Study On The Expression Of Aquaporin1, 5 In Acute Lung Injury Induced By Sea Water

Background and ObjectiveSeawater drowning is a major factor that cause disaster in the progress of voyage, work and travel on the sea. Boating accidents, and drownings related to aquatic sports such as diving are also more common in coastal regions. Sea water aspiration can lead to direct the alveolar epithelial and pulmonary capillary endothelial cells injury. Sea water aspiration also can induce acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), which are characterized by an acute inflammatory process in airspaces and lung parenchyma. These syndromes are manifestations of the loss of barrier function of the alveolar epithelial and pulmonary capillary endothelial cells.Aquaporins constitute a family of water channels that facilitate membrane water permeability in various tissues of animals. Aquaporins can clear up excess water in the lung tissue, which take an important role in the barrier function of the alveolar epithelial and pulmonary capillary endothelial cells. The effect of hyperosmolar fluid aspiration (seawater) on lung fluid balance and aquaporins has not been well studied. Very few studies have been addressed on the expression of gene and protein of aquaporin1, 5 or the changes of fluid transport function in lung during ALI/ARDS induced by sea water. It is unclear whether aquaporin1, 5 is related to the development of pulmonary edema. Whether do aquaporins reduce or increase after sea water drowning? The study aims to observe the aquaporin1 and aquaporin5 expression at mRNA and protein level in A549 cell line soaked respectively, and the aquaporin1 and aquaporin5 expression in rats with acute lung injury induced by seawater aspiration.Methods1. Microculture tetrazolium assay (MTT),microscope, immunofluorescence and RT-PCR were applied to detect growth curve, inhibition rate, cell morphological changes and the expression of aquaporin1 and aquaporin5 at the mRNA and the protein level in A549 cells treated or untreated with sea water.2. A total of 130 Wistar healthy male rats were randomly assigned into 3 groups: sea water aspiration group (n=60) which was reproduced by tracheotomy, intubatton and seawater aspiration (4ml/kg body weight), dexamethason intervention group (n=10)which was given intraperitoneal dexamethasone injection (1mg/kg) after sea water aspiration, control group (n=60) which underwent tracheotomy and intubatton only. All the indexes about ALI/ARDS were observed at 0.5h, 1h, 2h, 4h, 8h after treatment. Changes in partial pressure of oxygen in artery (PaO2 ) were determined by blood gas analysis. Pathological changes in pulmonary-tissue were observed by microscopy. Tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) levels in serum were determined using enzyme linked immunosorbent assay (ELISA). Pulmonary capillary permeability was measured with the Evan's blue intravenously method.3. RT-PCR and immunohischemistry were used to detect aquaporin1 and aquaporin5 expression at the mRNA and the protein level, respectively.Results1. After A549 cells were soaked for 15 minutes, cellular membrane crenulation was observed, while cellular organ injury and plasmatorrhexis was not found. The cell proliferation was suppressed after A549 cells soaked for 1,2,4,8 and 16min. However, there was no significant difference in cell proliferation rate between cells treated or untreated with sea water for 24 h.2. The expression of AQP1 at the mRNA and the protein level in the sea water group(0.78±0.07,27.3±2.7) was significantly higher than that in control group (0.41±0.04,18.6±1.5) after treatment at 0.5 h (P <0.01and P<0.05).3. The expression of AQP5 at the mRNA and the protein level in the sea water group (0.53±0.06,36.9±4.4)was significantly higher than that in control group(1.07±0.13,51.3±6.2)after treatment at 0.5 h (P <0.01and P<0.05).4. The PaO₂ and PaO₂ /FiO₂ of the sea water aspiration group decreased quikly after sea water aspiration and reached the lowest point at the 0.5h( 42.4±6.4,202.2±30.5). The PaO₂ and PaO₂ /FiO₂ of the sea water aspiration group was significantly lower than that of control group at 0.5h, 1h, 2h, 4h and 8h after injury (P<0.01).5. It was observed that there was hyperaemia in the capillary vessels of sea water aspiration group. Meanwhile, alveolar edema was also observed. No pathological changes were found in the control group.6. After sea water aspiration, the level of TNF-αin the serum increased quickly and reached 328.23±34.68 pg/ml at 1h, 340.24±30.25 pg/ml at 2h, then dropped slowly, but was still higher than that in the control group at each time point (P <0.01).7. After sea water aspiration, the level of IL-6 in the serum increased quickly and reached the highest point 1106.4±180.6 pg/ml at 8 h, and was higher than that in the control group at each time point (P <0.01).8. Pulmonary capillary permeability rised significantly (30.7±2.9μg/ml) after sea water aspiration, higher than that in the control group (18.4±1.7μg/ml) (P <0.01).9.The lung W/D increased and reached the highest point at 8h (5.86±0.43), and was significantly higher than that in the control group (P <0.01).10. The expression of AQP1 at the mRNA and the protein level in the sea water group was significantly higher than that in control group after treatment (P <0.01and P<0.05).11. The expression of AQP5 at the mRNA and the protein level in the sea water group was significantly higher than that in control group after treatment correspondingly(P <0.01and P<0.05).12.In the dexamethason intervention goup, TNF-α,IL-6 levels in plasma,alveolar and interstitia edema and W/D were lower than those in the sea water group, PaO₂ and PaO₂ /FiO₂ were higher than those in the sea water goup, and the expression of AQP1,5 at the mRNA and the protein level were higher than that in the sea water aspiration group(P<0.05).Conclusions1. Sea water was harmful, while not lethal to A549 cells in a short time.2. Cellular membrane crenulation was observed and the expression of AQP1, 5 both at the mRNA and the protein level were up-regulated after A549 was soaked, which suggestted that up-regulation of AQP1, 5 may contribute to the water exosmosing in A549 cells. 3. Sea water aspiration resulted in releasing of inflammation mediators (TNF-α,IL-6), pulmonary interstitial edema, PaO2 and PaO2 /FiO2 degression and ALI/ARDS.4. The expression of AQP1,5 at the mRNA and the protein in pulmonary-tissue of rats with acute lung injury induced by sea water increased, suggesting that AQP1,5 may play an important role in fluid transportation in ALI/ARDS.5. Dexamethasone could improve biological indexes, lung permeability, wet-to-dry weight ratio and up-regulate AQP1, 5, suggesting dexamethasone has an effect on protecting acute lung injury induced by sea water. The up-regulation of AQP1 and AQP5 might be one of the mechanisms of lung protection by the dexamethasone.