Bacterial endotoxin-induced endothelial cell injury and calcium overload associated with Toll-like receptor and calcium signal / 中华危重病急救医学

ABSTRACT

Objective 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).Methods 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.Results ① The mRNA levels of TLR4, MD2, and NF-κB gradually increased after LPS induction and peaked at 6 hours (2-ΔΔCt 23.52±2.88, 17.43±3.43, 18.13±2.99, respectively), which were statistically significant before the stimulation with LPS (2-ΔΔCt 7.02±2.81, 5.19±3.22, 8.11±1.42, allP < 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 1mg/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, allP < 0.05). Compared to LPS group, the mRNA levels of NF-κB and STIM1 were obviously inhibited in PDTC1 mg/L group and psiTLR 12 h group [NF-κB mRNA (2-ΔΔCt) 17.22±2.35, 13.24±3.54 vs. 30.16±2.06; STIM1 mRNA (2-ΔΔCt) 12.57±2.43, 12.21±2.46 vs. 25.12±2.02, allP < 0.05]. Conclusions 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.