Mitophagy In RLE-6TN Cells Injured By Cigarette Smoke Extract

ObjectivesThe aim of this study is to investigate whether Cigarette Smoke Extract(CSE)can induce mitophagy in RLE-6TN cells and discuss related mechanisms.Methods1.MTT method was used to determine the cell viability and choose the best condition to treat cells,then build oxidative stress model.2.The morphological changes of mitochondria in RLE-6TN cells were detected by transmission electron microscopy(TEM).3.Flow cytometry and Flexstation 3 were used to detect the level of Mitochondrial Membrane Potential(MMP),intracellular and mitochondria reactive oxygen species(ROS)and ATP.4.Using western blotting method and immunohistochemistry to detect the expression of LC3 B and P62.5.Using western blotting method to detect the expression of PINK1,Parkin,,Drp1 and Mfn2 in the total cellular protein and the expressions of PINK1,Parkin,Drp1 and Mfn2 in mitochondrial proteins/cytoplasmic proteins.6.The co-localization of Mito-Tracker Orange and EGFP-LC3 B plasmids to detect mitophagy in RLE-6TN cells.Results1.According to the MTT assay,we choosed 5%CSE to treat RLE-6TN cells for 12 h.2.CSE could induce the morphological changes of RLE-6TN cells: vacuolar cells increased significantly,increased phagosomes,mitochondria swelling and deformation were detected as well.3.CSE could induce mitochondrial damage in RLE-6TN cells.CSE can reduce mitochondrial membrane potential,and the level of intracellular and mitochondria ROS and intracellular ATP rise.4.CSE could induce autophagy in RLE-6TN cells.The LC3-II/ LC3-I ratio of CSE group andCCCP group were significantly higher than control group by Western blotting method.There was a statistically significant difference in the CSE group(P<0.05).The content of P62 in CSE group was decreased,but not statistically significant.Immunohistochemical staining method shows that: LC3 B and P62 mainly distributed in the cytoplasm.Compared with control group,the expression of LC3 B was significantly increased in CSE group;On the contrary,the expression of P62 in CSE group was significantly decreased.5.CSE could induce mitophagy in RLE-6TN cells.Western blotting method was used to detect total proteins.Compared with the Control group,the expression of PINK1 and Drp1 proteins in CSE group were increased(P ? 0.05),Mfn2 protein was decreased(P ? 0.05),and the expression of Parkin protein was decreased,but not statistically significant(P>0.05).Using western blotting method to detect mitochondrial proteins/cytoplasmic proteins.PINK1 and Mfn2 proteins did not express in cytoplasm but express in mitochondria.Compared with the control group,the expression of PINK1 and Mfn2 proteins in CSE group were decreased.Parkin and Drp1 proteins did not express in mitochondria but expressed in cytoplasm.Compared with the control group,Parkin and Drp1 proteins were increased in CSE group.6.The co-localization of Mito-Tracker Orange and EGFP-LC3 B was detected.Conclusion1.CSE can cause mitochondrial damage in RLE-6TN cells.2.CSE can cause autophagy in RLE-6TN cells.3.CSE can cause mitophagy in RLE-6TN cells.4.The PINK1-Parkin signaling pathway may participate in the mitophagy induced by CSE in RLE-6TN cells.