The Mechanisms Of BEAS-2B Cells And Mitochondria Damage Induced By Cigarette Smoke Condensate

Objective.A great many studies have shown that cigarette smoke can cause diseases in the organism,and the disease state is often related to the damage of mitochondria.The mitochondria plays an important role in mediating apoptosis.In the process of apoptosis,the extent of mitochondrial permeability transition pore is opened or closed abnormally,which give rise to the decrease of mitochondrial membrane potential,changes the expression level of protein related apoptosis and the oxidation and antioxidation level of cells.It is of great significance to further study the regulation mechanism of apoptosis by studying the specific mechanism of mitochondria in mediating apoptosis.This paper aims to explore the mechanisms of mitochondrial damage caused by cigarette smoke condensate in immortalized human bronchial epithelial cells(BEAS-2B),and provides a scientific basis for further study of the toxic mechanisms of CSC and the prevention diseases related to cigarette smoke.Methods1.BEAS-2B cells were exposed to CSC for 24 hours when the cells were fused to about 70%,the cells after exposure were the cells used in the experiments.2.Set up the solvent control groups,blank control groups,CSC exposure groups at the same time.Using the technology of morphological observation,scratch test,flow cytometric analysis,ELISA and CCK-8 to detect the cell apoptosis rate,morphological changes and functional changes of the cells.3.The technology of ELISA,RT-PCR and Western blot were used to detect the mitochondrial membrane potential and the expression of related proteins and genes.Resuits 1.The effects of CSC exposure on morphology and function of BEAS-2B cells.After CSC exposure,the activity of BEAS-2B cells was inhibited,and the survival rate of BEAS-2B cells decreased with the increase of CSC concentration.Compared with the blank control group BEAS-2B cells,the solvent control group cells had no obvious change in cellular morphology;there was no obvious change in cells and nuclear morphology of 0.02mg/mL CSC group cells,but a small number of intracellular vacuoles were observed,cell swelling,nucleolus deformation,nuclear anomalies,nucleolus increased were observed from the 0.04 mg/mL CSC group;the deformation of cell membrane and the needle like protrusions between cell membranes were observed in the 0.06 mg/mL CSC group cells,there were large vacuoles in the cytoplasm;cells and nuclei were significantly distorted,the cell membrane and cell nucleus were vague,and there were many characteristics such as nuclear fragmentation and multiple nucleoli,cell structure disintegration and cell area increased in the 0.08 mg/mL CSC group cells.Compared the concentration of CSC group with the solvent control group,the differences of migration distance of the cells were statistically significant(P<0.05).And there was no significant difference between the DMSO group and the normal BEAS-2B cell migration distance(P>0.05).2.Detection of ROS in each group after exposure.With the increase of the concentration of CSC,the fluorescence intensity of ROS in the cells was also enhanced by laser confocal microscopy.The results showed that the level of ROS in the DMSO group and the CSC exposure group were different,the difference was statistically significant(F=143.390,P ? 0.05).There was no significant difference between the DMSO group and the 0 groups(blank control group)(P>0.05).The difference of ROS levels between the groups with different concentrations of CSC was statistically significant(P < 0.05)3.SOD activity detectionCompared with DMSO group,the level of SOD activity in different groups was statistically significant(F=195.680,P < 0.05);Compared with DMSO group,the CSC group had significant difference(P < 0.05).With the increase of the concentration of CSC,the activity of SOD decreased gradually.4.Detection of apoptosis rateThe apoptosis rate was detected by flow cytometry with DMSO group as control.The results showed that compared with the control group,the apoptosis rate of DMSO group had no significant difference(P>0.05).The apoptosis rate of CSC group was higher than that of DMSO group(P < 0.05),and the apoptosis rate increased with the increase of CSC concentration.5.Detection of mitochondrial membrane potentialIt was observed that cells of red and green fluorescence rates were decreased significantly with CSC exposure concentration increased,suggesting that the decrease of mitochondrial membrane potential was caused by CSC.And the extent of membrane potential decreased with the increase of CSC concentration.6.Detection of mRNA relative level of Bcl-2,Bax,Cyt-C and caspase-3 gene in each group of cells.RT-qPCR detection results showed that the mRNA expression of the 0 group(blank control group)was no significant difference compared with the DMSO group(P>0.05).The mRNA expression of Bcl-2,Bax,Cyt-C and caspase-3 gene in different CSC concentration groups were significantly different from those in DMSO group(P < 0.05).Bcl-2 gene: the expression of the 0.02 group,the 0.04 group,the 0.06 group,and the 0.08 group had less expression than the DMSO group,the difference was statistically significant(P < 0.05),and while the increase of CSC concentration,the expression of Bcl-2 was decreased gradually.Bax,Cyt-C and caspase-3 gene: the expression of CSC group was higher than that of solvent control group,the difference was statistically significant(P < 0.05),and while the increase of CSC concentration,the expression of Bax,Cyt-C and caspase-3 increased gradually.7.Detection of Bcl-2,Bax and caspase-3 protein relative expression.The Bcl-2 protein relative expression in DMSO group compared with the control group,there was no significant difference(P>0.05),the expression of Bcl-2 protein in CSC group was lower than that in solvent control group,while the expression had significant difference(P <0.05),there was no significant difference in the expression of Bax and Caspase-3 in blank control group compared with DMSO group(P>0.05).The expression levels of Bax and actived caspase-3 in CSC group were higher significantly than those in solvent control group cells,and the difference was statistically significant(P < 0.05).Conclusion.CSC exposure induces oxidative damage in BEAS-2B cells,results in morphology and cell migration ability changes,leads to the imbalance of intracellular oxidation and antioxidation,membrane permeability increases under oxidative stress,results in up regulation of Bax expression and down regulation of Bcl-2 expression,brings about the release of cytochrome C and activation of Caspase-3,and ultimately lead to a vicious cycle of mitochondrial mediated apoptosis pathway.