Effects Of Mitochondrial Damage Induced By Cigarette Smoke Extract On The Outcome Of RLE-6TN Cells

Background Chronic obstructive pulmonary disease(COPD)is a common and frequently occurring respiratory disease that develops progressively and seriously endangers human health.The GOLD 2020 defines COPD is a common,preventable and treatable disease that is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases and influenced by host factors including abnormal lung development.WHO reported that,with the increasing prevalence of smoking in developing countries,and aging populations in high-income countries,the prevalence of COPD is expected to rise over the next 40 years and by 2060 there may be over 5.4 million deaths annually from COPD and related conditions.COPD is highly prevalent among Chinese adults and is the third leading cause of death.The potential mechanism of mitochondrial damage in COPD has been increasingly recognized as an important area of research.The dynamic balance of mitochondrial function and quantity plays an important role in cell biosynthesis,alveolar epithelial homeostasis and tissue regeneration after lung injury.Mitochondrial integrity is regulated by complex regulatory mechanisms,including mitochondrial biosynthesis,fusion and fission,and autophagic degradation processes.Mitophagy plays an important role in the quality control of mitochondria.Mitochondrial quality control is one of the key factors that determine the outcome of cells,is closely related to the outcome of cells such as aging,apoptosis and necrosis,but its underlying mechanism is still largely unknown.Objectives In this study,RLE-6TN cell line was selected as the research material,focusing on the influence of cigarette smoke extract(CSE)on mitochondrial structure and function,as well as its effects on the outcome of RLE-6TN cell,aming to further understand the mechanism of COPD and provide useful clues for seeking effective therapeutic targets.Methods 1.Groups:According to the culture conditions and different treatments,they were divided into the following four groups:(1)Control: DMEM treated RLE-6TN cells for 12 hours;(2)CSE: 5% CSE treated RLE-6TN cells for 12 hours;(3)CSE+SFN: 5?M SFN pretreated RLE-6TN cells for 12 hours,and 5% CSE treated for 12 hours;(4)CSE+NAC: 5% CSE and 1m M NAC treated RLE-6TN cells for 12 hours.2.Intracellular ROS detection: The fluorescence probe 2 ',7 '-dichlorofluorescein diacetate(2',7 '-dichloroacetate,DCFH-DA)was used to detect intracellular ROS production by flow cytometry.3.Mitochondrial membrane potential(MMP)detection: MMP was measured by a fluorescent,lipophilic,JC-1.4.Detection of mitochondrial morphology and distribution: Mitochondria were stained with Mitotracker Red to observe the changes of mitochondrial morphology and distribution under laser confocal microscope.5.Apoptosis assay: Annexin V-FITC apoptosis assay kit and flow cytometry were used to detect apoptosis.6.Cell cycle detection: cell cycle was detected by flow cytometry after PI dye staining.7.Caspase 9 activity detection kit was used to determine the activity of Caspase 9.8.SA-?-gal staining was used to observe cell senescence.9.The expression levels of Drp1 and p-Drp1;LC3 and p-PINK1;Caspase 9,Caspase 3,P53,Bcl2 and Bax;p-MLKL were detected by Western blotting.Results 1.CSE can improve the level of ROS in RLE-6TN cells.The results showed that CSE could induce ROS production in RLE-6TN cells(P?0.05),while SFN and NAC could significantly reduce ROS production induced by CSE in RLE-6TN cells.2.CSE can induced the decrease of mitochondrial membrane potential(MMP)in RLE-6TN cells.The flow results showed that CSE could reduce MMP in RLE-6TN cells.3.CSE can affect the morphology and distribution of mitochondria in RLE-6TN cells.Mitotracker Red was used to stain the mitochondria and observed under a laser confocal microscope.After dealt with CSE,the mitochondria of RLE-6TN cells were fragmented and showed perinuclear aggregation.Western blotting results showed that CSE did not change the protein expression of Drp1 in RLE-6TN cells,but p-Drp1 increased,indicating that CSE could induce mitochondrial division.4.Western blotting results showed that CSE could improve the ratio of LC3?/?,and the expression of p-PINK1 protein,which,combined with mitochondrial morphological changes,suggested that CSE could induce mitophagy in RLE-6TN cells.5.CSE induced apoptosis of RLE-6TN cells.Annexin V-FITC/PI double staining showed that CSE could induce apoptosis of RLE-6TN cells(P?0.05).6.CSE induced mitochondrial dependent apoptosis in RLE-6TN cells.The activity of Caspase 9 was increased significantly after CSE stimulation(P?0.05).Western blotting results showed that the protein levels of Caspase 9,Caspase 3 and P53 were significantly increased after dealt with CSE(P?0.05).Meanwhile,CSE could increase the protein level of Bax while decrease the protein level of Bcl 2.The opposite results were found after SFN and NAC treatment.7.After staining with SA-?-galactosidase,the results showed that CSE did not cause changes in SA-?-galactosidase activity in RLE-6TN cells.8.Western blotting results showed that CSE did not change the expression level of pMLKL in RLE-6TN cells.Conclusion CSE can damage the mitochondrial structure and function of RLE-6TN cells,and induce mitophagy;RLE-6TN cell apoptosis through mitochondrial pathway may be closely related to mitophagy.