Cigarette Smoke Extract Increases Mitochondrial Membrane Permeability Through Activation Of Adenine Nucleotide Translocator(ANT)in Lung Epithelial Cells

Chronic obstructive pulmonary disease(COPD)is a very common respiratory disease that seriously affects the personal health and quality of daily life of patients.Cigarette smoke,exposure to ambient air pollution,and repeated airway infections are all causative factors for COPD.Among them,cigarette smoke is one of the most important risk factors in the pathogenesis of chronic obstructive pulmonary disease.COPD is a clinical concept that involves complex pathological features and changes.It is generally believed that small airway obstruction is the most important pathological feature of COPD,and long-term airway inflammation affects the development of the disease.On the other hand,emphysema caused by destruction of the end-stage alveolar wall is also one of the pathological changes of COPD.In this study,we mainly studied the pathological changes of lung epithelial cells in the structure of the alveolar wall under the stimulation of cigarette smoke.It is generally believed that cigarette smoke can induce mitochondrial damage in alveolar epithelial cells,induce apoptosis of lung epithelial cells,and lead to COPD.However,the exact molecular mechanism for mitochondrial damage remains unknown.Mitochondrial membrane permeability is very closely related to the state of the cell itself.Mitochondria are unique organelles in eukaryotic cells and play an extremely important role in energy generation,information exchange,and material synthesis.Mitochondria are composed of outer membrane,inner membrane,inner and outer membrane gaps and mitochondrial matrix of phospholipid bilayer structure.The permeability of mitochondrial membrane is mainly affected by protein channels on the membrane.Common pore channel proteins include adenine nucleotide translocase(ANT),metalloproteinase,and phosphate carrier(phos-phatic carrier(Pi C),decoupling channel F1FO-ATP synthetase in the c-loop,voltage-dependent anion-selective channel(VDAC)on the outer membrane,and clophilin D(Cyp D)of the mitochondrial matrix These together affect the mitochondrial membrane permeability,ANT plays a major role,which regulates mitochondrial membrane potential changes and apoptosis.ANT accounts for 10% of total mitochondrial proteins and has the same family of proteins ANT1 to 4.It was originally discovered that the physiological function of ANT is mainly to catalyze ADP / ATP exchange on the inner membrane of mitochondria.Under normal physiological breathing conditions,proteins produced by ATP 4-oxidative phosphorylation in mitochondria are exported to the cytoplasm for cellular activity,while ADP 3-is imported into the mitochondrial matrix for continuous ATP synthesis.Therefore,ANT is an ADP 3-/ ATP-4 exchanger.In this rigorous exchange process,a net negative charge moves from the matrix to the cytoplasm,resulting in a difference in charge that is driven by a membrane potential across the inner mitochondrial membrane.ANT binds to its substrate with a relatively low affinity,while the high abundance of ANT can compensate for inefficient transportation.ANT also has intrinsic properties that mediate proton leakage.The difference in proton concentration between the mitochondrial membrane space and the mitochondrial matrix is called the mitochondrial membrane potential,which is the main driving force for ATP synthesis and is an important component of the normal physiological function of mitochondria.ANT-mediated proton leakage is closely related to mitochondrial membrane potential.The opening of ANT channels may cause a decrease in mitochondrial membrane potential.Apoptosis of lung epithelial cells is one of the molecular mechanisms that cause COPD.Cigarette smoke is generally believed to activate the lung epithelial cell apoptosisrelated protein family and start the apoptosis program.However,the mechanism of mitochondrial membrane permeability in the pathogenesis of COPD has not been studied.To study the effect of cigarette smoke on the permeability of mitochondrial membranes in lung epithelial cells,we treated lung epithelial cells A549 with 10% cigarette smoke extract(CSE)to simulate the stimulation of lungs by cigarette smoke,and studied the mechanism of ANT in this process.Part ? Toxicity of CSE on lung epithelial cells1.Prepare cigarette smoke extract to stimulate lung epithelial cells A549,using different concentrations of CSE(1%,5%,10%,30%,50%)and different times(0.5h,1h,2h,4h,8h)To detect mitochondrial autophagy in lung epithelial cells.As one of the indicators of mitochondrial health,mitochondrial autophagy is related to mitochondrial fusion and ATP production.The expression of Parkin protein related to mitochondrial autophagy was detected by Western Blot.The results showed that CSE induced increased mitochondrial division,decreased fusion,and fragmented mitochondria in lung epithelial cells.Moreover,CSE induces mitochondrial autophagy in lung epithelial cells in a concentration-and time-dependent manner.2.Decreased mitochondrial ATP synthesis can induce mitochondrial autophagy through the AMPK signaling pathway.In previous experiments,we found that CSE induces mitochondrial autophagy in lung epithelial cells.To further determine the cause of CSE-induced mitochondrial autophagy,we investigated ATP synthesis in lung epithelial cells stimulated by CSE.A ATP kit containing luciferase was used to detect the total ATP content of A549 cells after 10 hours of 10% CSE stimulation.The results showed that compared with the experimental group,the ATP synthesis in the 10% CSE group was significantly reduced.3.Use mitochondrial reactive oxygen fluorescent probes to label mitochondrial reactive oxygen species in lung epithelial cells,and detect changes in reactive oxygen species in lung epithelial cells A549 after 10 hours of 10% CSE stimulation.The results showed that 10% CSE can significantly increase the production of mitochondrial reactive oxygen species in lung epithelial cells.4.Use Seahorse XF24 Cell Energy Metabolometer to detect the effect of 10% CSE on the energy metabolism pattern of lung epithelial cells.After 10% CSE treatment for 6 hours,glycolysis and mitochondrial stress energy response tests were performed.The results show that after being stimulated by CSE,the cell's energy metabolism mode shifted from mitochondrial respiration mode to glycolysis mode;however,both glycolysis and mitochondrial oxidative phosphorylation ability were significantly inhibited.5.Normal membrane potential is conducive to the normal function of cells.The mitochondrial membrane potential is directly related to the permeability of the mitochondrial membrane,and the decrease in membrane potential is considered to be an antecedent phenomenon of apoptosis.The mitochondrial membrane potential was labeled with a JC-1 fluorescent probe,and changes in membrane potential of lung epithelial cells were detected after 10 hours of 10% CSE stimulation.The results show that 10% CSE can significantly reduce the mitochondrial membrane potential of lung epithelial cells.6.Lung epithelial cells were stimulated with Annexin V / PI dye for 6h after being labeled with 10% CSE,and the apoptosis of lung epithelial cells was analyzed by flow cytometry.The results show that CSE can significantly increase the proportion of lung epithelial cells.Part 2 Mechanism of CSE increasing mitochondrial membrane permeabilityIn the first part,it was found that the decrease of mitochondrial membrane potential is the core event of mitochondrial damage in pulmonary epithelial cells of chronic obstructive pulmonary disease.In order to clarify the mechanism of CSE changing membrane potential,in this part,we mainly studied Mitochondrial respiratory chain and ANT protein.1.To determine the structural integrity of the mitochondrial respiratory chain,Western Blot method was used to detect the expression of mitochondrial respiratory chain complex in lung epithelial cells after 10% CSE stimulation.The results showed that the expression of A549 mitochondrial complex III was significantly decreased.3.The function of the protein is related to its expression and modification.The expression of the respiratory chain was clarified in the previous section,but the function of the respiratory chain is not clear.To determine the effect of CSE on the mitochondrial respiratory chain of lung epithelial cells,we used the Seahorse XF24 cell energy metabolometer to detect the effect of CSE on the function of mitochondrial complexes in lung epithelial cells.The results showed that CSE inhibited the activity of mitochondrial complexes III and V of lung epithelial cells.4.ANT protein plays an important role in regulating mitochondrial membrane permeability.In order to study the mechanism of ANT protein affecting mitochondrial membrane permeability of lung epithelial cells under CSE stimulation.ANT-specific agonist Carboxyatractyloside and inhibitor ADP were used.The results show that CSE and ANT agonists act synergistically,but have opposite effects to ANT inhibitors.5.Bongkrekic acid,an ANT-specific inhibitor,was used to stimulate lung epithelial cells for 6 h with 10% CSE.Flow cytometry was used to detect the apoptosis of lung epithelial cells after CSE stimulation.The results showed that after 50?M ADP,the apoptosis in the CSE + ADP group decreased compared with the CSE group,indicating that inhibition of ANT can inhibit the apoptosis of lung epithelial cells caused by CSE.In conclusion:In summary,this study found that CSE can damage mitochondria of lung epithelial cells,induce a decrease in mitochondrial membrane potential,increase ROS,decrease ATP synthesis,and cause changes in its metabolic pattern.In the case of insufficient ATP produced by mitochondria,mitochondria of lung epithelial cells undergo autophagy to clear fragmented mitochondria.CSE increases mitochondrial membrane permeability by reducing the expression and activity of mitochondrial complex III and activating the ANT proton transport function,which leads to mitochondrial autophagy in lung epithelial cells and induces apoptosis.This study found that CSE can induce ANT proton channel function to open and cause mitochondrial damage in lung epithelial cells,which provides new basic research ideas for studying the pathogenesis of COPD.