Effect Of Airborne Fine Particulate Matters (PM_2.5) On Energy Metabolism Of SH-SY5Y Cells

Objective:The objectives of this study were to:?1?study the effect of Airborne Fine Particulate Matters(PM_2.5)on the energy metabolism of human neuroblastoma cells?SH-SY5Y?,?2?analyze their relationship with cell survival rate,oxidative stress levels,and apoptosis which may provide the basis for the understanding of PM_2.5.5 neurotoxicity.Methods:Cell culture and exposure:SH-SY5Y cells were routinely cultured as previous reports.When the cell confluence was approximately 80%,0.25%trypsin digestion was performed and the cells were inoculated.To study the dose-response relationship,SH-SY5Y cells were exposed to final concentrations of 0,20,80,and 320?g/m L PM_2.5for 24 hours.The time-effect of PM_2.5.5 and 3-tert-Butyl-4-hydroxyanisole?BHA?intervention effect on SH-SY5Y cells were studied with different dose of PM_2.5 and BHA treatments at three exposure duration?24,48,and 72h?.The control group and PM_2.5group were treated with 0 and 80?g/mL PM_2.5,and BHA intervention group was treated by 80?g/m L PM_2.5 and co-cultured with 20?mol/L BHA.The recovery of SH-SY5Y cells after PM_2.5.5 exposure at different times were further observed by culture the cell with additional 24 hours under fresh medium.Energy Metabolism Test:The Oxygen Consumption Rate?OCR?which reflects the mitochondrial respiratory function,and Extracellular Acidification Rate?ECAR?which is an indicator of cell glycolysis function,were analyzed by XFp Extracellular Flux Analyzer?Agilent Technologies Company?.Cell injury measurements:SH-SY5Y cell viability was measured by MTT assay.MDA content in cells was detected by TBA assay,and cell T-SOD activity,CuZn-SOD activity,and Mn-SOD activity were measured by xanthine oxidase assay.ROS levels,mitochondrial membrane potential changes and apoptosis levels were tested by Flow Cytometry?Beckman Coulter Company?.Results:The effects of PM_2.5.5 on Cellular Energy Metabolism:SH-SY5Y Cell Basal Respiration,ATP Production,Proton Leak,Maximum Respiratory,Spare Respiratory Capacity,Glycolysis and Glycolytic Capacity were showed a decreasing trend as the dosage increases?Ptrend<0.05?;the Basal Respiration and ATP Production were significantly different in each treated group compared with the control group?P<0.05?;Maximum Respiratory,Spare Respiratory Capacity,Glycolysis and Glycolytic Capacity were showed a significant differences between the control group and 80,320?g/m L PM_2.5 groups?P<0.05?.In 80?g/m L PM_2.5 group,Basal Respiration,ATP production,Proton Leak and Maximum Respiratory were gradually decreased with increasing exposure time;The difference of Basal Respiration,Maximum Respiratory and Glycolysis were statistically significant between the 48h and 24 exposure groups?P<0.05?.After 72 hours of exposure,the Basal Respiration,ATP production,Proton Leak,and Maximum Respiratory of cells were significantly different with the 24h exposure group?P<0.05?.The Spare Respiratory Capacity of the recovery group decreased significantly after 72h exposure compared with the corresponding concentration of PM_2.5 group?P<0.05?.Cytotoxicity of PM_2.5 on SH-SY5Y cell:With the increase of PM_2.5 exposure dose,SH-SY5Y cell survival rate,T-SOD activity,CuZn-SOD activity and Mn-SOD activity showed a downward trend?Ptrend<0.01?;MDA content,the ROS content,rate of decrease of mitochondrial membrane potential,early apoptosis rate and total apoptosis rate of cells showed an upward trend?Ptrend<0.01?;cell survival rate,T-SOD activity,Mn-SOD activity,ROS content in each dose groups,the mitochondrial membrane potential decrease rate,the early apoptosis rate and the total apoptosis rate of the cells were significantly different with the control group?P<0.05?.In addition,the MDA content and the CuZn-SOD activity of the 80,320?g/m L PM_2.5 group were significantly different with the control group?P<0.05?.In the 80?g/m L PM_2.5-treated group,the survival rate and Mn-SOD activity of SH-SY5Y cells gradually decreased with the increase of exposure time;the content of MDA,ROS,mitochondrial membrane potential,early apoptotic rate and total apoptotic rate gradually increased with the increase of exposure time.Moreover,the mortality rate increased gradually,and the difference was statistically significant compared with the 24h group?P<0.05?.The 24h-treated group showed the reduced viability of cells and the increased rate of mitochondrial membrane potential.Compared with the corresponding concentration of PM_2.5 group,the difference was statistically significant?P<0.05?.Increased MDA was detected in the cells of 48 h recovery group,and the decreased rate of the content and mitochondrial membrane potential were also observed comparing with the corresponding concentration of PM_2.5group.These difference were statistically significant?P<0.05?.The effect of Antioxidant BHA intervention:The significant increasing of the Basal Respiration,ATP production capacity,Maximum Respiratory and Spare Respiratory Capacity?P<0.05?were detected in BHA intervention group after 24 hour treatment;in BHA intervention 48h group,the significantly increasing of ATP production capacity,Proton Leak and Spare Respiratory Capacity?P<0.05?were showed.At the same time,BHA intervention significantly increased cell viability,T-SOD activity,Cu Zn-SOD activity and Mn-SOD activity,but decreased MDA content and ROS content?P<0.05?;BHA intervention in all time groups showed a significant reduction of the rate of mitochondrial membrane potential,early apoptosis as well as the total apoptosis?P<0.05?.Relationships between energy metabolism indicators and cell injury indicators:The Basal Respiration,ATP Production capacity,Proton Leak levels,Maximum Respiratory,Spare Respiratory Capacity,Glycolysis levels and Glycolytic Capacity of SH-SY5Y cells were positively correlated with cell viability;T-SOD activity,CuZn-SOD activity,and Mn-SOD activity;but negatively correlated with MDA value,ROS content,rate of decrease of mitochondrial membrane potential,early apoptosis rate of cells and total apoptosis rate.These association were all statistically significant?P<0.05?.Conclusion:PM_2.5 can induce the impairment of mitochondrial respiratory function of SH-SY5Y cells in a dose and time-dependent manner.Middle to high doses of PM_2.5 can cause a decrease in mitochondrial respiratory reserve and glycolytic function;energy damage persists after 24h exposure;PM_2.5 can also cause cell oxidative damage,decreased antioxidant capacity and apoptosis in a dose and time-dependent manner;antioxidant BHA can reverse PM_2.5 injury to mitochondrial respiratory function after 24h intervention.The disorder of PM_2.5-induced energy metabolism in SH-SY5Y cells may be related to oxidative damage.The disorders of energy metabolism may eventually lead to apoptosis.