psiTLR+LPS group (nmol/L: 50.78±8.05 vs. 109.43±20.21, P < 0.01) were both suppressed as compared with LPS group. While extracellular calcium peak level in psiTLR+psiSTIM+LPS group further decreased (nmol/L: 39.31±6.42 vs. 109.43±20.21, P < 0.01). (3) MD2 protein but not STIM1 or NF-κB can be detected in anti-TLR4 precipitates in control (ctrl-) by immunoprecipitation. MD2 protein level increased in anti-TLR4 precipitates in LPS group (ctrl+) and was suppressed in TLR4 inhibiting group (psiTLR). (4) The levels of TNF-α in PDTC 1 mg/L group were significantly lower than those of LPS group (ng/L: 0.60±0.24 vs. 1.77±0.66, P < 0.01). The levels of IL-6 in PDTC 0.1 mg/L, 1 mg/L group and psiTLR 12 h group decreased significantly lower than that of LPS group (ng/L: 232.10±63.54, 134.32±37.23, 284.23±56.14 vs. 510.22±89.23, all P < 0.05). Compared to LPS group, the mRNA levels of NF-κB and STIM1 were obviously inhibited in PDTC 1 mg/L group and psiTLR 12 h group [NF-κB mRNA (2): 17.22±2.35, 13.24±3.54 vs. 30.16±2.06; STIM1 mRNA (2): 12.57±2.43, 12.21±2.46 vs. 25.12±2.02, all P < 0.05].
TLR4, MD2, NF-κB signal and SOC calcium channel STIM1 mediate LPS induced-calcium influx and inflammatory mediators level in HUVEC cells. Extracellular calcium overload and inflammatory response by endotoxin induction can be effectively inhibited by down-regulation of TLR4, NF-κB and/or STIM1.
探讨Toll样受体4（TLR4）、髓样分化蛋白2（MD2）和基质相互作用分子1（STIM1）在调节细菌内毒素（LPS）诱导的人血管内皮细胞钙超载损伤及炎症反应中的作用。
采用杜氏改良Eagle培养基（DMEM）培养人脐静脉内皮细胞（HUVECs）。（1）采用逆转录-聚合酶链反应（RT-PCR）检测LPS刺激前及刺激后0.5、1、6、12、24小时HUVECs中TLR4、MD2和核因子κB（NF-κB）的水平。（2）用荧光探针fluo-3 AM负载HUVEC细胞，经LPS诱导并转染psiSTIM或psiTLR后，采用共聚焦显微镜检测细胞内钙峰值水平。（3）对转染TLR4抑制表达（psiTLR）12小时后再经LPS刺激6小时的HUVEC细胞，采用免疫沉淀（IP）和免疫印迹法检测MD2、STIM1或NF-κB蛋白水平。（4）将HUVEC细胞随机分为6组：对照组、LPS组、0.1 mg/L PDTC组、1 mg/L PDTC组、psiTLR 1 h组和psiTLR 12 h组。采用酶联免疫吸附测定（ELISA）检测上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平。采用RT-PCR检测STIM1和NF-κB的mRNA水平。
（1）LPS诱导后，TLR4、MD2和NF-κB的mRNA水平逐渐升高，6小时时达到峰值（分别为2：23.52±2.88、17.43±3.43、18.13±2.99），与LPS刺激前相比差异有统计学意义（分别为2：7.02±2.81、5.19±3.22、8.11±1.42，均P<0.05）。（2）LPS组细胞外钙内流显著高于对照组（nmol/L：108.13±22.33比41.57±13.19，P<0.01）。与LPS组相比，psiSTIM+LPS组（nmol/L：62.61±14.12比108.13±22.33，P<0.05）和psiTLR+LPS组（nmol/L：50.78±8.05比109.43±20.21，P<0.01）细胞外钙内流均受到抑制。而psiTLR+psiSTIM+LPS组细胞外钙峰值水平进一步降低（nmol/L：39.31±6.42比109.43±20.21，P<0.01）。（3）免疫沉淀法检测发现，对照组（ctrl-）抗TLR4沉淀物中可检测到MD2蛋白，但未检测到STIM1或NF-κB蛋白。LPS组（ctrl+）抗TLR4沉淀物中MD2蛋白水平升高，而TLR4抑制组（psiTLR）中MD2蛋白水平受到抑制。（4）1 mg/L PDTC组TNF-α水平显著低于LPS组（ng/L：0.60±0.24比1.77±0.66，P<0.01）。0.1 mg/L、1 mg/L PDTC组和psiTLR 12 h组IL-6水平显著低于LPS组（ng/L：232.10±63.54、134.32±37.23、284.23±56.14比510.22±89.23，均P<0.05）。与LPS组相比，1 mg/L PDTC组和psiTLR 12 h组NF-κB和STIM1的mRNA水平明显受到抑制[NF-κB mRNA（2）：17.22±2.35、13.24±3.54比30.16±2.06；STIM1 mRNA（2）：12.57±2.43、12.21±2.46比25.12±2.02，均P<0.05]。
TLR4、MD2、NF-κB信号及SOC钙通道STIM1介导LPS诱导的HUVEC细胞钙内流及炎症介质水平变化。下调TLR4、NF-κB和/或STIM1可有效抑制内毒素诱导的细胞外钙超载及炎症反应。
