Seawater Inhalation Induces Acute Lung Injury Though ROS Generation And The Endoplasmic Reticulum Stress Pathway

Background:Drowning is a public health problem which is very important but is often ignored by people.In the global scope,drowning is one of the most common causes of death.Seawater has complex Chemical composition,but also be rich in bacteria and algae,therefore,seawater drowning is more serious,and can caused seawater drowning-induced acute lung injury(SW–ALI)and seawater drowning-induced acute respiratory distress syndrome(SWARDS).When SW-ALI occurs,seawater can directly affect alveolar epithelial cells,induce apoptosis of epithelial cells,the production of inflammatory factors,alveolar structure disorder,alveolar and interstitial edema,leading to ventilation perfusion imbalance,impaired gas exchange,the body of hypoxemia and acidosis.When the body is subjected to adverse external stimuli,the function of protein folding in the endoplasmic reticulum(ER stress)is damaged and the endoplasmic reticulum stress is activated.At the same time,the level of reactive oxygen species(ROS)increased.Endoplasmic reticulum stress and reactive oxygen species play an important role in cell proliferation,apoptosis and transformation,but their role in SW-ALI/ SW-ARDS is still unknown.In this study,we investigated the role of ER stress and ROS in SW-ALI in rats,and explored the possible mechanism.Objective:By establishing the model of lung injury induced by seawater inhalation and the intervention of seawater on human alveolar epithelial derived A549 cells,we observed the effects of seawater on the injury and the apoptosis of alveolar epithelial cells,and investigated the role of ER stress and ROS in SW–ALI.Methods:1 Animal experiment: 65 male SD rats were randomly divided into:(1)normal control group,2h seawater group,4h seawater group,6h seawater group,8h seawater group;(2)Normal control group,seawater group,NAC group,NAC+ seawater group;(3)Normal control group,seawater group,4-PBA group,4-PBA+ seawater group.We established a rat model of SW-ALI in rats by intratracheal instillation of seawater.Pulmonary edema and lung injury were assessed by lung tissue wet dry weight ratio(W/D)and pulmonary histopathological examination.The apoptosis of lung tissue was detected by TUNEL staining.The expression of GRP78 and CHOP protein was detected by Western Blot.The expression of GRP78 was detected by immunohistochemical staining.2 Cell experiment:(1)stimulated A549 cells with 20%,40%,60%,80% seawater 4h.(2)stimulated A549 cells 2 h,4 h,6 h,8 h with 25% seawater.(3)Pretreatment with NAC or 4-PBA before adding seawater,Thap as positive control.A549 cell growth inhibition was assessed by CCK-8 assay.Cell apoptosis was detected by flow cytometry.Intracellular ROS levels were detected by reactive oxygen species fluorescent probe(DCFH-DA).The expression of GRP78,CHOP,p-JNK,p-PERK,p-IRE1,p-50ATF6,caspase-3 protein was detected by Western Blot.Results:1 Animal experiment: After seawater inhalation,the W/D value of lung tissue significantly increased,HE staining showed alveolar structure disorder,alveolar collapse,alveolar wall thickening,suggesting that seawater inhalation caused ALI.At the same time,the results of TUNEL staining showed that the apoptosis of alveolar epithelial cells was induced by seawater,and the pretreatment with NAC and 4-PBA could reduce the apoptosis of alveolar epithelial cells.Western Blot detection showed that seawater inhalation could increase the expression of GRP78 and CHOP protein.Immunohistochemical staining showed that 4-PBA pretreatment could decrease the expression of GRP78 protein.2 Cell experiment: CCK-8 assay and flow cytometry showed that seawater stimulation could inhibit the growth of A549 cells and promote apoptosis.DCFH-DA detection of intracellular ROS levels showed that seawater could cause an increase in ROS production.The results of Western Blot showed that seawater could increase the expression of GRP78 and CHOP.NAC pretreatment could not only reduce the level of ROS,but also reduce the expression of GRP78,p-50ATF-6,p-IRE1,p-PERK,CHOP,p-JNK and Caspase-3,and reduced the degree of cell growth inhibition and apoptosis induced by seawater.4-PBA pretreatment could not only reduce the expression of GRP78 and CHOP,but also reduce the expression of ROS and p-JNK,and reduced the cell growth inhibition and cell apoptosis induced by seawater in caspase-3.Conclusion: Seawater could cause injury and apoptosis of alveolar epithelial cells.The interaction between ER stress and ROS formed a vicious cycle,which mediated the apoptosis of alveolar epithelial cells and played an important role in the pathological process of SW-ALI.