Effect Of ROS/JNK Pathway On A549 Cells Apoptosis Induced By Inhalable Chrysotile Fibers

Objective:(1)To observe the effects of inhalable chrysotile fibers on the proliferation inhibition and apoptosis of A549 cells.(2)To observe the effect of active oxygen(ROS)and c-Jun amino terminal kinase(JNK)on the apoptosis of A549 cells stimulated by inhalable chrysotile fibers.(3)To observe the interaction between ROS and JNK signaling pathway in the apoptosis of A549 cells stimulated by inhalable chrysotile fibers,and to further explore the specific mechanism of apoptosis induced by inhalable chrysotile fibers.Methods:(1)The stimulated time and concentration of inhalable chrysotile fibers were selected by experiment:A549 cells were used as experimental subjects in vitro,different concentrations of chrysotile(12.5,25,50,100,200 μg/mL)were used to stimulated A549 cells at different time(12,24,48 h),while negative control group was set up at the same time,MTT method was used to evaluated cell viability.(2)Observation of morphological changes of A549 cells:Different concentrations of chrysotile(50,100,200 μg/mL)were used to treat A549 cells 24 h,while negative control group was set up.Hoechst 33258 reagent staining was used to observe and record cell morphological changes under fluorescence microscope.(3)To explore theeffect of ROS and JNK pathway on the apoptosis of A549 cells and the relationship between them:Different concentrations of chrysotile(50,100,200 μg/mL)were used to stimulated A549 cells 24 h,and set up negative control group,JNK inhibition group(SP600125 intervention)and ROS inhibition group(NAC intervention).Annexin V-FITC /PI double staining was performed to analysis cell apoptosis;JC-1 fluorescence probe was used to measure the decrease of mitochondrial membrane potential in each group.The distribution of cell cycle in each group was detected by PI staining.DCFH-DA fluorescence probe was used to detect the production level of ROS in each group.Western blot was used to detect and analysis the changes in the expression of p-JNK protein,JNK protein and cleaved caspase-3 protein.Results:(1)Inhalable chrysotile fibers inhibits the proliferation of A549 cells,and presents a dose and time effect relationship:When chrysotile stimulated A549 cells for 12 h,the cell survival rate of chrysotile group decreased(P< 0.05).Stimulated by chrysotile for 48 h,the cell survival rate decreased significantly(P <0.01),the cell survival rate of the 100 and 200 μg/mL chrysotile group was the most obvious.And when A549 cells were stimulated by chrysotile for 24 h,the survival rates of the 50,100 and 200 μg/mL chrysotile groups survival rates were(74.86 + 6.95)%,(70.04 + 6.52)%,(60.78 +3.97)%.In order to observe the experimental results,inhalable chrysotile fiber with concentration of 50,100 and 200 μg/mL were selected for thefollow-up experiments of A549 cells 24 h.(2)Compared with the negative control group:Stimulated by different concentrations of chrysotile,the overall fluorescence intensity of the cells increased.There were many bright blue apoptotic cells that were densely stained.The cell morphology was narrowed and round,and some of them showed vacuolar changes.Some of the apoptotic bodies with more fragmentation and the shape of the crescent(horseshoe)cell nucleus were observed.(3)The apoptosis rate of 50,100 and 200 μg/mL chrysotile group were(23.05±3.44)%,(31.67±1.55)% and(64.04±2.78)%.At the same time,the level of ROS increased,the mitochondrial membrane potential decreased,the expression of p-JNK protein and cleaved caspase-3 protein increased,and the cell cycle stagnated in the G2/M phase.(4)After inhibiting the JNK pathway,the expression of p-JNK protein decreased(P <0.05).Apoptosis rate of JNK inhibition group with chrysotile concentration of 50,100,200 μg/mL were(16.28±1.83)%、(25.20±3.11)%、(52.82±4.57)%.At the same time,the decrease of mitochondrial membrane potential and the increase of cleaved caspase-3 protein expression were inhibited(P < 0.05).(5)When the concentration of chrysotile were 50 or 100 μg/mL,the inhibition of JNK pathway had no significant effect on cell cycle(P > 0.05).When the concentration of chrysotile was 200 μg/mL,the inhibition of JNK pathway could reduce G2/M phase arrest(P < 0.05).(6)When chrysotile concentration was 50 and 100 g/mL,inhibition of JNK pathway could not inhibit ROS production(P > 0.05).When the concentration of chrysotile group was200 g/mL,inhibition of JNK pathway could reduce ROS production(P <0.05).(7)After inhibiting the production of ROS,ROS production levels drop(P < 0.05).Apoptosis rate of ROS inhibition group with chrysotile concentration of 50,100,200 μg/mL were(16.28±1.83)% 、(25.20±3.11)%、(52.82±4.57)%.At the same time,the decrease of mitochondrial membrane potential and the increase of cleaved caspase-3protein expression were inhibited(P < 0.05).Conclusion:(1)Inhalable chrysotile fibers induced A549 cells apoptosis.(2)The inhalable chrysotile fibers induced A549 cells apoptosis through ROS/JNK pathway.(3)In the process of inducing apoptosis of A549 cells induced by inhalable chrysotile fibers,the inhibition of the JNK pathway in a certain concentration range can reduce the production of ROS.