SIRT1Regulates The Human Alveolar Epithelial A549Cell Apoptosis Induced By Pseudomonas Aeruginosa Lipopolysaccharide

Background&Aims:Pseudomonas aeruginosa, an increasingly prevalent opportunistic and virulent pathogen, is the most common Gram-negative bacterium and produces many virulence factors, including exotoxins and enzymes (for example LPS, etc), involved in infectious diseases when the immunity of the host is low. Lipopolysaccharide (LPS) is a key factor in the virulence produced by P. aeruginosa and plays an important role in mediating the interactions between the bacterium and its host. Studies have showed that LPS can induce cells apoptosis and increase the generations of the intracellular reactive oxygen species. Literatures reported that ROS were one of crucial apoptotic factors that could cause oxidative stress and subsequent cell apoptosis. Sirtuinl (SIRTl) is a multi-functional transcriptional regulation factor which is an NAD+-dependent class III protein deacetylase and belongs to the silent information regulator (Sir2) family. SIRT1plays important roles in regulating cell apoptosis or cell survival, endocrine signaling, cell differentiation, metabolism, caloric restriction (CR) and chromatin remodeling. SIRT1is considered a type of apoptosis inhibitor through deacetylating p53to inhibit apoptosis. In the present study, the apoptosis models induced by Pseudomonas aeruginosa LPS was established. Our study aims to indentify the role of SIRT1in regulating the human alveolar epithelial A549cell apoptosis induced by LPS and the role of ROS during this process.Methods:1. The cell viability was examined in A549cells that were treated with LPS at different concentrations and different times by MTT method.2. The levels of SIRT1protein were examined in A549cells that were treated with LPS at different concentrations and different times by Western Blot. The expressions of SIRT1protein in A549cells were detected by immunofluorescence.3. The apoptosis models induced by Pseudomonas aeruginosa LPS was established. According to the result of threshold experiment, the concentration of LPS is10μg/ml and the treatment hour of LPS is48hour. The proportion of apoptotic A549cells was measured using Hoechst33258staining and an Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s protocol. Intracellular ROS levels were quantified to determine the oxidative stress to the A549cells in response to LPS stimulation using flow cytometry. Meanwhile, the protein levels of SIRT1、p53、acetylation of p53、Bax and Bcl2were further explored by western blot analysis.4. A549cells were pretreated with20μM Res for1h before LPS treatment. The rate of apoptotic A549cells was measured using Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s protocol. Intracellular ROS levels were quantified using flow cytometry. The protein levels of SIRT1、p53、acetylation of p53、Bax and Bcl2were further explored by western blot analysis.5. A549cells were pretreated with5mM NAM for1h before LPS treatment. The rate of apoptotic A549cells was measured using Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s protocol. Intracellular ROS levels were quantified using flow cytometry. The protein levels of SIRT1、p53、 acetylation of p53^Bax and Bcl2were further explored by western blot analysis.6. A549cells were pretreated with5mM NAC for1h before LPS treatment. The rate of apoptotic A549cells was measured using Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s protocol. Intracellular ROS levels were quantified using flow cytometry. The protein levels of SIRT1、p53、acetylation of p53、 Bax and Bcl2were further explored by western blot analysis.Results:1. A549cell viability was decreased compared with the control group in a dose-and time-dependent manner. A549cell viability was decreased significantly when the cells were treated with10μg/ml LPS for48h.2. In our study, we detected SIRT1in the nuclei of A549cells using immunofluorescence. Western blot analysis showed that LPS treatment significantly reduced the level of SIRT1expression in a dose-dependent manner. The protein levels of SIRT1expression increased at6、12h (but there is no statistical significance) and decreased at24、48h. There is obvious statistical significance at48h.3. According to the result of threshold experiment, the concentration of LPS is10μg/ml and the treatment hour of LPS is48hour. The results show that the LPS induced A549cell apoptosis. Moreover, we found that LPS elevated ROS generation in the A549cells in a time-dependent manner. And the Western blot showed that LPS decreased SIRT1protein expression and stimulated p53protein expression and p53protein acetylation.4. To further determine the role of SIRT1in regulating the A549cell apoptosis induced by LPS, we choose the Res as the activator of SIRT1to pretreatment cells. The cells were divided into four groups, including a DMSO group (control), a Res group, a LPS treating plus DMSO group (DMSO+LPS), and a LPS-supplemented Res group (Res+LPS). A549cell viability was examined when the cells were treated with Res (20μM). The results suggested that exposing A549cells to Res did not affect cell viability. Our findings demonstrate that Res induces SIRT1protein overexpression in A549cells. The A549cell apoptosis induced by LPS was alleviated by Res and that ROS generation was decreased by Res in the A549cells. The effects of Res on the activation of the SIRT1pathways were rescued by mediating p53deacetylation in the A549cells.5.To further determine the role of SIRT1in regulating the human alveolar epithelial A549cell apoptosis induced by LPS, we chose NAM as an inhibitor of SIRT1. Our findings demonstrate that SIRT1protein expression in the A549cells is decreased by NAM in a dose-dependent manner. A549cell apoptosis is aggravated by NAM while ROS generation is increased. The effects of LPS on inhibiting SIRT1pathways were aggravated by NAM in the A549cells.6. To determine the role of ROS during the A549cell apoptosis induced by LPS, NAC was chose as an antioxidant. The data indicate that ROS generation was decreased in the A549cells and that cell apoptosis was alleviated by NAC. In addition, western blotting demonstrated that NAC restored SIRT1expression during LPS treatment.Conclusions:1. Our findings suggested that A549cells apoptosis was induced by LPS. The generation of intracellular ROS was increased by LPS.SIRT1inhibition by LPS resulted in LPS-induced A549cells apoptosis through regulation of p53.2. Resveratrol attenuates LPS-induced A549cell apoptosis through SIRT1-mediated deacetylation of p53.3. However NAM aggravates the A549cell apoptosis induced by LPS through SIRT1-mediated regulation of p53.4. Furthermore, pre-treating the A549cells with NAC decreased ROS generation, alleviated the LPS-induced A549 cell apoptosis, activated the SIRT1and inhibited p53.5. SIRT1plays important role in regulating the human alveolar epithelial A549cell apoptosis induced by Pseudomonas aeruginosa LPS. SIRT1inhibits the A549cells apoptosis through deacetylation of p53. LPS can induce the A549cells apoptosis through inhibiting SIRT1.