| Objective:The airway epithelial cell-line A549 was used as the research object to investigate the potential mechanism of airway epithelial cell apoptosis and abnormal accumulation in occurrence of asthma,while airway microenvironment is under the influence of airway pressure and inflammatory factors.The small GTPase Rac1,a protein that is widely distributed in most of the cells of the body,which regulates the apoptosis during airway abnormalities in asthmatic cells.So,the effect of Rac1 on apoptosis and phagocytosis will also be analyzed in this study.Finally we want to explore the potential targets for the treatment of asthma.Methods:1 Cell culture: The airway epithelial cells A549 obtained from ATCC were cultured in DMEM medium with 10% fetal bovine serum according to the standard procedure.2 Select curcumin as a stimulating factor to induce apoptosis of airway epithelial cell A549: The apoptotic status of A549 was investigated under diverse concentration conditions(30μmol / L,50μmol / L,70μmol / L)and time condition(12 hours,24 hours and 48 hours).3 Flow cytometry was used to detect cell apoptosis: The apoptotic A549 cells were stained with Annexin V and PI double staining method,and the apoptotic cells were observed by flow cytometry within 4 hours according to the instructions of the kit.4 Hoechst nuclear staining was used to observe the morphology of apoptotic cells:Hoechst nuclear staining solution was used to observe the apoptosis in apoptotic cells according to the specific standard procedure.The apoptotic condition of A549 cells was observed by fluorescence microscopy.5 A549 cell phagocytosis: A549 cells with good condition were selected and seeded in a6-well cell culture plate at 4x105 cells per well,5% CO2 and incubated overnight at 37℃.Apoptotic cells that were induced for 48 hours were collected according to the correct procedure.The treated apoptotic cells were added to the six-well culture plate twice as much as normal cells and incubated at 37℃for 90 min at room temperature for flow analysis and fluorescence quantitative PCR analysis.6 Real-time fluorescence quantitative PCR analysis: A549 cells were subjected to phagocytosis and the mRNA expression levels of IL-33 and RAC1 were determined by real-time fluorescence quantitative PCR.7 Rac1 inhibitors inhibit phagocytosis:The Rac1 inhibitor NSC23766 was added to theculture system at a concentration of 50 μmol / L and 100 μmol / Ld,respectively,24 hours prior to phagocytosis.Results:1 Curcumin can induce apoptosis in A549 cells:Curcumin were used to stimulated normal A549 cells for 24 hours,50% of cells apoptosis,of which about 20% of late apoptosis.And90% of the cells were apoptotic when the time was 48 hours.2 Rac1 inhibitors can enhance curcumin-induced apoptosis in A549 cells: Added Rac1 inhibitor in the apoptosis induction test of curcumin significantly enhanced the apoptotic effect of A549,and the effect of this enhancement was more pronounced with the increase of inhibitor concentration,indicating its dose-dependent effect.3 Airway epithelial cells A549 can engulf apoptotic cells: The results of flow cytometry analysis after phagocytosis showed that normal A549 cells could engulf apoptotic A549 cells,thus demonstrating the phagocytosis of A549 cells.4 The level of IL-33 expression was increased during phagocytosis: After the phagocytosis,the real-time fluorescence quantification analysis showed that IL-33 mRNA level was significantly increased,but the expression of Rac1 did not change significantly.5 Rac1 inhibitors can inhibit the phagocytosis of A549 cells.Conclusion:This study shows that curcumin can effectively induce the apoptosis of A549 cells,and the role of Rac1 inhibitor shows that Rac1 plays a significant negative regulatory role in the process of apoptosis.At the same time,A549 cells have the ability to swallow the same apoptotic cells,and Rac1 played a positive regulatory role in its phagocytosis. |
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