PMID- 28625263
OWN - NLM
STAT- MEDLINE
DCOM- 20181018
LR  - 20181018
IS  - 2095-4352 (Print)
VI  - 29
IP  - 2
DP  - 2017 Feb
TI  - [Bacterial endotoxin-induced endothelial cell injury and calcium overload 
      associated with Toll-like receptor and calcium signal].
PG  - 150-155
LID - 10.3760/cma.j.issn.2095-4352.2017.02.011 [doi]
AB  - 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-kappaB (NF-kappaB) 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-kappaB 
      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-alpha (TNF-alpha) 
      and interleukin-6 (IL-6) were detected by enzyme linked immunosorbent assay 
      (ELISA) in supernatant. The mRNA levels of STIM1 and NF-kappaB were detected by 
      RT-PCR. RESULTS: (1) The mRNA levels of TLR4, MD2, and NF-kappaB gradually increased 
      after LPS induction and peaked at 6 hours (2(-DeltaDeltaCt): 23.52+/-2.88, 17.43+/-3.43, 
      18.13+/-2.99, respectively), which were statistically significant before the 
      stimulation with LPS (2(-DeltaDeltaCt): 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-kappaB 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-alpha 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-kappaB and STIM1 were 
      obviously inhibited in PDTC 1 mg/L group and psiTLR 12 h group [NF-kappaB mRNA 
      (2(-DeltaDeltaCt)): 17.22+/-2.35, 13.24+/-3.54 vs. 30.16+/-2.06; STIM1 mRNA (2(-DeltaDeltaCt)): 
      12.57+/-2.43, 12.21+/-2.46 vs. 25.12+/-2.02, all P < 0.05]. CONCLUSIONS: TLR4, MD2, 
      NF-kappaB 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-kappaB and/or STIM1.
FAU - Xiao, Jianguo
AU  - Xiao J
AD  - 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: 
      srj127@sina.com.
FAU - Song, Qing
AU  - Song Q
FAU - Li, Tanshi
AU  - Li T
FAU - Sun, Rongju
AU  - Sun R
LA  - chi
PT  - Journal Article
PL  - China
TA  - Zhonghua Wei Zhong Bing Ji Jiu Yi Xue
JT  - Zhonghua wei zhong bing ji jiu yi xue
JID - 101604552
RN  - 0 (Endotoxins)
RN  - 0 (Lipopolysaccharides)
RN  - 0 (NF-kappa B)
RN  - 0 (Toll-Like Receptors)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Calcium
MH  - *Calcium Signaling
MH  - Endotoxins
MH  - Humans
MH  - Lipopolysaccharides
MH  - NF-kappa B
MH  - Toll-Like Receptors
MH  - Tumor Necrosis Factor-alpha
EDAT- 2017/06/20 06:00
MHDA- 2018/10/20 06:00
CRDT- 2017/06/20 06:00
PHST- 2017/06/20 06:00 [entrez]
PHST- 2017/06/20 06:00 [pubmed]
PHST- 2018/10/20 06:00 [medline]
AID - 10.3760/cma.j.issn.2095-4352.2017.02.011 [doi]
PST - ppublish
SO  - 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.