Endotoxin Tolerance In Rat Pulmonary Inflammation Model Selectin, Vascular Cell Adhesion Molecular Changes And The Regulatory Mechanisms Of Nf-��b

OBJECTIVESevere endotoxemia could cause the acute respiratory distress syndrome (ARDS) .which has a high mortality. Endotoxin tolerance(ET) is defined as a state in which endotoxin triggered responses are at least partially abrogated by prior exposure to endotoxin. However, its mechanism remained unclear. In this experiment, we studied the kinetic expression of E —selectin, P —selectin and vascular cell adhesion molecules - 1 (VCAM—1) in serum, bronchial alveolar lavage(BALF) and lung tissue among the endotoxin tolerance rats and normal control (NC) rats, in order to understand their functions on alleviation of the increased permeability of pulmonary micro vascular or recruitment neutrophil to lung tissue and lung inflmmation upon LPS stimulation in ET rats. To further investigate the molecular mechanism of NF-k B by studying its activation and sub-unit components in the ET and NC rats.METHODS(1)Establishment of rats model of ET and endotoxemia initiated inflammation: Eight -two Sprague-Dawley male rats weighing between 170 and 200g were randomized into ET group and NC group. Endotoxin tolerance was induced by four daily intraperitoneal injections of 0.6mg/kg/day Escherichia coli LPS(serotype 055:B5). NC group received intraperitoneal injections of the same volume saline. On the fifth day, rats were injected with high dose of LPS(6mg/kg) to induce endotoxemia and lung inflammation. In the first part experiment, blood, left BALF and right lung tissue were collected before and 1, 2, 6, 24 hours after the high dose injection of LPS(five rats for each time point) , blood routine test and white cell counting and differentiating of BALF were immediately done. The right part of lung tissues was placed in liquid nitrogen cane quickly. The serum , supernates of BALF and deep frozen lung tissuewere stored in -80°C refrigerator for further measuring. In the second part of this experiment, right lung tissue were collected before and 2> 6> 24, 48, 72 hours after the high dose injection of LPS(six rats for each time point), then placed in liquid nitrogen cane quickly. The deep-frozen lung tissue was stored in -80°C refrigerator for further measuring.(2)The measurement of E —selectin , P —selectin and VCAM—lin serum , BALF and homogenates of lung tissue was done by enzyme-linked immunosorbent assay(ELISA).(3)The activation and sub-unit components of NF-k B in the lung tissues of the ET and NC rats were measured by Electrophoretic mobility shift assays (EMSA) .(4)Software SPSS 10.0 was utilized for statistical analysis. T test was used for comparison between two groups. Dunnett' s test was adopted in comparison of each time point within group. RESULTS(1) The expression of serum E-selectin was increased dramatically at lh point in both ET group and NC group, however , there was no significant difference (70. 31 ±10. 67pg/mL Vs 54.95+13. 10 pg/mL, P>0. 05). The serum E-selectin was decreased in both ET and NC groups at 2h point, it was apt to express stablely at 2h , 6h, 24h points, and there was the same level as that before LPS stimulation.(2) The E-seletin expression of BALF was obvious increased at lh after received high dose of LPS in NC group, and it was higher than that before LPS stimulation. The increase of E-selectin of BALF in ET group occurred at 2h point, the expression was delayed obviously, the peak was at 6h point (P<0. 01). It was significantly higher compared with that of NC group (98. 97±40. 49pg/mL Vs 38. 64± 17. 20 pg/mL, P<0. 05).(3) The expression of lung tissue E-selectin in NC group at lh and 2h points increased dramatically after LPS stimulation, however, there was no significant difference between two groups. The expression of lung tissue E-selectin in ET group increased slowly. It was higher at 6h and 24h points than those before LPSstimulation.(4)In NC group, the expression of serum p-selectin was significantly increased at 2h point after LPS stimulation (63. 02 ± 14. 63 pg/mL), it was significantly higher than that before LPS stimulation and that of ET group (53. 91 ±4. 86pg/mL). In ET group, the expression of serum P-selectin at 2h point was mild, furthermore, there was no significant difference compared with that before LPS stimulation. At 6h and 24h points, the expression of serum P-selectin was higher in NC group than those of ET group.(5) One hour after LPS stimulation, the decrease of P-selectin of BALF in ET and NC groups was observed, there was significant difference compared with that before LPS stimulation (P<0.01), also, the decrease was obvious in ET group. At 2h point after LPS stimulation, the recovery of BALF P- selectin appeared in ET and NC groups, however, there was no significant difference between before and after LPS stimulation (P>0.05).(6) There was no difference in P-selectin expression of lung tissue before LPS stimulation. The high expression was occurred in those two groups, it was higher in NC group than that of ET group. At 2h point, the P-selectin of lung tissue decreased, there was no significant difference between thses two groups, it recovered at 6h point. At 24h point, the expression of ET group was stable, but the expression of NC group remained unstable.(7)The obvious increase VCAM-1 expression in ET and NC groups showed at lh point after LPS stimulation, moreover, there was significant difference within group between before and after LPS stimulation. But, there was no difference between NC and ET group (4.92 ± 0. 31ng/mL Vs 4. 86 ±0. 28 ng/mL, P>0. 05).(8) The expression of VCAM-1 in BALF at 0, 1, 2, 6h points in ET group was lower than those of NC group, there was significant difference between NC and ET group at lh point (P<0.01). The increase of expression in ET group was observed at 24h point, it was higher than that of NC group.(9)Before LPS stimulation (Oh point), the expression of VCAM-1 of lungtissue at some extent was shown in both groups. It was higher in NC group compared with that of ET group ( P<0. 05). At 2, 6h points, the expression of VCAM-1 of lung tissue in NC group was obviously increased. There was significant difference between that of 6h point after LPS stimulation and that before LPS stimulation (Oh) (P<0.01). It was higher in NC group at 6h point than that of ET group. The increase of expression VCAM-1 of lung tissue in ET group. The expression of VCAM-1 of lung tissue in ET group appeared at 6h and 24h points, moreover, it was stable. But the tendency of decrease at 24h point of VCAM-1 in NC group showed. (10) After high dose LPS stimulation in NC group, activation of NF- k B dramatically increased, reaching summit during 2-6 hours. In ET group, there was no change in the translocation and DNA-binding activity of NF-k B after high dose LPS stimulation. (ll)There was little DNA-binding activity of NF-k B before 6mg/kg of LPS stimulation in normal control group, and p50 homodimers predominated. The p65/p50 heterodimers was increased sharply after LPS stimulation, however, p65/p50 heterodimers remained at a constant level as before the stimulation in ET group, percentage of p50 homodimers was higher in ET group than that of NC group. The ratio of p50 homodimers to p65/p50 heterodimers decreased from 1.500 + 0.341 to 0. 684±0. 092 in NC group.CONCLUSIONS(1) The decrease of expression of serum E—selectin and delay of expression of E—selectin of bronchial alveolar lavage fluids and lung tissue may be associated with decrease and alleviation of systemic and pulmonary inflammation in endotoxin tolerance rats upon LPS stimulation in ET rats.(2)The lower expression of P—selectin in lung tissue and serum in ET group compared with NC group might be related with the decrease or inhibition of systemic and pulmonary inflammation in endotoxin tolerance rats upon LPS stimulation. However, the initial low expression of P—selectin in BALF in ET and NC groups ,the detail...