The Role Of Mitochondrial Fusion Changes In PC12 Cells Damage Induced By PBDE-47

Objective:The developmental neurotoxicity of polybrominated diphenyl ethers（PBDEs）has received extensive attention in the world.However,its toxic mechanism has not yet been clarified.A large amount of studies have suggested that the neurotoxic mechanisms of PBDEs are closely related to mitochondrial damage.Our recent studies showed that 2,2’,4,4’-tetrabromodiphenyl ether（PBDE-47）decreased the expression levels of regulatory proteins of mitochondrial fusion and fission in PC12 cells,accompanied by mitochondrial membrane potential（MMP）and adenosine triphosphate（ATP）reduction,cell apoptosis increase and cell survival decrease.However,it is unclear that the role of the mitochondrial fusion and fission inhibitions plays in this toxic process.This study aimed to clarify the role of mitochondrial fusion changes in PC12 cells damage induced by PBDE-47,and to provide theoretical basis for further elucidating the neurotoxic mechanisms of PBDEs.Methods:Two different approaches were used to promote mitochondrial fusion level in PC12 cells,including:（1）mitochondrial fusion promoter M1 treatment.PC12cells were divided into control group,20μmol/L PBDE-47 treatment group,20μmol/L PBDE-47+5μmol/L M1 combined treatment group and 5μmol/L M1 treatment group.（2）adenovirus-mediated mitofusion 2（Mfn2）overexpression（Ad-Mfn2）.PC12 cells were divided into control adenovirus（Ad-Null）treatment group,PBDE-47+Ad-Null treatment group,PBDE-47+Ad-Mfn2 treatment group and Ad-Mfn2 treatment group.Western blotting was used to determine the expression levels of mitofusion 1（Mfn1）,mitofusion 2（Mfn2）,fission 1（Fis1）and phosphorylation dynamin related protein 1（Serin616）(^ser616p-Drp1)proteins.Laser confocal microscopy was used to detect the morphological alteration of mitochondria stained by MitoTracker?Deep Red FM.Flow cytometry or fluorescence microscope was used to measure the fluorescence intensity of JC-1 to evaluate the changes in MMP.The ATP detection kit was used to measure the ATP level in PC12 cells.Western blotting or immunofluorescence was used to determine the expression level or intracellular distribution of active cysteine containing aspartate specific protease-3（active Caspase-3）protein.Cell Counting Kit-8（CCK-8）was used to measure the survival rate of PC12 cells.Results:（1）Compared with the control group,the expression levels of Mfn1,Mfn2and ^ser616p-Drp1 proteins in the PBDE-47 treatment groups were significantly decreased（P<0.05）.Compared to the PBDE-47 treatment groups,the expression levels of Mfn1,Mfn2 and ^ser616p-Drp1 proteins in the M1+PBDE-47 or Ad-Mfn2+PBDE-47combined treatment groups were significantly increased（P<0.05）.（2）Confocal results showed that the mitochondria were mainly filamentous and interconnected into a network in the control group.Compared with the control group,the PBDE-47 treatment groups demonstrated more fragmented,punctate and spherical mitochondria.Compared to the PBDE-47 treatment groups,the number of abnormal mitochondrial in the M1+PBDE-47 or Ad-Mfn2+PBDE-47 combined treatment group was significantly reduced.（3）Compared with the control group,the MMP and ATP levels of the PBDE-47treatment groups were significantly decreased（P<0.05）.Compared to the PBDE-47treatment groups,the MMP and ATP levels of the M1+PBDE-47 or Ad-Mfn2+PBDE-47 combined treatment group were significantly increased（P<0.05）.（4）Compared with the control group,the expression level of active Caspase-3 protein in the PBDE-47 treatment groups was significantly increased（P<0.05）.Compared to the PBDE-47 treatment groups,the expression level of active Caspase-3 protein in the M1+PBDE-47 or Ad-Mfn2+PBDE-47 combined treatment groups was significantly decreased（P<0.05）.Immunofluorescence assay showed that the staining of active Caspase-3 in each treatment group was consistent with the expression of active Caspase-3.（5）Compared with the control group,the survival rate of PC12 cells in the PBDE-47 treatment groups was significantly decreased（P<0.05）.Compared to the PBDE-47 treatment groups,the survival rate of PC12 cells in the M1+PBDE-47 or Ad-Mfn2+PBDE-47 combined treatment group was significantly increased（P<0.05）.Conclusions:Promoting mitochondrial fusion attenuates PBDE-47-induced mitochondrial morphological and functional abnormalities,alleviates apoptosis,and facilitates survival in PC12 cells.