The Function And Relevant Mechanism Of SPAK On Seawater Inhalation Induced Acute Lung Injury

Background:Drowning is a very important public health problem; however, we often neglected it.It has just after road traffic injury become the second leading cause of unintentional injury,especially among young children. Water inhalation can induce acute lung injury (ALI) andthe acute respiratory distress syndrome (ARDS) by damaging the barrier functions ofalveolar epithelial cells, leading to lung edema and inflammatory reactions. Previousstudies has shown that the occurrence and exacerbation of seawater instillation inducedacute lung injury (SWI-ALI) were partly because phosphorylation of NF-κB andactivation of inflammation mediators which were mainly triggered by the hyperosmolaritychallenge. However, the upstreams of NF-κB in SWI-ALI have not been well studied.STE20-related protein proline/alanine-rich kinase (SPAK) is a member of STE20family of kinases. SPAK is very conservative between species and ubiquitously expressedin many organs. The main function of SPAK is phosphorylating and activatingNa+-K+-2Cl cotransporter (NKCC) which is a very important ion transporter that couldmediate the inflammation reactions in many conditions. What is more, SPAK also has important influence on proliferation and differentiation of some kinds of cells, and themediation of inflammatory in intestinal. As shown in a lot of experiments, SPAK has beenproven that could activate p38pathway, which has a vital role in mediating theinflammation caused by the exposure of hyperosmosis. Studies conducted on mousecolitis model induced by addition of hypertonic dextran sodium sulfate showed that theoverexpression of SPAK exacerbated experimental colitis in mice and the reason might bethat overexpression of SPAK leaded to epithelial barrier dysfunction which then leaded tothe increased production of inflammatory cytokines. So we presumed that SPAK mightalso give rise to the unbalance of inflammation reaction during SWI-ALI.Objective：(1) To observe the expression of SPAK in lung tissue by copying the seawaterinhalation induced acute lung injury models.(2) To clarify the pathway that regulating the expression of SPAK.(3) To explore the role that SPAK plays in seawater inhalation induced acute lunginjury through interfering SPAK expression by siRNA technology.Methods：Part1:30healthy rats were anesthetized with1.5%sodium pentobarbital followed byintratracheal administration seawater (4ml/kg body weight) into both lungs via a20-gaugeintravenous catheter through the tracheae within4min. The rats were kept in supine and30°head-up tilt during the experiments. At the end of the animal experiment, they wereeuthanized by bleeding to death at the predetermined points of time (0hour,1hour later,3hours later,6hours later and12hours later) and then the lungs were harvested for theexperiments described below.a. We measured the lung W/D ratios to evaluate the severity of pulmonary edema. Aftergetting and recording the wet weight and dry weight of the lung, W/D ratios weregotten by divided the lung wet weight by the dry weight. b. The lung tissues of the same part harvested from every rat were fixed, embedded, andfinally cut into5μm thick. After that, the slices were stained with hematoxylin andeosin (HE) before visualization under a light microscope.c. Levels of TNF-α and IL-1β in the lung tissues were tested by ELISA kits.d. The expression of SPAK in lung tissue was detected and observed byimmunohistochemical staining, and we also verified the result by western blot method.e. The change of transcription of SPAK was detected by RT-PCR method.Part2:We selected rat alveolar macrophage cell line, NR8383cells, in our experiment.Twenty-four hours before the experiment started, the cells were centrifuged andserum-free medium. The total cells were distributed to three groups: normal, seawatertreatment group and PDTC pretreatment plus seawater group. The medium of seawatertreatment group was replaced with medium containing25%seawater. The cells of PDTCpretreatment group was pretreated with PDTC (200μM)30minutes before seawaterexposure. Four hours later, the supernatants and cells were separated by centrifugating.a. The influence of PDTC pretreatment on the expression of SPAK was detected bywestern blot.b. First, down-regulated the expression of SPAK. Then measured changes of TNF-α andIL-1β in supernatants.Results:Part1:a. The W/D ratio of the lung tissue increased significantly (P <0.001) after the seawaterinhalation. This demonstrated that seawater inhalation could cause pulmonary edemaevidently.b. The results of HE straining showed that, after seawater challenge, the wall of alveolarbecame thick, many blood cells and fluid exudated, and alveolar structures weredamaged.c. The secretion of inflammatory cytokines TNF-α and IL-1β increased significantly increased (P <0.05) after seawater inhalation, and reached the maximum about sixhours later.d. The results gotten from immunohistochemical straining demonstrated that, comparedwith the normal group, there were more cells were stained into brown in seawaterinhalation groups. This indicated that the expression of SPAK increased significantlynot only in the cytoplasm but also in nucleus.e. The results of western blot showed that seawater inhalation could significantlyincrease the expression of SPAK, up to around2.8times.f. The results of PCR showed that the transcription of SPAK of seawater inhalationgroups was significantly plentiful than in normal group.Part2:g. Seawater stimulation increased the phosphorylation of NF-κ B in NR8383cells, andPDTC pretreatment could inhibit this reaction obviously.a. Seawater stimulation could increase the release of TNF-α, IL-1β and many othercytokines. In the SPAK knockdown cells, seawater stimulation could also cause therelease of inflammatory cytokines. However, compared with contrast group, therelease of inflammatory cytokines was restrained to some extent.Conclusion:In this study, we demonstrated that the transcription and translation of SPAK wasup-regulated under hyperosmotic seawater stimulation. And these changes played roles inthe inflammatory reactions, and deteriorated the injury of lung. By inhibition theactivation of NF-κB, the expression of SPAK was down-regulated. This indicated thatNF-κB might participate the regulation of SPAK. When down-regulate the expression ofSPAK by siRNA, the release of inflammatory factors was significantly inhibited.