Roles Of Apoptosis And Autophagy Induced By ERS In PBDE-47-induced Neurotoxicity

Part 1: Roles of apoptosis and autophagy impairment induced by ERS in developmental neurotoxicity of PBDE-47 in ratsObjective: 2,2’,4,4’-tetrabromodiphenylether(PBDE-47),one of the more toxic homologues of polybrominated diphenyl ethers(PBDEs),has been confirmed as a developmental neurotoxicant,but its toxicity mechanism has not been completely elucidated.This study was to explore the roles of apoptosis and autophagy induced by endoplasmic reticulum stress in hippocampal damage caused by PBDE-47 in rats.Methods: sixty neonatal female Sprague-Dawley rats were randomly divided into 6groups.On the eighth day after birth,2 groups of them were injected by intraperitoneal two times a day to give 4-phenylbutyric acid(PBA),a inhibitor of endoplasmic reticulum stress,for a total dose of 150 mg/kg bw continued for 3 weeks,and at the tenth day,they were administered with corn oil and the 10 mg/kg bw PBDE-47 in single gavage respectively;similarly,animals in the other 4 groups received corn oil,1 mg/kg bw,5 mg/kg bw and 10mg/kg bw PBDE-47 respectively.At the eighth weekend,rats of control group,10 mg/kg bw PBDE-47 treated group,150 mg/kg bw PBA treated group and 10 mg/kg bw PBDE-47+150 mg/kg bw PBA group were administrated Morris water maze test to examine their spatial learning and memory ability,then all animals were sacrificed,body weight and brain weight were recorded,cortex and hippocampus of rats were observed in HE staining and Nissl staining,expression level of GADD153/C/EBP homologous protein(CHOP)in hippocampus tissue was observed with immunohistochemistry method and expression levels of proteis related to ERS,autophagy and apoptosis were detected by Western blotting.Results: PBDE-47 accelerate Adolescent rats’ s body weigh gain and reduce the organ coefficient of brain.The results of Morris water maze experiment showed that 10 mg/kg bw PBDE-47 damaged the learning and memory ability,the exposed group needed more time and longer distance to find the platform(P<0.05).histology detection showed that PBDE-47 caused the disordered arrangement of the cells in hippocampal dentate gyrus and CA3 region,decrease and even disappearing of deeply stained glial cell as well as the reduction of Nissl body.The 5 mg/kg bw and 10 mg/kg bw PBDE-47 treatment increased the expression levels of the apoptosis related protein Cyt c and unfolded protein response related protein Inositol-requiring enzyme 1(IRE1)significantly(P<0.05).Both the expression of CHOP examinated by Western Blotting and the immunohistochemistry method was accordance with the level of cell apoptosis.1 mg/kg bw PBDE treatment up regulated the expression level of Beclin 1 and down regulated the expression level of p62,while 5 mg/kg bw and 10 mg/kg bw PBDE treatment up regulated the expression level of Beclin 1,p62 and the ratio of LC3-Ⅱ to LC3-Ⅰ significantly(P<0.05).Compared with 10mg/kg bw PBDE treatment group,the expression levels of unfolded protein response related proteins(IRE1,glucose-regulated protein 78(GRP78)and CHOP),autophagy related proteins(Beclin 1 and p62)and apoptosis related protein Cyt c in hippocampal tissue of 10 mg/kg bw PBDE+150 mg/kg bw PBA treatment group were down regulated(P<0.05).Conclusions: PBDE-47 could damage the hippocampal neuron and disturb the learning and memory ability through the endoplasmic reticulum mediated apoptosis.As a chemical chaperone,PBA can enhance autophagy and inhibite apoptosis to protect hippocampal neuron against damage caused by PBDE-47.Part 2: Roles of apoptosis and autophagy impairment induced by ERS in toxicity of PBDE-47 to PC12 cellsObjective: To observe whether endoplasmic reticulum stress inhibitor PBA can antagonize autophagy and apoptosis induced by PBDE-47,and improve cell survival rate,so as to provide basis for further study of neurotoxicity mechanism and prevention of PBDE-47.Methods: According to the previous experimental results of our team,the PC12 cells was divided into control group,20 μmol / L PBDE-47 exposure group,1 mmol / L PBA treatment group and 20 μmol / L PBDE-47 + 1 mmol / L PBA intervention group.The effects of PBDE-47(20 μmol / L)and PBA(1 mmol / L)on endoplasmic reticulum stress-related proteins(GRP78,IRE1 and chop),autophagy related proteins(Beclin 1,p62 and LC3),and apoptosis related proteins(caspase 3 and poly ADP ribose polymerase(poly ADP ribose polymerase))were detected by Western blot,PARP)expression level.Results: compared with the control group,20 μ mol / L PBDE-47 treatment decreased the survival rate of PC12 cells(P<0.05)and upregulated the expression of unfolded protein response related proteins IRE1,GRP78,CHOP and autophagy related protein p62.At the same time,the activation of apoptosis related protein caspase 3 and its downstream shear product PARP were increased.1 mmol / L PBA could antagonize the above effects induced by PBDE-47(P<0.05).Conclusion: a certain dose of PBDE-47 can lead to the increase of endoplasmic reticulum stress level in PC12 cells,resulting in autophagy disorder and increased apoptosis level.PBA intervention can restore the autophagy level of PC12 cells by inhibiting endoplasmic reticulum stress,thus reducing the apoptosis level of PC12 cells.