| Background:Drowning is one of the most common causes of death and remains serious social and medical issue. It is estimated that drowning results in around four hundred thousand deaths each year and seawater drowning account for a large part. Despite decades of intensive research, the mortality rate remains high. Seawater aspiration can also bring about acute lung injury(ALI) or acute respiratory distress syndrome(ARDS). The seawater aspiration-induced ALI/ARDS is characterized by up-regulation of inflammatory mediators, severe dyspnea, serious hypoxemia and edema. In seawater aspiration-induced ALI animal models, the inflammatory cells which have infiltrated into alveolar spaces release several molecules, such as pro-inflammatory cytokines, reactive oxygen species, contributing to the pulmonary inflammatory process. In addition, stimulation of seawater induces the apoptosis of the pulmonary epithelial cells, which lead to the impairment of lung barrier function.Autophagy controls the degradation of long-lived proteins, protein aggregates, and invading microbes and may be involved in the regulation of the inflammatory response. Moreover, several studies indicated that autophagy interact closely with apoptosis. However, autophagy and its relationship with inflammation and apoptosis in seawater aspiration-induced ALI are still unknown. In addition, dexamethasone treatment has been proved to have therapeutic effects in seawater aspiration-induced ALI, but its pharmaceutical effect on autophagy regulation is poorly understood. Our study aims to investigate the expression of autophagy related proteins through establish the seawater aspiration-induced ALI model in rats. Moreover, we also investigate the effect of dexamethasone on autophagy and its relationship with inflammation and apoptosis. Objective:1. To investigate the expression of pro-inflammatory cytokines, autophagy and apoptosis related proteins.2. To investigate the pharmaceutical effect of dexamethasone on autophagy and its relationship with inflammation and apoptosis. Methods: Part1:40 rats were randomly divided into 5 groups: normal group, 1h, 3h, 6h, 12 h seawater group. At different time point, Pa O2, Pa CO2, Sa O2, TNF-α, IL-1β, IL-8, W/D of lung tissue were detected. In addition, autophagy related protein ATG5, Beclin1, LC3Ⅱand apoptosis protein Bcl-2, Bax, cleaved caspase-3 were detected by Western blot.Part2:A549 cells were divided into 5 groups: normal group, 6h seawater group, rapamycin+6h seawater group, dexamethasone+6h seawater group, 3-MA+6h seawater group. The transfection of GFP-LC3 was detected by immunofluorescent staining. TNF-α, IL-1β, IL-8 were detected by ELISA. In addition, autophagy related protein ATG5, Beclin1, LC3Ⅱand apoptosis protein Bcl-2, Bax, cleaved caspase-3 were detected by Western blot. Results: Part1:Pa O2 and Sa O2 were significantly decreased and reached its deepest point at 1h after seawater stimulation. Pa CO2 and W/D were significantly increased and reached its peak at 1h after seawater instillation. Moreover, seawater instillation for 6h significantly induced pulmonary edema, infiltration of inflammation cells in lung tissue and expression of pro-inflammatory mediators. In addition, autophagy related protein ATG5, Beclin1, LC3Ⅱand apoptosis protein Bcl-2, Bax, cleaved caspase-3 were also significantly increased after seawater stimulation. Part2:In cell experiments, rapamycin and dexamethasone induced the increase of transfection of GFP-LC3 and the expression of autophagy related protein ATG5, Beclin1, LC3Ⅱafter seawater stimulation, and inhibited the expression of apoptosis protein Bcl-2, Bax, cleaved caspase-3 and pro-inflammatory mediators. However, the effect of 3-MA was reverse.Conclusions: 1. seawater instillation induced the expression of pro-inflammatory mediators, autophagyrelated proteins and apoptosis proteins. 2. dexamethasone inhibited seawater induced apoptosis and inflammation by increasingthe expression of autophagy related proteins. |
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