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.

Objective:To establish a stable fragment-induced penetrating liver injury model in landrace pigs and evaluate the characteristics of deep tissue injury.Methods:According to the different positioning methods of aiming points, twelve healthy adult landrace pigs were divided into group A (the relative height "h" of the aiming point and the highest point of the body surface on the tracing line was set to 5 cm) and group B ("h" was set to 6 cm). Ultrasonography was used to determine the direction of fragment projection, and an experimental ballistic gun was used to project high-velocity fragments to cause injury to animals. The vital signs of the two groups were monitored, and whole blood cell count, blood gas analysis, and liver and renal function were tested. Damages to the liver and adjacent organs, as well as the amount of bleeding and survival time were analyzed.Results:For the overall analysis of the two groups, the liver hit rate of fragment simulating projectiles was 100% (right anterior lobe and right lateral lobe injury), the hit rate of other organs in the abdominal cavity was 25% (3/12), and the incidence of hemothorax or pneumothorax was 8% (1/12). The wounds were mainly characterized by liver lacerations, with total or partial disconnection of the distal liver lobe. There was no significant difference in wound length and bleeding amount between groups A and B [wound length (cm): 9.8±1.7 vs. 11.2±3.8, bleeding amount (g): 597.0±477.1 vs. 1 032.0±390.3, both P > 0.05]. The depth of liver parenchymal laceration in group B with the aiming point closer to the anterior median line was significantly longer than that in group A (cm: 2.8±0.4 vs. 1.9±0.6, P = 0.015). Mean arterial pressure (MAP), pH value, residual arterial blood base (BE), hemoglobin (Hb) and hematocrit (HCT) levels decreased after the fragment-induced injury, and then reached a trough level [MAP (mmHg, 1 mmHg ≈ 0.133 kPa): 87.0±33.6, pH: 7.26±0.15, BE (mmol/L): -6.65±8.48, Hb (g/L): 9.86±1.10, HCT: 0.309±0.029, all P < 0.05] in the first hour. Blood lactate (Lac), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) levels increased over time, and reached a peak level [Lac (mmol/L): 10.21±4.40, LDH (U/L): 1 417.0±223.3, AST (U/L): 234.5 (162.5, 357.5), both P < 0.05] at 1 hour after injury. Pearson's correlation analysis showed that the total amount of bleeding was correlated with the depth of liver parenchyma laceration ( r = 0.684, P = 0.014). The Kaplan-Meier survival curve showed that the 3 hours survival rate in group A was higher than that in group B, but the difference was not statistically significant [83.3% (5/6) vs. 33.3% (2/6), P > 0.05]. Conclusions:The high-velocity fragment-induced penetrating liver injury model established by striking landrace pigs closer to the anterior median line with fragment simulating projectiles is reproducible and the degree of damage is controllable, and the model is applicable to further relevant research of hepatic ballistic trauma.