Hepcidin Produced By Astrocytes Involved In LPS-induced Neuronal Apoptosis And Iron Metabolism

Objective: Iron is one of the essential elements in human, to maintain thedevelopment and physiological function of brain iron levels is very important.Brain iron metabolism has strict regulation, but in the inflammation and otherpathological conditions, brain iron metabolism changed evidently. Hepcidin isa peptide hormone primarily known as the key regulator of iron homeostasis.Although hepcidin is predominantly expressed in the liver, it is also detectablein the brain. Hepcidin was localized in the nerve tissue and functionalproperties suggesting that it plays an important role in maintaining ironhomeostasis in the brain. However, the effect of hepcidin in iron metabolismand whose effect in neurons’ apoptosis induced by LPS still needed to befurther clarified.In this study, we analyzed the role of microglia and astrocytes andhepcidin produced with astrocytes in iron metabolism and whose effect inneurons’ apoptosis induced by LPS and provided further proof tounderstanding the molecular mechanisms of brain iron homeostasis neurons’apoptosis induced by LPS.Methods:1Primary cultures of neurons, microglia and astroglial1.1Primary cultures of rat cortical neurons：The pregnancy (E16-E18) SD rat were killed, taken out of fetal rats，bilateral brain cortex which were digested with papain (2mg/ml in DMEM),37℃for30min, washed with DMEM plus serum and dissociated by passingthe tissue through imported pipette tip, then made cell suspension culture fluidwith planting the cell concentration was adjusted to1×105/cm2. Thentransferred to a6-well or24-well cell culture plate which was coated withpoly-L-lysine, the plating medium is DMEM containing10%fetal bovine serum,1%penicillin and streptomycin(1%P/S). After6h to8h of initial plating,the culture medium was completely replaced with feedingmedium(Neurobasal medium supplemented with2%B27) and changedculture medium every3days.1.2Primary cultures of mixed glial cells:The cerebral hemispheres from new born rats (within24hours) werecleaned by removing the meninges and blood vessels, minced, and weredigested with papain (2mg/ml in DMEM),37℃f or30min, washedwithDMEM plus serum and dissociated by passing the tissue through importedpipette tip, then made cell suspension culture fluid with planting the cellconcentration was adjusted to1×105/cm2. Then transferred to75cm2cellculture flask was coated with poly-L-lysine, the plating medium is DMEMcontaining10%fetal bovine serum,1%penicillin and streptomycin(1%P/S).According to the cell metabolism, changed culture medium every2~3days.1.3Purified astrocytes and microgliaAstrocytes: Primary cultures for9–10days were shaken vigorously18~20h with a rotary shaker to remove non-astrocytic cells. The resultantastrocytic monolayer was detached and dissociated by0.125%trypsintreatment and then transferred to a6-well or24-well cell culture plate whichwas coated with poly-L-lysine, adding complete medium,37℃,5%CO2theconstant temperature incubator culture, changed culture medium every2~3days.Microglia: Microglia were obtained from primary cultures during days9–11of culture. Cells were detached by gentle shaking of the flasks2h, collecting suspension at1000r7min/min centrifugation, plus quantitativecomplete medium again triturated into cell suspension, the seeding densityof1×105/cm2in poly lysine pre treated with6-well or24-well cell cultureplate,37℃5%CO2incubator after30min removed, completely liquidchange1times, with the removal of non-adherent cells.1.4Immunocytochemistry:Remove the different treatment of the cell immunocytochemistry and immunofluorescence method to identify the cell purity.2Neuronal apoptosis was analysed by using Hoechst33258, MTT and Flowcytometry.3Proteins were detected by western-blot.4IL-6, hepcidin mRNA in different cells were detected by RT-PCR.Results:1Primary cultures and pure of different cells1.1Primary cultures and pure of cortical neuronsNeurons were adherent and round after inoculation for6hours, then thecells grew few of protrusions from the cell body. After1days the cells grewprojections, and cell bodies round or oval, plump, halo obvious. After4-6d thecell bodies increased significantly, and began to gather, a mesh-like growthprojections are connected. Immunocytochemistry staining showed that thepurity of neurons was up to95%.1.2Primary cultures and pure of mixed glial cellsCells cultured1-2d, the number and morphology of glial cells did notchange, but the number of neurons decreased; Cells cultured3-4d, the numberof astrocytes significantly increased, cell body spreading gradually into layers.Neuronal death was accelerating, cell disintegration products increased; Cellscultured5-6d began to appear layered growth, the lower cells wasastrocytes,and the soma was large, flat, and the refraction was less; the topis microglia; cells cultured9-11d layered growth more significantly; aftercultured11d, the number and morphology of astrocytes and microglia did notchange significantly.1.3Primary cultures and pure of astrocytesPrimary cultures for9–10days were shaken vigorously18~20h with arotary shaker. The resultant astrocytic monolayer was detached anddissociated by0.125%trypsin treatment and then transferred to a6-well or24-well cell culture plate which was coated with poly-L-lysine. GFAPanti-body staining showed that the purity of neurons was up to95%. 1.4Primary cultures and pure of microgliaPrimary cultures for9–11days were shaken vigorously2h with a rotaryshaker to separate microglia cells. Microglia were more adherent in30min,after30min the medium was changed to remove non-adherent cells.Microglial cells were round, irregular; the first2d cell bodies retraction wereseen, a few cells extended protrusions, mostly unipolar, after cultured3-4dthe most cells into stationary state, the cell body long and narrow, andextending protrusion. CD11b anti-body staining showed that the purity ofneurons was up to98%.2Microglia and astrocytes to participate in the LPS-induced neuronalapoptosis processLPS treated microglia for24h, the conditioned medium was namely CM1;CM1treated astrocytes, namely CM3.2.1Neuronal apoptosis was detected by using Hoechst33258Compared with the control group, LPS treated neuron for6h, neuronalapoptosis rate did not change significantly; CM1treated neurons6h, neuronalapoptosis rate was20.9%, compared with the control group, there was nosignificant difference; CM3treated neurons6h, neuronal apoptosis wassignificantly increased, reaching36.7%(P<0.01).2.2Neuronal viability was detected by MTTCompared with the control group, LPS treated neuron for6h, nosignificant changes in cell viability; CM1treated neurons6h, also no alsodecreased; CM3treated neurons6h, neuronal viability decreased significantly(P<0.01)2.3Neuronal apoptosis was analysed by FCMCompared with the control group, LPS treated neuron for6h, neuronalapoptosis rate did not change significantly; CM1treated neurons6h, neuronalapoptosis rate was5.65%, compared with the control group, there was nosignificant difference; CM3treated neurons for6h, neuronal apoptosis wassignificantly increased, reaching22.6%. CM3compared with the CM1group,the apoptosis rate increased significantly.(P <0.01) 3Changes of iron concentrations3.1Measured the iron concentrations in neuronsIron concentrations in neurons measured by ICP-MS. LPS treated neuronfor6h, iron concentrations did not increase; CM1treated neurons6h, ironconcentrations did not increase significantly; CM3treated neurons6h, ironconcentrations significantly increased (P<0.01). Results imply thatLPS-induced neuronal apoptosis may be associated with an increase in theiron concentrations and microglia and astrocytes are involved in this process.3.2Changes of Tfr1Compared with the control group, LPS treated neurons for6h, Tfr1without significant changed; CM1group, Tfr1was up to18.3%(P<0.01),CM3group Tfr1was up to35.8%(P<0.01), there was a significant difference.3.3Changes of Fpn1Compared with the control group, LPS treated neurons for6h, Fpn1without significant changed; CM1group, Fpn1protein was down to6.52%(P<0.05), CM3group, Fpn1was down to25.4%(P<0.01), there wasdifferences.3.4Changes of DMT1Compared with the control group, LPS treated neurons for6h, DMT1protein with no significant changed; CM1group, DMT1protein was up to26.3%(P<0.001), CM3group, DMT1protein was up to119.5%(P<0.001),there was a significant differences.3.5Changes of H/L-FerritinCompared with the control group, LPS treated neurons for6h, H-Ferritinwith no significant changed, CM1group, H-Ferritin was up to27.8%(P<0.001), CM3group, H-Ferritin was up to72.4%(P<0.001), there was asignificant difference. Compared with the control group, LPS treated neuronsfor6h, L-Ferritin with no significant changed, CM1group, L-Ferritin was upto22.9%(P<0.001), CM3group, L-Ferritin was up to68.9%(P<0.001), therewas a significant difference. 4LPS activated microglia to release IL-6which induced astrocytes to expresshepcidin4.1LPS-induced hepcidin expressionIn the control group, hepcidin mRNA expression in the three cells wasvery low, LPS (100ng/ml) dealed with the three cells24h, hepcidin mRNAexpression did not change significantly.4.2IL-6-induced hepcidin expressionIL-6(10ng/ml) deal with three cells24h, hepcidin mRNA expression inastrocytes was significantly elevated; hepcidin mRNA was no significantchanged in neurons and microglia.4.3LPS-induced IL-6mRNA expressionLPS treated microglia24h, IL-6mRNA expression was significantlyincreased, in astrocytes also increased, but far below the microglia; whereasnot detected the IL-6mRNA expression in neurons.4.4CM1and IL-6were treated astrocytes24h, hepcidin mRNA expressionwere significantly increased; adding anti-IL-6antibody in CM1, and thentreated astrocytes24h, hepcidin mRNA expression was significantly inhibited.5The expression of iron-related proteins, after knockout of hepcidinBecause of purified astrocytes and C6had no differences, so C6insteadof purified astrocytes. IL-6treated C624h, the conditioned medium wasCM2’; IL-6treated shCtrl-C624h, conditioned medium was CM2’(+); IL-6treated shHepcdin-C624h, conditioned medium was CM2’(-). And then IL-6,CM2, CM2’(+) and CM2’(-) treated neurons6h, to detected iron-relatedproteins in neurons.5.1Measured the iron concentration in neuronsCompared with the control group, iron had increased in IL-6group, buthad no significant difference, iron concentration had increased significantly inCM2’, CM2’(+) group; and compared with the CM2’(+) group, CM2’(-)groupdecreased significantly. The fact descripted that knockout of hepcidininhibited IL-6-induced neuronal iron concentration increasing, and suggestingthat hepcidin was a factor of inflammation induced increasing in ironconcentration 5.2Changes of Tfr1Compared with the control group, Tfr1were raised39.4%(P<0.01) and69.7%(P<0.01) in IL-6and CM2group, there was a significant difference.And compared with CM2’ group, Tfr1protein was down to39.6%in CM2’(-)group (P<0.001), CM2’(+) group had no significant change.5.3Changes of Fpn1Compared with the control group, Fpn1were down to12.9%(P<0.05)and37.3%(P<0.05) in IL-6and CM2group, there was significant difference.And compared with CM2’treatment groups, Fpn1was up to124.4%(P<0.001)in CM2’(-), Fpn1was up to30.1%(P<0.001) in CM2’(+) group, there was asignificant difference.5.4Changes of DMT1Compared with the control group, DMT1were raised17.1%(P<0.05)and39.6%(P<0.05) in IL-6and CM2groups, there were significantdifferences. And compared with CM2’ groups, DMT1was down to62.3%(P<0.001) in CM2’(-), DMT1was down to27.1%(P<0.01) in CM2’(+) group,there was a significant difference.5.5Changes of H/L-FerritinCompared with the control group, H-Ferritin had no significant changesin IL-6and CM2groups. Compared with the CM2, H-Ferritin was down to62.3%(P<0.001) in CM2’(-) group, H-Ferritin was down to19.3%(P<0.05) inCM2’(+) group, there were significant differences. Compared with the controlgroup, L-Ferritin with no significant changes in IL-6and CM2groups.Andcompared with CM2’ groups, L-Ferritin was up to160.0%(P<0.05) in CM2’(-)group, L-Ferritin was up to71.3%(P<0.05) in CM2’(+) group, there weresignificant differences.6Neuronal apoptosis was changed after knockout of hepcidinCompared with the control group, IL-6treated neurons for6h, neuronalapoptosis was not significantly increased, and apoptosis significantlyincreased in CM2group (P<0.01). Compared with CM2’, neuronal apoptosiswas significantly decreased in CM2’(-) group. Conclusions:1Microglia/astrocytes activation is required for apoptosis of neuronsinduced by LPS.2Apoptosis of neurons induced by LPS is related to iron overload inneurons.3Microglia are the primary initial responders to LPS and produceinflammatory mediators such as IL-6that induces hepcidin expression ofastrocytes.4Hepcidin produced by astrocytes increases iron concentration in neuronsby inhibiting neuronal iron exporter.5Iron increase mediated by hepcidin of astrocytes is a factor ofLPS-induced neuronal apoptosis.