The Effects Of NFAT5and Angâ…¡ And Receptor AT1in Seawater Aspiration Induced The Acute Lung Injury In Rats And Its Mechanism

Part1The effect of NFAT5in seawater inhalation induced the acute lunginjury and its mechanismBackground:Seawater aspiration induced ALI is one of the major causes of death in the maritimeaccidents and offshore production operation. Most patients would die from suffocationinduced by laryngeal airway reflex spasm after seawater aspiration and victims wouldsuffer from seawater inhalation induced acute lung injury (SWI-ALI) and seawaterinhalation induced acute respiratory distress syndrome (SWI-ARDS). There are limitedreports about the mechanism of seawater aspiration induced ALI at home and abroad andthe theoretical basis and molecular mechanism remains to further elucidate. Furthermore,effective clinical therapy system about seawater aspiration induced ALI has not yet formed.Therefore, research of the mechanism of seawater aspiration induced ALI is important tothe choice of clinical and drug therapy. Nuclear factor of the activated T cell5(NFAT5), belonging to NF-κB/Rel family, isthe only one transcription factor related to osmotic pressure that has been researched inmammals and it is expressed in many tissues. NFAT5plays a vital role in many diseasessuch as scheroma and inflammatory bowel disease (IBD) via activating the expression ofpro-inflammatory cytokines. Since the seawater is hypertonic, we investigated the role ofNFAT5in seawater aspiration-induced ALI.Objection:To explore the expression the expression and function changes of NFAT5and NF-κBin rat seawater aspiration-induced ALI model. To verify the important role of NFAT5inseawater inhalation lung injury, we used siRNA technology inhibiting NFAT5expressionfurther observation NF-κB and inflammatory factor changes at the cellular level.Method:In vivo:Sprague-Dawley (SD) rats, weighing190-210g, were randomly divided into controlgroup and3different time seawater group, including inhaling3,6and12hour seawater,respectively. Before seawater administration (4ml/kg body weight) through the trachea,rats were narcotized using3%pentobarbital sodium by the means of injecting into ratsabdominal cavity. After the rats appear shortness of breath, nose and mouse with cyanosis,mosist crackles in whole lungs, outflow of pink foam liquid from nose and mouse,0.5mlartery blood from right internal carotid artery was taken at indicated time points to analyzethe blood gases Arterial blood gas shows PaO2<50mmHg, SaO2<45%, and PaO2/FiO2(oxygenation index)<150mmHg tips seawater inhalation lung injury model wassuccessfully. Then the rats were sacrificed and lung tissue embedded in paraffin,hematoxylin-eosin staining (HE) staining, wet/dry ratio (W/D) detection, homogenizedlung tissue by reverse transcription PCR (RT-PCR) and Western blot to detect NFAT5andNF-κB expression changes in the lung tissue, as measured tumor necrosis factor (TNF-α),interleukin-1β (IL-1β) and interleukin-8(IL-8) levels change by enzyme linked immunosorbent (ELISA) in lung tissue.In Vitro:NR8383cells were used in our experiments. Cells were cultured in F12mediumcontaining20%fetal bovine serum and were stimulated by12.5%,25%,37.5%seawaterfor6h after cells covering bottle bottom. At the end of experiment, culture medium werecollected and used for determination of pro-inflammatory cytokines and NFAT5. The cellswere also treated with NFAT5siRNA and the expression of proinflammatory cytokinesand p-NF-κB were detected.Results:In vivo:The rats had been administrated seawater, had a significantly reduced partial pressureof oxygen (PaO2) and increased partial pressure of carbon dioxide (PaCO2). HE stainingshowed that lung tissue structural disorder, lung inflammatory cells and visibility withinthe alveolar exudate, accompanied with local atelectasis. The ratio of W/D increasedrapidly, the IL-1β, IL-8and TNF-α expression increased significantly. In addition, theexpression of NFAT5significantly increased after seawater stimulation.In Vitro:The expression of NFAT5and p-NF-κB were most obvious in25%seawater group.In addition, the expression of pro-inflammatory cytokines and p-NF-κB were reducedafter the inhibition of NFAT5by siRNA technology.Conclusion:NFAT5plays a vital role in the process of seawater aspiration-induced ALI. Theprobable mechanism might be regulation of p-NF-κB. The expression of proinflammatorycytokines and lung injury were reduced after suppressing the expression of NFAT5. Part2AngⅡ and receptor AT1mechanisms research in acute lung injuryinduced by seawater inhalation and the protective effect of losartan on acutelung injury induced by seawater inhalationBackground:Several researches have shown that renin-angiotension system (RAS) was closelyrelated to physiological and pathological processes in lung tissues. The lung vesselspossess local RAS system and this system can be activated and play an important role inthe early stage of ALI. AngiotensionⅡ (AngⅡ), a key effector of RAS, mostly expressedin endothelial cells and performed effect through binding to specific AngⅠtype (AT1)receptor and AngⅡ(AT2) type receptor, especially AT1receptor. AngⅡparticipate in theregulation of the circulatory system via activating the sympathetic system, increasingblood pressure and maintaining the balance of fluid and electrolyte. Furthermore, AngⅡcan also regulate the pathophysiological process of some diseases through paracrine,autocrine and intracellular interaction. There are also some other studies which shown thatAng Ⅱ may be a kind of proinflammatory peptide. The expression of manyproinflammatory genes were related to the interaction between AngⅡand AT1receptor.Moreover, it has shown that losartan, an AT1receptor selective inhibitor, can alleviate ALIinduced by LPS, acid aspiration, sepsis and ventilation via some potential mechanisms.However, the role of AngⅡand AT1receptor have not been reported in seawateraspiration-induced ALI. Therefore, we investigated the role of AngⅡand AT1receptor andthe effect of losartan in seawater aspiration induced ALI.Objective:1. To explore the expression of AngⅡand AT1receptor in the model of seawateraspiration-induced ALI.2. To investigate the effect of losartan in seawater aspiration-induced ALI. Method:Sprague-Dawley (SD) rats were weighed and divided into5groups in the same way,including control group, inhaling seawater group(3h,6h,12h) and low(5mg/kg),medium(15mg/kg), high(30mg/kg) dose losartan therapy group. Different doses oflosartan treatment groups were given to intraperitoneally30min before seawateradministration, and the other treatment conditions were the same as the seawater groups.Rats appear to be breathing wheezing, shortness of breath, cyanosis of ear and nose, ralescovered the whole lung by auscultation, the nose and mouth with pink bubble liquid. Theright side of the neck to take0.5ml within the corresponding time points to detect arterialblood gas analysis, the result shows PaO2<50mmHg, SaO2<45%, and PaO2/FiO2(oxygenation index)<150mmHg tips seawater inhalation lung injury model wassuccessfully. The severity of lung injury was assessed by wet to dry weight ratios,histopathological and myeloperoxidase (MPO) activity, as well as ELISA assays forinflammatory cytokines.Results:In this study, Seawater aspiration induced injuries of the lung alveolar structures,increasing of PaCO2and decrease of PaO2. AngⅡand AT1were up-regulated and theNF-κB were activated after seawater administration. In addition, the expression ofpro-inflammatory cytokines, MPO, However, losartan pre-administration significantlyreduced these injuries.Conclusion:Seawater aspiration induced the acute lung injury and increased the expression ofAngⅡand AT1. Pre-treatment of losartan significantly reduced lung injuries throughinhibition of phosphorylation NF-κB and pro-inflammatory cytokines expression.