The Effect Of Fas/fasl Pathway On Pbde-47-induced Apoptosis In Sh-sy5y Cells

Polybrominated diphenyl ethers(PBDEs) is an important and newly found Persistent Organic Pollutants(POPs), which have been widely used in electronic equipments, cars, buildings and decoration materials and textile in the form of brominated flame retardants. The environmental epidemiological investigation demonstrated that PBDEs exist in various environmental media. Human exposure to PBDEs mainly through respiratory tract, gastrointestinal tract, and skin. PBDEs have been found in human tissues such as blood, milk, and adipose tissue. In vitro and in vivo experimental studies preliminarily suggested that health hazard caused by PBDEs included endocrine disrupting, hepatotoxicity, reproductive toxicity, neurotoxicity and mutagenicity. Nervous system, adipose tissue, thyroid and reproductive system are the main target organs for PBDEs. Exposure to PBDEs in early neural developmental stage may affect sensorimotor activity, learning and memory, and development of independent behavior.Apoptosis, a programmed cell death model under genetic control, contributes to selective elimination of cells in physiological and pathological situations. Some studies indicated that 2,2',4,4',5-pentabromodiphenyl ether(BDE-99)might cause apoptosis in Human Astrocytoma cells. Our primary experimental results found that 2,2',4,4'-tetrabromodiphenyl ethers(PBDE-47) might disrupt intracellular calcium homeostasis via increasing calcium concentration([Ca²⁺]i)in SH-SY5Y cells and then cause apoptosis. Moreover, our early studies indicated that high mRNA expressions of caspase12, caspase3, cytochrome c, and DAPK were closely related to apoptosis. These research indicate that PBDE-47-induced apoptosis might through three classic apoptosis pathways—the mitochondrial, endoplasmic reticulum, and death receptor pathways. So far which is the main pathway and mechanisms in PBDE-47-induced apoptosis are not clear. Further studies are needed.The Fas and FasL-mediated apoptosis relys on the functional Fas protein on the cell membrane by way of the combination of Fas and FasL, which further activate ensuing apoptosis-related protein such as Caspase3 and then induce apoptosis. It has been reported that H2O2 treatment increased [Ca2+]i in L-02 cells and induced apoptosis with higher expression of specific Fas mRNA transcription and Fas protein. These indicated that activation of Fas death gene may mediate peroxide-triggered L-02 cell apoptosis. H2O2-induced cytoplasmic Ca2+ imbalance may induce over-expression of Fas death genes, activate death receptor signal pathways and cause apoptosis.In order to provide some useful clues for further study of PBDE-induced neurotoxicity, we used human neuroblastoma SH-SY5Y cells to explore the effects of PBDE-47 on the expressions of Fas, FasL, Caspase3, and Caspase8.This paper consists of two partsPart I Effects of PBDE-47 on mRNA expression levels of Fas, FasL, Caspase3, and Caspase8 in SH-SY5Y cells in vitroObjective To investigate the effects of PBDE-47 on mRNA expression levels of Fas, FasL, Caspase3, and Caspase8 in SH-SY5Y cells in vitro. Methods SH-SY5Y cells were exposed to different concentrations of PBDE-47(0, 1, 5, 10μmol/L) for 24 h, and the mRNA expression levels of Fas, FasL, Caspase3, and Caspase8 were measured by using Real time PCR. Result The mRNA expression levels of Fas in 5, 10μmol/L PBDE-47-treated groups were higher than those of the control group(P<0.05). The mRNA expression levels of FasL in 10μmol/L PBDE-47-treated group was higher than those of the control group and 1μmol/L PBDE-47-treated group(P<0.05). The mRNA expression levels of Caspase3, Caspase8 in 5, 10μmol/L PBDE-47-treated groups were higher than those of the control group and 1μmol/L PBDE-47-treated group ( P<0.05 ) . Conclusion PBDE-47 may increase the mRNA expression levels of Fas, FasL, Caspase3, and Caspase8 in SH-SY5Y cells in vitro.PartⅡEffects of PBDE-47 on protein expression levels of Fas, FasL, Caspase3, and Caspase8 in SH-SY5Y cells in vitroObjective To investigate the effects of PBDE-47 on protein expression levels of Fas, FasL, Caspase3, and Caspase8 in SH-SY5Y cells in vitro. Methods SH-SY5Y cells were exposed to different concentrations of PBDE-47 (0, 1, 5, 10μmol/L) for 24 h, and the protein expression levels of Fas, FasL, Caspase3, and Caspase8 were detected by Western blot. Result The protein expression levels of Fas in 5, 10μmol/L PBDE-47-treated groups were higher than those of the control group and 1μmol/L PBDE-47-treated group(P<0.05). The protein expression levels of FasL in 10μmol/L PBDE-47-treated group was higher than those of the control group and 1μmol/L PBDE-47-treated group(P<0.05). Compared with control group, the proenzyme protein expression levels of Caspase3 in 10μmol/L PBDE-47-treated group and Caspase8 in 5, 10μmol/L PBDE-47-treated groups were obviously decreased(P<0.05). Conclusion PBDE-47 may increase the protein expression levels of Fas and FasL, and decrease the proenzyme protein expression levels of Caspase3 and Caspase8. Combined with the first part about mRNA expression levels of Fas, FasL, Caspase3, and Caspase8, we could concluded that Fas/FasL pathway may plays an important role in PBDE-47-induced apoptosis of SH-SY5Y cells in vitro.