Lead-induced Astrocyte Endoplasmic Reticulum Stress Responses In The CNS

ObjectiveHeavy metal lead is a widespread environmental toxicant;never reversible neurotoxic and learning memory effects from chronic exposure to low levels of lead are a problem of significant magnitude in children and infants.The absorptivity to lead of alimentary canal in children is higher than that in adults,and excretion rate of children is lower than that of adults.In addition,the agenesis of blood and brain barrier in children,so lead concentrates easily in children's neural system.The sensitivity to lead that development of brains for 0 to 6 years old of children is 30 times higher than that of adults.Nearly 30～40%of children in many cities of china have the trouble of lead poisoning which is almost 10 times of children in America.Studies indicate that when the concentration of Pb in the blood increases from 0.48μM to 0.97μM,the IQ of these children averagely decreases 2～6 points.90%of brain cells are glia cells,which are neural sustaining cells.The important functions of glia include that myelination,promoting development of neuron,rehab and rebirth of neuron,etc.The primary cells of glia are astrocyte,whose functions includes that helping the development and movement of neuron,forming of synapse, accommodation of neurotransmitter transfer and energy,maintaining the stabilization of central nerve system.Some studies considered that maybe lead damaged astrocyte firstly and then damaged endodermis so that to pass the blood brain barrier easily,and damaged the brain function ultimately.It is well known that stress can bring about alterations of the endoplasmic recticulum(ER)homeostasis which cause ER stress.Unfolded protein reaction(UPR) is an important genomic response to ER stress by synthesis ER chaperones proteins such as glucose regulated proteins 78(GRP78),which plays critical roles in cell survival as part of UPR.When the ER function is severely impaired,the organelle elicits apoptosis signal activated by growth arrest and DNA damage inducible gene (GADD153)and other signals.Some studies have shown that most of Pb could be isolated to the astroglial cells when rat children were exposed to a higher level of Pb in the environment,while at the same time,a kind of new protein which could bind to Pb was found.In addition,some scholar have considered that astroglia could cumulate a great deal of Pb by ER stress so that to protect the more sensitive neuron.All these evidences indicate that cells are capable of activating tolerance mechanisms to the nonphysiologic metal,i.e.astroglia cells could increase expression of certain protein and chelate with Pb when cells were exposed to Pb,which were as the Pb cumulated pool to protect the normal function of CNS.To evaluate the impact of acetate Pb induced stress in ER;we investigate the effects of acetate Pb on morphological,cell proliferation,cell-cycle and the expressions of cyclinD1,GADD153 and GRP78.In order to discuss if GRP78 is the target protein on the ER for Pb,we observe the chelate of GRP78 with Pb and some certain signal pathways through which Pb induces the express of GRP78.Methods1.Wistar rat brain astrocyte cells were primary cultured in medium with or without acetate Pb which blocked by PD98059(MEK1 inhibitor)or Wortmannin (PI3K inhibitor),acetate sodium substituted acetate Pb as the comparison.2.The hallmark of the primary culture astrocyte was estimate by observing the expression of glia fibrillary acidic protein(GFAP)in the astrocyte through the method of immunochistochemistry(IC).3.The effect of acetate Pb on the cell proliferation in astrocyte was estimated by MTT analysis.4.The effect of acetate Pb on the cell-cycle in astrocyte was estimated by FCM analysis.5.The protein expressions of cyclinD1 and GADD153 induced by acetate Pb were accessed by western blotting.6.The chelate of GRP78 with Pb was examined by immunoprecipitation(IP), western blotting and atomic absorption spectrophotometer(AAS). 7.The localizations transformation of GRP78 was observed with colloid gold immunoelectron microscopy.8.The changes of the activity of ERK and PKB,and the signal pathway which GRP78 was induced by Pb were determined by reverse transcription PCR(RT-PCR) and western blotting.Results1.By immunohistochemical staining and observing under the microscope,more than 95%primary culture cells were positively stained with GFAP as witnessed by the brown DAB reaction product in the cytoplasm,indicating that they were astroglia.2.MTT assay showed that:Treated astrocyte cells with acetate Pb,the cell proliferate rate decreased time dependently.When 1.0μM acetate Pb treated cells for 8 day,the cell proliferate rate decreased statistics(P＜0.05).3.FCM assay showed that:Treated astrocyte cells with acetate Pb for 8,12,15 or 30 day,the cell-cycle progression was inhibited at G1 phase(P＜0.01).4.Western blot showed that:(1)Treated astrocyte cells with acetate Pb,CyclinD1 protein expression decreased time dependently(P＜0.05).(2)1.0μM acetate Pb treated astrocyte for 30 day,GADD153 protein began to be expressed.(3)Acetate Pb could induce the increasing expression of GRP78 dose and time dependently.0.2,1.0 and 10μM acetate Pb treated astrocyte cells for 24h,the GRP78 protein expression would be increased,1.0 and 10μM acetate Pb group have statistics significance(P＜0.01).1.0μM acetate Pb treated cells for 24 h,the expression of GRP78 increased significance,and the peak was at treated for 4～8 day(P＜0.01), nearly at 12 day,the expression of GRP78 began to decrease which was still higher than the normal level(P＜0.05).PD98059 and Wortmannin could inhibit the effects of acetate Pb treatment,and the effect of Wortmannin was better.(4)Acetate Pb could increase activation of ERK dose and time dependently.0.2, 1.0 and 10μM acetate Pb treated astrocyte cells for 30 min,the activation of ERK would be increased,1.0μM acetate Pb group has the most statistics significance(P＜ 0.05).When 1.0μM acetate Pb treated cells for 5min,the activation of ERK began to increase and the peak was at 30min,and then the level of ERK activation gradually decreased to the normal level.PD98059 could prevented the increasing of ERK activation induced by acetate Pb(P＜0.05).(5)Acetate Pb could increase activation of PKB dose and time dependently.0.2, 1.0 and 10μM acetate Pb treated astrocyte cells for 30min,the activation of PKB would be increased,1.0μM acetate Pb group has the most statistics significance(P＜0.05).When 1.0μM acetate Pb treated cells for 5min,the activation of PKB began to increase and the peak was at 30 min,and then the level of PKB activation gradually decreased to the normal level.Wortmannin could prevented the increasing of PKB activation induced by acetate Pb(P＜0.05).5.Immunoprecipitation and method of AAS showed that:GRP78 could chelate with Pb ion which came into cells tightly.The immunoprecipitated GRP78 protein was analyzed by Western blotting which could detect the protein was a single protein of 78KD.The immunoprecipitated content was detected by AAS,Pb content began to increase when cells were treated by acetate Pb for 24 h,and the peak was at 8 d(P＜0.01),and then Pb content decreased gradually,but still higher than the normal(P＜0.05).6.Colloid gold immunoelectron microscopy assay showed that:1.0μM acetate Pb treated cells for 24 h,GRP78 protein expression began to remarkably increase whose location transferred from ER to the cytosol around the nuclei.10μM acetate Pb treated cells for 10 h,cells would have the similar changes.7.RT-PCR analysis showed that:(1)Acetate Pb could induce the increasing of mRNA expression for GRP78 dose and time dependently.PD98059 and Wortmannin could inhibit the effects of acetate Pb treatment,and the effect of Wortmannin was better.(2)Acetate Pb could upregulate mRNA expression of ERK₂(P＜0.05),which was prevented by PD98059 effectively. Conclusion1.Acetate Pb could induce the UPR in the primary culture astrocyte ER,and could down-regulate the expression of protein cyclinD1 and the stagnation of cell-cycle.A long time of exposing to Pb could induce the expression of GADD153, but we still couldn't observe apoptosis.2.Acetate Pb up-regulated GRP78 mRNA and protein expression through PI3K/PKB and MEK1/ERK signal transduction pathway.3.GRP78 in astrocyte could strongly chelate with Pb ion dose dependently.4.Acetate Pb activated MEK/ERK and PI3K/PKB by phosphorylating the kinases immediately rather than increasing their protein synthesis.