The Role And Mechanism Of The ERS And UPR Signal Pathway In PBDE-47-induced Neurotoxicity

Polybrominated diphenyl ethers (PBDEs) are bromined flame retardants, which arewidely acted as a kind of important industry additive of flame retardant and added intosynthetic materials with large molecular. PBDEs are widespread used in electronics,electrical, chemical, transportation, construction materials, textiles, petroleum and miningand other areas. PBDEs can release from products and enter the environment, causingenvironment pollution. By now, many countries have detected and gained PBDEs fromenvironmental medium, including the air, water, soil, food as well as the human tisssuessuch as blood, milk and fat. As a kind of persistent organic pollutant (POPs), the level ofPBDEs in the environment is increased annually. The residual and the toxicity of PBDEsare likely to cause serious environmental pollution and harm to human health, which hascaused worldwide concern.In recent years, both in vivo and in vitro studies confirmed that PBDEs can causeneurotoxicity, reproductive toxic, hepatotoxicity, immunotoxicity and carcinogenicity.Infants could be exposed through breast milk intake and fetal exposure can occurtransplacentally. In addition, because of their child-specific hand-to-mouth behavior,frequent ground contact (resulting in the ingestion of house dust), and relatively weakdischarge capacity, children are exposed to larger amounts of PBDEs than are adults, whichleads to higher body burden in infant and toddlers than those in other age groups. Hence,developing nerutoxicity especially causes worldwild concern relative to other toxicities generated by PBDEs. In the critical period of brain development, the animals subjected tosimilar dose of PBDEs homologue to humans’ exposure caused irreversible damage inbrain function, manifesting disorder of independent behaviour, descent of the ability oflearning and memory, and these changes appeared to worsen with age. At present, PBDEsinduced morphological and functional abnormalities of brain are the research hotspot,however, the underlying mechanisms are still not clear.PBDEs can increase reactive oxygen species (ROS) and induce intraneuronaloxidative stress. Oxidative stress is the beginning factor in cascade reaction of cellsapoptosis. Many studies showed that PBDEs congeners can cause apoptosis mediated byoxidative stress in nerve cells. Abnormal and excessive apoptosis could inevitably generatethe serious consequences of changing neurons structure and functions and trigger toxicity innerve cells. Therefore, cells apoptosis may be a key factor of neurotoxitic effects caused byPBDEs.Endoplasmic reticulum signal transduction pathway is one of the three classicapoptosis pathways. Endoplasmic reticulum is the site for protein folding, assembly andtransportation. When cells suffered different intensity of stimulation such as oxidativestress, unfolded or misfolded protein will accumulate in endoplasmic reticulum and this cancause different degree of the endoplasmic reticulum stress (ERS), which will trigger theunfolded protein response (UPR). Sustained or excessive endoplasmic reticulum stress willcontinue to produce more unfolded proteins and disturb internal environment ofendoplasmic reticulum, finally, apoptosis will ensue mediate by the UPR signal pathwayAlthough there is no report on whether the endoplasmic reticulum stress and UPR isinvolved in the neurotoxicity of PBDEs. But newly researches found that after PBDEsexposure, hippocampus of rats was merged pathological changes in endoplasmic reticulum.Furthermore, metabolic products of PBDEs can changed the expressions of genesimplicated in UPR signal pathway such as GRP78, CHOP, GADD34and XBP1. Accordingto these findings, PBDEs may cause neurotoxicity mediated by endoplasmic reticulumstress and UPR signal pathway.On basis of above reasons, this study will employ PBDE-47, which is a typicalpredominant congner of PBDEs, and SH-SY5Y cells, which posses characteristic of nerve cells to conduct experiments in vitro. Using the methods including flow cytometry, Hoechst33258for cells nuclei staining, real time RT-PCR and Western blot to detect the productionof ROS in cells, cells apoptosis, the mRNA and protien expression levels of genes such asGRP78, IRE1, XBP1, JNK, CHOP and so on. In addition, using small interfering RNA(siRNA) to knockdown IRE1gene then detecting apoptosis and the expression alterationsof related genes. To investigate wheather ERS and UPR is associated with the toxicityinduced by PBDE-47and the role of IRE1pathway in the neurotoxicity caused byPBDE-47in nerve cells. Try to provide foundational data and theory for preventing andcontroling the damage caused by PBDEs.Part1PBDE-47induced oxidative stress damage and UPR as well asactivated IRE1pathway in SH-SY5Y cellsObjective: The aim of the present study is to identify whether PBDE-47can causeneurotoxicity in SH-SY5Y cells via endoplasmic reticulum stress and UPR and to provide aclue for studying the mechanisms of PBDEs neurotoxicity.Methods: SH-SY5Y cells were exposed to different concentrations of PBDE-47(0,1,5,10μmol/L) for3-24h and then the ROS production was measured by flow cytometryand the expressions of the genes involved in UPR and IRE1apoptotic pahway includingGRP78, IRE1, CHOP, JNK, p-JNK, Bcl-2and Bax were detected by real time RT-PCR andWestern blot.Results: Compared with the control, certain concentration of PBDE-47significantlyincreased the ROS level and the mRNA expression levels of GRP78and IRE1within3-24h in SH-SY5Y cells (p <0.05), which showed a concntration-effect relationship. After cellswere treated with10μmol/L PBDE-47, the protein results showed that IRE1and CHOPexpression levels at24h were higher than those at0,3,6and12h (p <0.05). The proteinexpressions of IRE1, CHOP and phosphorylation of JNK in cells treated with differentconcentrations of PBDE-47notablely increased in comparison with those in the controls,respectively (p <0.05), which also demonstrated a concentration-response manner at24h.In contrast, the Bcl-2protein decreased markedly (p <0.05) and the Bax/Bcl-2ratioincreased significantly in cells under the same conditions relative to those in the control (p <0.01).Conclusions: PBDE-47can cause neurotoxicity by inducing SH-SY5Y cells apoptosisvia endoplasmic reticulum stress and UPR, and this may be triggered by oxidative stressmediated by ROS. The IRE1signal transduction of UPR plays a vital role in theneurotoxicity caused by PBDE-47.Part2The role and mechanism of the IRE1signal pathway in toxic effectinduced by PBDE-47Objective: IRE1is an important transmembrane protein with fundamental regulationof cells apoptosis and decides cells suviral or death. The aim of the present studies is toinvestigate the role and mechanism of the IRE1apoptotic pathway in neurotoxic effectinduced by PBDE-47.Methods: After knocking down the IRE1gene with targeting siRNA sequence andbeing exposed with10μmol/L PBDE-47for24h, then the apoptotic rates was detected byflow cytometry, the morphological changes of cells nuclear using nuclei staining wasobserved by inverted fluorescence microscope, and the mRNA and protien expressionlevels of genes such as GRP78, IRE1, XBP1, CHOP, JNK and p-JNK involved in UPR andIRE1apoptotic pathway was measured by real time RT-PCR and Western blot.Results: Compared with the control group, cells treated with PBDE-47showed thecharacteristic features of apoptosis including cell volume shrinkage and irregularmorphological change as well as a significantly reduction of survival cells number. Cellstreated with PBDE-47and stained by Hoechst33258nuclear staining revealed nuclearchromatin condensation and fragments, karyorrhexis, plasma membrane blebbing, and evenapoptotic bodies (p <0.05). These results regarding apoptotic changes especially emergedin cells in which IRE1was silenced and treated with PBDE-47(p <0.05). The FCManalysis results showed that the apoptotic rates higher than that in the control (p <0.01)and further increased when combined with IRE1gene siRNA (p <0.05). PBDE-47increased the mRNA expression levels of spliced XBP1, unspliced XBP1, JNK, CHOP andBax markedly compared with those in the control, respectively (p <0.05or p <0.01), butdecreased Bcl-2significantly (p <0.05). Knockdown of IRE1and treatment with PBDE-47 decreased the mRNA levels of spliced XBP1, JNK, CHOP and Bcl-2compared withnegative siRNA+PBDE-47groups (p <0.05or p <0.01), but increased mRNA level ofBax alone(p <0.05). It also significantly increased the ratio of Bax/Bcl-2in mRNA levels(p <0.01). After knocking down IRE1, PBDE-47significantly decreased the proteinexpressions of CHOP, JNK and Bcl-2compared to those in the negative siRNA+PBDE-47groups (p <0.05), but increased Bax protein level alone (p <0.05). It also significantlyincreased the ratio of Bax/Bcl-2in protein levels (p <0.01).Conclusions: IRE1has bifunction of both protection against ERS and pro-apoptosismediated by activating spliced XBP1, JNK and CHOP. Significant increase in the ratio ofBax/Bcl-2by down regulation of Bcl-2as well as up regulattion of Bax is the main reasonfor increased apoptosis induced by knockdown of IRE1and treatment with PBDE-47. Therole of IRE1signal pathway to protcet cells from neurotoxity effect is more important at24h.