| ObjectiveThis study is to investigate the effect of icaritin on cigarette smoke-mediated oxidative stress in human lung epithelial cells via activation of PI3K-Akt and Nrf2signalingMethod(1)After stimulated the A549cells with different concentration of CSE(2.5%,5%,10%) for24hours, observe the cellular morphology and test the cell viability by CCK-8in order to choose a appropriate concentration of CSE,(2)After pretreated the A549cells with different concentration of ICT(1u M,10μ M,100μ M) for24hours, observe the cellular morphology and test the cell viability by CCK-8in order to choose a appropriate concentration of ICT,(3)Pretreate the A549cells with ICT(10μM)for1hours and then stimulate the cells with CSE(5%,10%),24hours later, observe the cellular morphology and test the cell viability by CCK-8, compared with stimulated by CSE only,(4)Cultured A549cells are divided into6groups:A:normal control groupB:model group stimulate by CSEC:pretreated by ICT groupD:pretreated by ICT for1hour then stimulated by CSE groupE:pretreated by Akt inhibitor for1hour, pretreated by ICT for1hour and then stimulated by CSE groupF:after the transfection of Nrf2siRNA for48hours, pretreated by ICT for1hour then stimulated by CSE group(5)Test index as follows1)Observe the cellular morphology of every group under the invert microscope.2)Test the ROS of every group cells by flow cytometry assay,3)Test the GSH of every group cells by ELIASA reader. 4)Test the protein of Nrf2and P-Akt of every group by western blot assays5)Test the GCLM mRNA of every group by real-time PCRResults(1)CSE exposure reduced cell viability of A549cells in a dose-dependent manner,(2)Low concentration ICTcan increase cell viability,but high concentration ICT didn’t affect cell viability.(3) ICT attenuates CSE-induced cytotoxicity in A549Cells(4) ICT exerts antioxidant properties by scavenging CSE-induced ROS(5) ICT restores CSE-depleted GSH levels in A549cells.(6) ICT induced the expression of GCLM, nuclear translocation of Nrf2and activated Akt in CSE-treated A549cells(7) SiRNA-mediated knockdown of Nrf2resulted in downregulation of GCLM mRNA expression.(8) LY294002partially reverses ICT-induced nuclear translocation of Nrf2, GCLM gene transcription, and ROS scavenging in CSE-treated A549cells.Conclusion1.CSE can lead to cell injury and death by oxidative stress2. ICT attenuates CSE-induced cytotoxicity.3. Icaritin attenuates cigarette smoke-mediated oxidative stress in human lung epithelial cells via activation of PI3K-AKT and Nrf2signaling... |
