| Background and ObjectiveAcute lung injury (ALI) is a critical clinical syndrome with progressive dyspnea, refractory hypoxemia and high mortality, which pathological features mainly include inflammatory reaction and alveolar-capillary membrane injury result from severe infection, trauma, shock, etc.Endotoxima is a key etiological factor for ALI. Pulmonary microvascular endothelial cells (PMVEC) and overexpression of intercellular adhesion molecular-1 (ICAM-1) play key roles in the pathogenesis of lipopolysaccharide (LPS)-induced ALI. So far, the therapeutic methods for ALI have not been improved. Respiratory support is still one of major methods for treating ALI. The studies have shown that liquid ventilation, especially aerosol therapy with perfluorocarbon (PFC) has more advantages than conventional mechanical ventilation (CMV) in the treatment of ALI. However, its mechanism has not been completely elucidated. Especially the anti-inflammatory effect of PFC is still controversial and the protective effects of PFC on PMVEC remain unclear. Therefor, in this study we will observe the effects of aerosolized perfluorocarbon on pulmonary microvascular permeability in the a model of LPS-induced ALI in vivo, evaluate the impact of PFC on LPS-induced ICAM-1 expression in cultured PMVECs of rat, and then investigate the influence of PFC on LPS-activated TLR4 signal transduction in vitro. The purposes of this study are to confirm whether there is anti-inflammatory effect of PFC for ALI, to elucidate the protective mechanism of PFC on PMVEC, and then reveal the anti-inflammatory mechanism of aerosolized PFC on LPS-induced ALI.Methods1. A rat model of ALI was established by jugular injection of LPS at a dose of 6mg/kg. Sixty-four SD rats were randomly divided into four goups: N, LPS, CMV and PFC. Group N remained untreated as normal control, group LPS was injured by LPS injection as ALI model. After establishment of lung injury by LPS, group CMV received the conventional mechanical ventilation, group PFC received both the conventional mechanical ventilation and inhalation of aerosolized PFC at a dose of 10ml/kg/h. After 2 hours treatment and another 6 hours observation, the artery blood and lung tissue were collected. The level of PaO2 in artery blood and the wet/dry ratio of lung tissue were measured. The pulmonary microvascular permeability and MPO activity of lung tissue were detected. The level of ICAM-1 mRNA and protein expression in lung tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot, respectively. The histological changes of lung tissue were observed under optical microscope.2. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripheral lung tissue pieces used for cell culture were examined. Used rat pulmonary artery smooth muscle cells and human umbilical vein endothelial cells as control, CD34, lectin from Bandeiraea simplicifolia and factorⅧrelated antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morphology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively.3. Rat PMVECs cultured by peripheral lung tissue-sticking method were seeded in Transwell chambers until confluence, and divided randomly into 4 groups: group C remained untreated as blank control; group F was incubated with PFC; group L was incubated with 100ng/ml LPS; group LF was incubated with both 100ng/ml LPS and PFC. After treatment for 3, 8 and 24 hours, the cell morphology of group F was observed with optical microscope. Then the cells of each group were harvested, respectively. The cell activity in group F was determined by MTT assay. The mRNA and protein expression levels of ICAM-1 in each group were detected by RT-PCR and flow cytometry (FCM), respectively.4. Rat PMVECs were cultured, grouped and treated as mentioned above. Twenty-four hours later, the level of TLR4 protein expression and its affinity with FITC-LPS in group F and goup C were detected by FCM. The level of TRAF6 protein expression and the activities of p38MAPK and NF-κB were determined by Western Blot and EMSA. Results1. Compared with group N, PaO2 decreased and W/D ratio, pulmonary microvascular permeability, MPO activity, ICAM-1 mRNA and protein expression, pathomorphological score of lung tissue in group LPS increased significantly (P<0.01).There was no significant difference between group CMV and group LPS (P>0.05). However, compared with group CMV and group LPS, the above indexes in group PFC improved significantly (P<0.01-0.05).2. Histological sections showed that tissue pieces were scissored from periphery lung lobes accurately. The cultured cells had features of binding lectin from Bandeiraea simplicifolia and positive immunocytochemical staining with CD34 antibody, but negative for factorⅧrelated antigen. Weibel-Palade bodies were not observed in the cultured cells.3. The cell morphology and activity in normal rat PMVECs did not have significant change between before and after PFC treatment (P>0.05), despite at 3, 8 or 24 h. Both mRNA and protein expression levels of ICAM-1 had no significant difference between group F and group C (P>0.05). Group L had significantly higher the mRNA and protein expression levels of ICAM-1 in a time-dependent manner than group C (P<0.01-0.05). In comparison with group L, the mRNA and protein expression levels of ICAM-1 in group LF were not suppressed significantly by treatment at 3h-point (P>0.05), however, they markedly decreased at 8h-point and 24h-point (P<0.01-0.05).4. The TLR4 protein concentration and FITC-LPS affinity on cell membrance had no significant difference between group N and group F (P>0.05). The levels of TRAF6 protein expression, the activity of p38MAPK and NF-κB in group L were significantly higher than those in group C (P<0.01-0.05). There was no significant defference in TRAF6 protein expression between group LF and group L. But compared with those in group L, the activity of p38MAPK and NF-κB in group LF decreased significantly (P<0.05).Conclusion1. Aerosol therapy with perfluorocarbon significantly protects lung from LPS-induced injury in rats by the suppression of intercellular adhesion molecular-1 expression and polymorphonuclear leukocyte accumulation in lung tissue and the subsequent attenuation of pulmonary microvascular permeability.2. Peripheral lung tissue-sticking is a convenient, economical and efficacious method for rat PMVEC cultivation. FactorⅧrelated antigen and Weibel-Palade body are not ideal indexes for ascertaining rat PMVEC. The combination of peripheral lung tissue section, CD34 immunocytochemical staining and lectin from Bandeiraea simplicifolia binding assay is a simple and reasonable comprehensive method for identifying rat PMVECs.3. The cell morphology and viability of normal rat PMVECs are not significantly impaired by PFC treatment Perfluorocarbon suppresses the mRNA and protein expression of ICAM-1 induced by LPS which suggests a potential mechanism that PFC protects PMVECs from LPS-induced injury via its biological anti-inflammatory effect.4. The interaction between TLR4 and LPS on PMVECs surface membrane is not altered by PFC. The inhibition of PFC on TRAF6 and NF-κB activation seems to be the cause of attenuated ICAM-1 overexpression induced by LPS.
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