Alteration Of Claudin Expressions In Pseudonomas Aeruginosa-induced Acute Lung Injury And Its Significance

Part Ⅰ:Alteration of Claudin Expressions in Pseudonomas aeruginosa-induced Acute Lung InjuryObjective We established rat model of acute lung injury(ALI) to investigate claudin expression alterations in Pseudonomas aeruginosa(PA)-induced ALI and it’s potential significance.Methods Sprague Dawley rats were randomized into normal group(N group), control group(C group) and PA group(P group), and three time points(3h,9h and24h) were set in C group and P group. Samples were collected before and3h,9h,24h after instillation of normal saline(NS) and PA. Arterial blood gas analysis, levels of total proteins in bronchoalveolar lavage fulid(BALF) were examined. Furthermore, the pathological changes of lung were observed under light microscope and lung injury scores were evaluated. The mRNA and protein levels of claudin-3,-4,-18and-5were determined by RT-PCR and Western Blot respectively.Results The arterial partial pressure oxygen(PaO2) declined and total concertrations of proteins in BALF significantly increased at different time points after PA instillation compared with N group and C group, especially P-24h. Mild to severe pathological changes happened after instillation of PA. P-3h showed a little inflammatory infiltration; P-9h showed alveolar septal widening, more infiltration of neutrophils; P-24h showed widespread of alveolar septal widening and massive neutrophils infiltration with microvascular expansion, hemorrhage and partial atelectasis. Lung injury scores of P-24h was significantly higer than those of P-3h and P-9h. Claudin-4,-18and-5mRNA levels increased significantly24h after PA instillation without increase in claudin-3mRNA while protein levels of claudin-3,-4,-18and-5showed no significant change.Conclusion1.Rats intratracheally injected with PA showed obvious patho physio logic changes of which P-24h suffered most severe lung injury.2.Claudin-4,-18and-5mRNA levels increased significantly24h after PA instillation in pathophysiologically identified most severe lung injury, which may be a protective mechanism to enhance barrier function in early stage of ALI. Part Ⅱ:Significance of claudin expression alterations in Pseudonomas aeruginosa-induced Acute Lung InjuryObjective In this study, we explored the possible significance of claudin expression alterations in BALF in PA-induced ALI.Methods The ultrastructure of alveolar epithelial cells, pulmonary vascular endothelial cells and TJs structures were observed under electron microscopy. The mRNA levels of surfactant protein(SP-A, SP-B, SP-C) and von Willebrand factor(vWF) were determined by RT-PCR. The concertrations of claudin-3,-4,-18and-5were determined by ELISA and the relationship between concertrations of claudins and lung injury scores were analyzed by spearman correlation.Results Ultrasructural changes were observed after instillation of PA. P-3h showed mild alveolar epithelial damage and normal TJs. P-9h showed severe disruption of epithelial cells, especially ATI with sloughed membrane in the air space. Meanwhile, TJs of alveolar epithelial cell were not tight enough. P-24h showed even more severe disruption of alveolar epithelial and endothelial cells with the abundant cellular debris and a loss of completeness of alveolar-capillary barrier. TJs were not easy to be find. The mRNA expression of SP-A, SP-B, SP-C and vWF showed no significant difference in N group, C group and P group. The concertrations of clauidn-3,-4and-18in BALF in N group and C group were low while they increased in a time-dependent manner after PA instillation. The concertration of claudin-5had already been high after3h of PA instillation and there were only little increase in the next few hours. The concertrations of claudin-3,-4and-18had positive correlation with the severity of lung injury while clauidn-5did not.Conclusion1.The concertrations of clauidn-3,-4and-18in BALF increased in a time-dependent manner after PA instillation correlated with the severity of lung injury, which might be a potential biomarker for alveolar epithelial disruption in early stage of PA-induced ALI.2.The concertration of clauidn-5in BALF increased at all three time points after PA instillation irrelated with the severity of lung injury, which could not used as biomarker for endothelial disruption in early stage of ALI.