| Objective:Acute lung injury (ALI) is a kind of lung tissue injury which cause by many etiological factors,such as serious infection,trauma,shock,its clinical manifestation is the serious breathing difficulties. The damaged pulmonary vascular endothelial cell is the main target cell in the ALI,its activation can be considered an important indicator of ALI.It is important step to induce ALI that the activation of endothelial cell can increase leukocyte adhering and migration,and this process is closely related with the expression of adhesion molecules.E-sel- ectin is a kind of adhesion molecules which is only limited to express on the surface of activated endothelial cell,so it is a good marker of endothelial cell activation.Untill now,the studies about whether E-selectin expression can become a marker of ALI, it can be used as early indicators to reflect the severity of ALI,and find E-selectin antagonist can help reduce the occurrence and development of ALI,are seldom.The purpose of ours experiment is,according to the duplicate rat ALI model with lipopolysaccharide (LPS),to investigate the expression of E-selectin in lung tissue, the controlled mechanism of the transcription factor NF-kappaB to expression of E-selectin and effect of NF-kappaB inhibitor pyrrolidine dithiocabamate (PDTC),and to explore how E-selectin effect on the process of ALI and the diagnostic value.All of these studies may provide theory and new method in the treatment of ALI.Methods:96 healthy femina Wistar rats were provided by Experimental Animal Center of Hebei Medical University.Used the method of injecting lipopolysaccharide into abdominal cavity to copy the model of ALI. 96 rats were randomly divided into four groups:Intraperitoneal injection of normal saline instead of LPS in normal control group (N group);first PDTC was injected into abdominal cavity,then normal saline was also injected after half an hour in PDTC control group (P group);intraperitoneal injection of LPS(3mg/kg) in ALI group (L group);first PDTC was injected into abdominal cavity,then LPS(3mg/kg) was also injected after half an hour in PDTC group (P +L group).Rats were sacrificed and taked lung tissue in 2,4,8 and 12 hours after mading in models.We maded pathological section by hematoxylin eosin (HE) stain,then used optical microscope to observe the pathologic change of lung tissue in order to judge the success of copied ALI model.Wet/dry ratio of lungs was determined, the protein expression of E-selectin and NF-kappaBp65 was determined by immunohistochemical method,then the gene expression of E-selectin mRNA was assessed by rever transcription- polymerase chain reaction (RT-PCR).The datas were analyzed by student't test,one-way-analysis of variance(ANOVA) and linear correlation.Result:1 Pathologic change of lung tissue:In N group and P group the tissue structure of lung was intact;in L group the interval of pulmonary alveolus became thicker and many inflammatory cells barged into the lung tissue,and as the time rolled aroud the degree of lung tissue injury increased;in P+L group,the lung tissue had same representation too,but the degree of injury decreased.2 Wet/dry ratio of lung tissue:The wet/dry ratio in N group and P group at different time were 2h group 4.27±0.029,4h group 4.29±0.050,8h group 4.28±0.038,12h group 4.26±0.047 and 2h group 4.26±0.046,4h group 4.28±0.014,8h group 4.27±0.052,12h group 4.28±0.028,they had no significant difference (P>0.05).The W/D ratio at different time in L group were 2h group 4.70±0.081,4h group 4.94±0.082,8h group 5.50±0.074,12h group 5.80±0.071,they were more than N group,and as the time rolled aroud the W/D ratio unceasingly increased, L group at different time had significant difference (P<0.01).The W/D ratio at different time in P+L group were 2h group 4.43±0.058,4h group 4.52±0.057,8h group 4.96±0.058,12h group 5.31±0.058,they were lesser than L group,but they were more when compared with N group,three groups at same time had significant difference (P<0.01).3 Protein expression of E-selectin in lung tissue:We did not see E-selectin positive cell in N group and P group at different time.There all had positive expression of E-selection protein in each slice in L group, coloring rate of vascular endothelial cells at different time were 2h group 18.22±2.68%,4h group 31.90±4.70%,8h group 24.34±4.77%,12h group 19.28±3.27%, the protein expression of E-selectin was the highest at 4 hours,then as the time rolled aroud it unceasingly decreased, L group at different time had significant difference (P <0.01).There all had positive expression of E-selection protein in each slice in P+L group, coloring rate of vascular endothelial cells at different time were 2h group 13.78±3.96%,4h group 22.45±4.03%,8h group 16.36±2.79%,12h group 14.63±2.58%, they were lesser than L group, two groups at same time had difference (P<0.05), among they had significant different in 4 hours and 8 hours group (P<0.01).4 Gene expression of E-selectin in lung tissue:We did not see the gene expression of E-selectin in N group and P group at different time.The E-selectin mRNA at different time in L group were 2h group 0.3885±0.028,4h group 0.8202±0.026,8h group 0.6914±0.013,12h group 0.5543±0.011, the E-selectin mRNA was the highest at 4 hours,then as the time rolled aroud it unceasingly decreased, L group at different time had significant difference (P<0.01).In P+L group the E-selectin mRNA at different time were 2h group 0.2976±0.037,4h group 0.5969±0.017,8h group 0.5105±0.022,12h group 0.4582±0.021, they were lesser than L group, two groups at same time had significant difference (P<0.01).5 Protein expression of NF-kappaBp65 in lung tissue:The NF-kappaBp65 positive cells in five areas of campus visualis in N group and P group at different time were 2h group 162.33±6.95,4h group 167.33±5.96,8h group 164.17±8.61,12h group167.33±8.80 and 2h group 166.67±5.65,4h group 162.83±5.85,8h group 161.33±8.33,12h group 165.17±7.25, they had no significant difference (P > 0.05).The NF-kappaBp65 positive cells at different time in L group were 2h group 609.67±23.87,4h group 375.33±12.61,8h group 231.83±15.74,12h group 210.50±10.55, they were more than N group,the NF-kappaBp65 positive cells in 2h group were the most,also in optical microscope brown granules more expression in the nucleus ,as the time rolled aroud the positive cells unceasingly decreased, L group at different time had significant difference (P<0.01).The NF-kappaBp65 positive cells at different time in P+L group were 2h group 365.50±10.17,4h group 266.83±11.99,8h group 195.33±15.42,12h group185.17±13.76, they were lesser than L group,but they were more when compared with N group, three groups at same time had significant difference (P<0.01).6 The correlation analysis:There was a positive correlation between NF-kappaBp65 and the protein expression of E-selectin (r=0.7851, P<0.01),the NF-kappaBp65 positive cells and the gene expression of E-selectin had a significant positive correlation(r=0.8116, P<0.01).Conclusion:1 Two hours after injecting LPS,we can see the pathologic change of lung tissue, the model of ALI is copied successfully.When rats were induced ALI,E-selectin that it represent the activation of endothelial cell appeared in the lung tissue and as the time rolled aroud the expression of E-selectin unceasingly increased,it was the highest at 4 hours. This shows that the activation of endothelial cell occur at the earlier period of ALI,the high expression of E-selectin on endothelial cell may start that leukocyte adhered to the surface of endothelial cell, this process may be one of ALI pathogenesy.2 E-selectin express on the surface of activated endothelial cell,this specific may be the early diagnosis index of acute lung injury;if E-selectin unceasingly increased,illness would become more serious and prognosis would become worse,so it may be a useful marker for monitoring the severity and prognosis of infection-induced ALI.3 When rats taked place ALI, the transcription factor NF-kappaB is activated, the expression of NF-kappaBp65 increase,the increased of NF-kappaBp65 correlated with the higher expression of E-selectin. NF-kappaB inhibitor PDTC can inhibit NF-kappaB not to be activated and can impede that NF-kappaBp65 displace into karyon,meanwhile the expression of E-selectin decrease. So this shows that the expression of E-selectin closely relates to the accommodation of NF-kappaB.4 After rats are gave intervention by PDTC, we can look at that the pathologic change of lung tissue improve,the quantity of NF-kappaBp65 displaced into karyon decrease and the expression of E-selectin decrease,these confirm that the high expression of E-selectin after endothelial cell is activated is significance in ALI. After giving PDTC,it is prevented that leukocyte adhered to the surface of endothelial cell,they also would cause protective effect on ALI,which providing theoretical knowledge and new method in the treatment to ALI.
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