Effect Of Cigarette Smoke Extract On Mitochondrial Division Of Mouse Myoblast Cells

Objective:Chronic obstructive pulmonary disease(COPD)is a common preventable and treatable disease characterized by persistent respiratory symptoms and irreversible airflow restriction.Skeletal muscle dysfunction(SMD)is one of the most prominent extrapulmonary comorbidities of COPD,which not only affects patients’ exercise ability but also worsens their quality of life,and is an important index to predict the mortality of COPD patients.Smoking is considered to be one of the main causes of COPD.Oxidation-antioxidant imbalance and abnormal airway inflammation caused by cigarette smoke participate in and accelerate the occurrence and development of COPD lung diseases,but its role in skeletal muscle remains unclear.Mitochondrial dynamics plays an indispensable role in regulating cell energy metabolism and cell growth activities.The imbalance of mitochondrial dynamics will lead to the decrease of ATP,the increase of ROS,the improper processing of calcium,and directly lead to respiratory dysfunction and lung diseases.Previous studies have found that muscle cell mitochondrial division is enhanced and mitochondrial dynamics is unbalanced in muscle atrophy.In this study,the effects of cigarette smoke extract(CSE)on mitochondrial division of mouse myoblasts C2C12 were observed by creating a COPD muscle cell model,and the mitochondrial dynamin related protein 1(Drpl)in relation to the above changes.By detecting intracellular reactive oxygen species(ROS),intracellular mitochondrial morphological and functional changes,cell apoptosis rate,and Drp-1 expression,the potential molecular mechanism of skeletal muscle dysfunction in COPD will be explored,in order to provide more treatment and rehabilitation directions for skeletal muscle dysfunction in COPD.Methods:CSE extraction was performed 30 minutes before the experiment.Toxicity experiment was carried out with CCK-8 method to determine appropriate concentration and intervention time.C2C12 cells were divided into four groups,labeled as Control,2.5%CSE,5%CSE,and 10%CSE.All group were cultured by DMEM high-glucose medium containing 10%fetal bovine serum.When cells reached logarithmic growth stage,different concentrations CSE were used to intervene cells.Inverted fluorescence microscope and transmission electron microscope were used to observe the cell growth and mitochondrial mophology changes after CSE intervention.Cell transfection,realtime-PCR,western blot and flow cytometry were used to observe the relationship between CSE intervention and Drp1 expression,mitochondrial morphology and functional changes,ROS production and apoptosis.Results:1.When the concentration of CSE was 12.5%,all effect time are toxic to cells.When effect time was 48h,all concentration of CSE have toxicity to cells.The longer the CSE intervention time,the more the cell number decreased.The shape of cells in 5%CSE group and 10%CSE group tended to be round and the intercellular space increased.Transmission electron microscopy showed that the number of mitochondria increased in the cells stimulated by CSE,and the difference was statistically significant(P<0.05).However most of the mitochondria became shorter and presented short rod-like length,reduced mitochondrial crista,and showed vacuolar changes.Under inverted fluorescence microscope,the green fluorescence of cells in 5%CSE and 10%CSE stained with Mito-Tracker Green was significantly lower than that in control and 2.5%CSE,and the difference was statistically significant(P<0.05).In the 5%CSE and 10%CSE stained with JC-1,the red and green fluorescence increased,and the difference was statistically significant(P<0.05).Flow cytometry analysis showed that ROS production and apoptosis rate were increased in CSE intervention group compared with control group,and the difference was statistically significant(P<0.05).After CSE stimulation,both mRNA and protein levels of Drpl were increased,and the difference was statistically significant(P<0.05).2.After cell transfection with Drp1 knockdown,the cell number,mitochondrial morphology and function changes,cell ROS production and cell apoptosis were improved in the same CSE intervention condition,and the differences were statistically significant(P<0.05).Conclusion:CSE inhibited mouse myoblasts to varying degrees in a concentration and time dependent manner.The cell survival rate decreased with the extension of time.CSE resulted in abnormal mitochondrial morphology,decreased mitochondrial function,increased ros production and apoptosis.In this process,CSE is achieved through the upregulation of Drp1 expression and the imbalance of mitochondrial dynamics.