The Study Of Pyocyanin Cause Alveolar Type â…¡ Epithelial Cell Apoptosis And Related Pathways

Background : Pseudomonas aeruginosa pneumonia is one of the most common hospital acquired pneumonia, the alveolar pulmonary edema is the major cause of the high fatality rate. Alveolar epithelial cells plays an important role in the occurrence of alveolar edema and lung reabsorption of water. A study shows pseudomonas aeruginosa alveolar epithelial cells can be induced to apoptosis, and pyocyanin(PCN)is one of the most important toxic metabolite of pseudomonas aeruginosa, so whether can also cause alveolar epithelial cell apoptosis? In the event of apoptosis, through which pathway? This study use pyocyanin stimulating human type II alveolar epithelial cell line A549 cells, observe alveolar type II epithelial cell activity and apoptosis at different toxin concentrations and different incubation time, detect the important biological markers of apoptosis pathways by the application of protein and nucleic acid detection technology,in order to further clarify the important factor of lung injury caused by pseudomonas aeruginosa, as well as the possibility of a apoptosis signaling pathway, so as to provide theoretical basis for optimal control of pseudomonas aeruginosa pneumonia.Objective:This study tries to observe in different toxin concentration and incubation time of alveolar type â…¡ epithelial cell activity and apoptosis, is determined by MTT,flow cell and protein immunoblot technology to detect cell apoptosis, and applies the q PCR from the level of nucleic acid detection of protein expression. To explore PCN in inducing apoptosis of A549 cells and its related molecular mechanisms.Methods: 1. Determined by MTT method to detect the selection of different concentrations(5ug/ml, 10ug/ml, 25ug/ml and 50ug/ml) PCN effect on A549 cell activity.2. According to the result of determined by MTT, select 25ug/ml PCN as experiment concentration, respectively, to stimulate A549 cell 6h, 12 h, 24 h,application of flow cytometry to detect the ratio of A549 cells apoptosis, survival and necrosis.3. Using the application of Western blot method to detect 25ug/ml PCN stimulus A549 cell 0 h, 6 h, 12 h,24 h after, the protein expression of Caspase-3.4. q PCR method is applied to detect 25 ug/ml PCN stimulus A549 cell 0 h, 6 h,12 h, 24 h after, caspase 3, caspase- 8, caspase- 9 RNA expression changesResults: 1.when PCN stimulating A549 cell,the higher the concentration of the stimulus, the longer the time, the lower the cell activity,a dose dependent and time dependent.2. Using 25ug/ml PCN stimulate A549 cells, cell apoptosis rate increased after24 h, the survival rate is reduced, the necrosis rate has no obvious change. It is statistically significant.3. Using 25ug/ml PCN stimulate A549 cells, starting from the stimulation of 24 h, the expression of Caspase-3 precursor protein is reduced.4. 25 ug/ml PCN stimulus A549 cells for 24 h, the expression of Caspase 3 RNA,Caspase- 9 RNA transcription increased obviously, Caspase- 8 RNA has no obvious changeConclusion:1. A certain concentration of PCN stimulate A549 cells in a certain time,can be directly caused damage of the A549 cells, and this damage is mainly due to the caused apoptosis of A549 cells and necrosis is not the main reason.2. When PCN stimulation of A549 cells,the apoptosis is caused by Caspase- 9protein pathways(mitochondrial pathway) cause, which lead to increase in the number of activated Caspase 3 protein, thus resulting in cell apoptosis.