Role Of Autophagy In PBDE-47-induced Mitochondrial Damage In PC12 Cells

Objective: In recent years,the neurotoxicity of brominated flame retardants(Polybromodiphenyl ethers,PBDEs)has been paid more and more attention,but the exact mechanism of PBDEs neurotoxicity is still not very clear.The aim of this study was to investigate the effect of PBDE-47(one of PBDE congeners)on autophagy and mitochondrial function in adrenal medullary rat pheochromocytoma cell(PC12 cells),as well as the possible role of autophagy in mitochondrial damage,and to provide a basis for further elucidating the neurotoxicity mechanism of PBDE-47.Methods: After treated with different concentrations of PBDE-47(1~40 μmol/L)for 24 h,the viability of PC12 cells was determined by CCK-8 assay,and the result was the basis of the setting of the dose of the follow-up experiment.After treated with different concentrations of PBDE-47(1 μmol/L,10 μmol/L and 20 μmol/L)for 24 hours.the autophagosomes and mitochondrial structures were observed by transmission electron microscopy;the ATP detection kit was used to determin the effect of PBDE-47 on ATP levels in PC12 cells;the MMP(mitochondrial membrane potential)of PC12 cells was detected by flow cytometry after JC-1 staining;the expression levels of autohagy relevant proteins Atg7(autophagy related gene 7),LC3(microtubule-associated protein 1 light chain 3),P62,and the expression level of p-AMPK(phosphorylated AMP-activated protein kinase),p-ULK1(phosphorylated Unc-like kinase1),in the AMPK-ULK1 upstream autophagy pathway,as well as the expression levels of mitohagy relevant proteins PINK1(PTEN induced putative kinase 1),Parkin were detected by Western blottingting.The expression level of autophagy protein LC3 II was measured by Western blottingting after treated with 20 μmol/L PBDE-47 coupled with inhibitor chloroquine(CQ).Results: Compared with the control PBDE-47 group,CCK8 results showed a significant dose-response relationship from the 5 μmol/L PBDE-47 group(P < 0.05),and cell viability was less than 70% from 30 μmol/L PBDE-47 group.So,1~20 μmol/L concentrations were chosed for follow-up experiments.The results of electron microscopy showed that there was no obviously abnormal change in the cells of the control group;1 μmol/L PBDE-47 group was similar to that of the control group,but the swollen mitochondria and a large amount of autophagosomes could be found in the 10 μmol/L and the 20 μmol/L PBDE-47 group has more.Compared with the control group,the levels of ATP and MMP were evidently decreased in 10 μmol/L and 20 μmol/L PBDE-47 group(P < 0.05);the protein expression levels of Atg7,LC3 II and P62 was dramatically increased(P < 0.05).After CQ treatment,compared with CQ group,the expression of LC3 II was significantly increased in CQ and PBDE-47 group(P < 0.05).Compared with the control group,in 10 μmol/L and 20 μmol/L PBDE-47 groups,the expression levels of p-AMPK and p-ULK1 protein were significantly increased(P < 0.05);the expression of PINK1 and Parkin protein was remarkably lower(P < 0.05).Conclusions: A certain of PBDE-47 can induce autophagy of PC12 cells through AMPK-ULK1 pathway and promote mitochondrial damage by down-regulating the expression of PINK1/Parkin to interfere with mitochondrial clearance by autophagy pathway.