| BackgroundIron is an essential element for the central nervous system(CNS)to maintain its normal physiological function.It plays an important role in oxygen transport,DNA synthesis,myelin production,mitochondrial respiration and neurotransmitter synthesis.However,intracellular free ferrous ion could react with endogenously generated hydrogen peroxide to produce reactive oxygen species(ROS).Therefore,ferrous ion deposit could induce oxidative stress(OS)and inflammatory response in neural cells,thus causing neurological injury.So iron homeostasis in neural cells is indispensible for the proper function of CNS.Iron accumulation has been previously documented in intracerebral hemorrhage(ICH),brain ischemia,traumatic brain injury(TBI)and several neurodegenerative diseases(such as Alzheimer's disease,Parkinson's disease,Huntington' s disease etc.),indicating the vital role of ferrous ion deposit-induced neurological injury in the development and progression of these diseases.Tert-Butylhydroquinone(tBHQ)is a metabolite of butylated hydroxyanisole,a widely used food antioxidant.tBHQ was reported to be capable to activate the Nrf2/ARE pathway,which is the key pathway to regulate the intracellular redox state.Under normal condition,Nrf2 is anchored in the cytoplasm by Kelch-like ECH-associated protein-1(Keap1),which could also induce Nrf2 ubiquitination.The ubiquitinated Nrf2 could be degraded rapidly,thus keeping the activity of Nrf2/ARE pathway at the physiological level.Upon OS,the Keapl conformation changes so that its ubiquitination effects on Nrf2 vanish.The Nrf2 escapes the Keapl-mediated degradation,leading to the accumulation of newly synthesized Nrf2 in the cytoplasm.Meanwhile,Nrf2 translocates into the nucleus,where it binds with the antioxidant response element(ARE)to induce the expression of heme oxygenase-1(Hmox-1),glutathione transferases(GSTs),glutathione peroxidase-1(Gpxl),NAD(P)H:quinone oxidoreductase-1(Nqol)and other antioxidant enzymes.tBHQ could alter the conformation of the Keap1-Nrf2 complex,which could inhibit Keapl-mediated Nrf2 ubiquitination,thus increasing the stability of Nrf2.tBHQ has been reported to exert protective effects in different models of CNS disease.In addition,tBHQ has been proved for human use.However,the effects of tBHQ on ferrous ion-induced neurological injury remain to be further investigated.The Peroxiredoxins(Prdxs)belong to the thioredoxin system,and they are a family of thiol peroxidases,which play an antioxidant role by scavenging intracellular ROS.Additionally,Prdxs have been reported to exert anti-inflammatory effects in several inflammatory diseases.There are 6 isozymes in the mammalian Prdx subfamily,named Prdx 1-6.Among them,Prdx2 is the most abundant in CNS,making it the primary candidate to fight against OS in the brain.Previous studies have found that Prdx2 could offer protection in brain ischemia,Alzheimer' s disease and Parkinson disease.However,its effects on ferrous ion-induced neurological injury still remain unclear.PC12 cells and neurons resemble in some attributes,such as morphology,physiology,biochemistry etc.Therefore,PC12 cells have been widely used in the in vitro study on neurophysiology,neurotoxicology,neuroprotection and neurocognition.Previous study has found that ferrous ion could induce lipid peroxidation and cell death in PC12 cells.So in our study,we will use ferrous sulfate(FS)-treated PC12 cells as an in vitro model to study ferrous ion-induced neurological injury.There are two parts in this thesis.In the first part,PC12 cells are treated with tBHQ prior to FS stimulation,and then the effects and mechanism of tBHQ on FS-induced cell injury and apoptosis are detected.After that,an animal model in which rats received intrastriatal stereotaxic injection of FS is used to study the function of tBHQ on ferrous ion-induced neurological injury in vivo.In the second part,the effects of FS on Prdx2 expression in PC12 cells are firstly detected.Then PC12 cells were infected with lentivirus to knock down Prdx2,followed by FS treatment,and then the function of Prdx2 on ferrous ion-induced neurological injury is evaluated.We hope that these experiments could provide some novel therapeutic targets for multiple CNS diseases which are accompanied with iron deposit,such as stroke,TBI and neurodegenerative diseases.PART ?:THE EFFECT AND MECHANISM OF tBHQ ON FERROUS ION-INUCED NEUROLOGICAL INJURYObjectiveTo determine whether tBHQ could alleviate the ferrous ion-induced neurological injury both in vitro and in vivo.Using in vitro experiments to determine that whether the neuroprotective effect of tBHQ is dependent on the Nrf2/ARE pathway.Methods1.Cell culture:Rat PC12 cells were routinely maintained in DMEM supplemented with 10%horse serum and 5%fetal bovine.2.Determination of the working concentration and treatment period of FS:PC12 cells were exposed to different concentrations(1 ?mol/L,2 ? mol/L,5 ?mol/L,10 ? mol/L and 20 ? mol/L)of FS for 24 h,and then subjected to CCK-8 assay to examine their viability.We chose the minimum concentration that could decrease the cell viability by over 50%as the working concentration.Then we used the working concentration of FS to stimulate PC12 cells for different periods(3h,6h,12h,24h,36h),and the cell viability was detected by CCK-8 assay.After that,we chose the optimal period according to the results.3.Grouping for cell experiment:After the optimal working concentration and treatment period of FS were confirmed,the cells were divided into the 4 following groups according to different treatments:Ctrl group,tBHQ treatment group(Treated with 40 ?mol/L tBHQ for 40h),FS stimulation group(Stimulated with 10 ?mol/L FS for 24h)and tBHQ+FS group(Pretreated with 40 ?mol/L tBHQ for 16h before co-treatment with 40?mol/L tBHQ and 10?mol/L FS for another 24h).4.Measurement of intracellular ferrous ion level:After treatment,cells from each group were lysed.The protein concentration of each group was determined by the BCA assay,and then the concentration of ferrous ion in the lysis buffer was detected using the Ferrozine assay.5.Detection of cell injury and apoptosis:Lactic dehydrogenase(LDH)release assay and flow cytometry were used to detect PC12 cell injury and apoptosis,respectively.6.Detection of apoptosis-related protein:Western Blot was used to detect the expression of apoptosis-related protein,Bax,Bc1-2,Cytochrome C and Cleaved Caspase-3 in PC12 cells.7.Detection of OS-related marker:2',7'-Dichlorofluorescin diacetate(DCFH-DA)staining was applied to measure the intracellular ROS content-The levels of intracellular malondialdehyde(MDA)and ?-Histone H2A.X were detected to evaluate lipid peroxidation and DNA oxidative damage in PC12 cells,respectively.8.Detection of inflammation-related index:The levels of nuclear factor kappa B(NF-?B)p65 in nuclear protein,as well as I kappa B ?(I ? B ?)and Cyclooxygenase-2(COX-2)in total protein were detected using Western Blot.The contents of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)in cellular supernatant were measured with ELISA.9.Detection of Nrf2 protein nuclear translocation and gene transcription:The nuclear and cytoplasmic levels of Nrf2 protein were detected using Western Blot.The level of Nrf2 mRNA in each group was tested by qRT-PCR.10.Detection of the expressions of Nrf2 downstream enzymes:Heme oxygenase-1(Hmox-1),NAD(P)H quinone oxidoreductase-1(Nqo1)and glutathione peroxidase 1(Gpx1)belong to Nrf2 downstream enzymes.Their expressions were detected by Western Blot.11.Determination that whether the protective effect of tBHQ is dependent on the Nrf2/ARE pathway:PC12 cells were transfected with Nrf2 siRNA,and Western Blot was used to examine the efficiency of Nrf2 siRNA.Subsequently,LDH release assay was applied to determine that whether Nrf2 knockdown could abolish the protective effect of tBHQ.12.Animal grouping:According to different treatments,Wistar rats were randomly dived into 4 groups:Sham group,tBHQ treatment group,FS injury group and tBHQ+FS group.For tBHQ treatment group and tBHQ+FS group,the tBHQ injection was performed at 24h before intracerebral stereotaxic injection.The the normal saline that contains 30mmol/L tBHQ was intraperitoneally injected 3 times at 3.33mL/kg separated by 8h intervals.For Sham and FS injury group,equal volume of normal saline which did not contain tBHQ was injected in the same way.13.The construction of animal model:Intracerebral stereotaxic injection of FS or normal saline was performed at 24h after the first intraperitoneal injection.10?L normal saline containing 1mmol/L FS was stereotaxically injected into the right striatum of rats from FS injury group and tBHQ+FS group.For Sham group and tBHQ treatment group,equal volume of normal saline that did not contain FS was injected in the same way.14.Detection on the effect of tBHQ intraperitoneal injection on Nrf2 gene transcription and protein nuclear translocation in rat striatum:Nuclear and cytoplasmic protein,as well as the total RNA were isolated from the right striatum of the rats in Sham and tBHQ treatment group.The level of Nrf2 in nuclear and cytoplasmic protein was detected with Western Blot,and the Nrf2 mRNA was measured by qRT-PCR.15.Detection of the markers for lipid peroxidation and inflammatory response in rat striatum:The content of 4-HNE in rat striatum was measured by ELISA.The expressions of NF-?B and ionized calcium-binding adapter molecule 1(Iba1)were detected by immunohistochemistry.16.Detection of cell apoptosis and lesion volume in rat striatum:Cell apoptosis in rat striatum was detected by TUNEL staining.For the measurement of lesion volume,fixed rat brain was placed on a shaped container which was specially designed for rat brain sectioning.The brain was cut coronally through the needle entry site,and then serially sliced(2 mm thickness)anterior and posterior to the needle entry plane.Photographs of the serial slices were taken and the lesion area was measured using ImageJ.The lesion volume was calculated by summing the lesion area in each section and multiplying the distance between sections.17.Detection of rats' neurological deficits:The neurological deficits were evaluated using a 24-point neurological scoring scale.Results1.Selection on the concentration and time period of FS treatment:Higher concentration of FS induced lower viability of PC12 cells.10 ? mol/L FS treatment for 24 h could decrease the viability by 55%.Exposure of PC12 cells to 10? mol/L FS resulted in a quick decrease in the viability within 24 h.However,the degree of reduction in viability from 24 h to 36 h significantly decreased.Therefore,a dose of 10 ? mol/L FS treatment for 24 h was applied in our following experiments.2.FS treatment significantly increased the level of ferrous ion within PC12 cells:Ferrozine assay showed that 10 ?mol/L FS treatment for 24 h could increase the level of intracellular ferrous ion for more than 10 times.However,tBHQ had no effects on the level of intracellular ferrous ion in FS-treated PC12 cells.3.tBHQ alleviated the FS-induced cell injury and apoptosis in PC12 cells:FS increased the LDH content in the culture medium,while tBHQ diminished that effect.FS stimulation raised the apoptosis rate of PC12 cells from 3.75%to 13.30%.However,tBHQ pretreatment alleviated FS-induced apoptosis,decreasing the apoptosis rate to 9.08%.4.tBHQ inhibited the FS-induced increase in the expression of apoptosis-related proteins in PC12 cells:FS treatment upregulated the expression of Bax and downregulated Bcl-2 expression,thus increasing the expression ratio of Bax/Bcl-2.Moreover,FS could induce the expression of Cytochrome C and Cleaved Caspase-3.tBHQ pretreatment inhibited the FS-induced increase in the expression ratio of Bax/Bcl-2 and the expressions of Cytochrome C and Cleaved Caspase-3.5.tBHQ alleviated the FS-induced OS in PC12 cells:DCFH-DA staining showed that tBHQ could suppress the FS-induced ROS production.Additionally,tBHQ diminished the FS-induced increase in the content of MDA and y-Histone H2A.X,suggesting tBHQ alleviated the FS-induced lipid peroxidation and DNA oxidative damage.6.tBHQ alleviated the FS-induced inflammatory response in PC12 cells:The nuclear translocation of NF-?B p65 was evaluated by detecting its protein level in nuclear fraction.FS increased the level of NF-? B p65 in nuclear protein and induced the phosphorylation of I?B?.However,tBHQ inhibited the FS-induced NF-? B p65 nuclear translocation and I ? B ?phosphorylation.The expressions of COX-2,TNF-? and IL-1 ? in PC12 cells were increased after FS treatment,and those effects could be inhibited by tBHQ.7.tBHQ promoted the nuclear translocation of Nrf2 but diminished the FS-induced increase in Nrf2 transcription and expression:Treatment with tBHQ increased the nuclear fraction of the Nrf2 protein and reduced the Nrf2 protein expression in the cytoplasm.FS stimulation increased the Nrf2 expression levels in both the nuclear fraction and the cytosolic fraction,but those effects could be inhibited by tBHQ pretreatment.The results of qRT-PCR showed that tBHQ treatment had no effects on the gene transcription of Nrf2.However,FS significantly increase the level of Nrf2 mRNA,and that effect was inhibited by tBHQ pretreatment.8.tBHQ increased the expression levels of Nrf2 downstream enzymes,but reduced the FS-induced increase in their expression levels:tBHQ treatment induced the expressions of Nrf2 downstream enzymes-Hmox-1,Nqol and Gpxl,while FS could increase their expressions to a greater extent.However,tBHQ pretreatment diminished the FS-induced increase in the expressions of Hmox-1,Nqo1 and Gpx1.9.The protective effects of tBHQ on FS-treated PC12 cells are dependent on the Nrf2/ARE pathway.PC12 cells were transfected with Nrf2 siRNA.The results of Western Blot and qRT-PCR showed that Nrf2 siRNA could reduce the level of Nrf2 protein and mRNA in PC12 cells.The results of LDH release assay showed that FS had damage effects on both Ctrl siRNA and Nrf2 siRNA-transfected cells,but the damage in Nrf2 siRNA-transfected cells was more severe,indicating that the Nrf2/ARE pathway is of vital importance for PC12 cells to combat the ferrous ion-induced neurotoxicity.Additionanlly,Nrf2 siRNA transfection could abolish the protective effect of tBHQ on PC12 cells.This result suggested that the protective effects of tBHQ on FS-stimulated PC12 cells were dependent on the Nrf2/ARE pathway.10.Intraperitoneal injection of tBHQ induced Nrf2 nuclear translocation,but had no effects on the gene transcription of Nrf2 in rat striatum:tBHQ increased the level of Nrf2 in nuclear protein,which was isolated from rat striatum.Meanwhile,the protein level of cytosolic Nrf2 was reduced by tBHQ.The results of qRT-PCR showed that tBHQ did not affect the Nrf2 mRNA level in rat striatum.11.Intraperitoneal injection of tBHQ alleviated the FS-induced OS and inflammatory response in rat striatum:Intracerebral injection of FS increased the level of 4-HNE,a byproduct of lipid peroxidation,in rat striatum.tBHQ diminished the FS-induced increase in 4-HNE level.Immunochemistry showed that intracerebral FS injection induced the activation of NF-?B and microglia,while those effects could be inhibited by intraperitoneal injection of tBHQ.12.Intraperitoneal injection of tBHQ alleviated the FS-induced cell apoptosis in striatum and rat neurological deficits,but not the lesion volume:As compared with the FS injury group,the tBHQ+FS group had lower TUNEL-positive cell rate and neurological deficit score.However,the lesion volume between these two groups had no significant difference.Conclusion1.Ferrous ion stimulaition could induce neural oxidative and inflammatory injury,thus causing neural cell apoptosis.2.tBHQ mitigates the ferrous ion-induced neural oxidative and inflammatory injury,and alleviates cell apoptosis and neurological defiits,thus exerting neuroprotective effects.3.The neuroprotective effects of tBHQ are dependent on the Nrf2/ARE pathway.PART ?:THE EFFECT AND MECHANISM OF PEROXIREDOXIN 2 ON FERROUS ION-INUCED NEUROLOGICAL INJURYObjectiveTo determine the effects of ferrous ion stimulation on Prdx2 expression in PC12 cells,as well as the effects of Prdx2 knockdown on ferrous ion-induced OS,inflammatory response,cell injury and apoptosis in PC12 cells.Methods1.Detection on the effects of FS on Prdx2 expression in PC12 cells:Immunofluorescence and Western Blot were applied to detect the Prdx2 expression in PC12 cells.The level of Prdx2 mRNA was measured with qRT-PCR.2.Construction of the PC12 cell line with stable expression of Prdx2 shRNA:Lentiviruses that expressed green fluorescence protein(GFP)and Prdx2 shRNA or Ctrl shRNA were transfected into PC12 cells.At 24h after transfection,cells were passaged at 10-times dilution.Once the cells attached,the culture medium was changed to the complete medium that contained 1 ? g/mL puromycin.The medium was changed every 3 days,and the cell line with stable expression of Prdx2 shRNA could be acquired after 1 week.The expression of GFP in the stable cell line was observed using fluorescence microscopy,and the levels of Prdx2 mRNA and protein were detected by qRT-PCR and Western Blot,respectively.3.Grouping for cell experiment:There are 4 groups in cell experiments:Ctrl group,FS stimulation group(PC12 cells were treated with 10 ?mol/L FS for 24h),FS+Prdx2c group(PC12 cells with stable expression of Ctrl shRNA were treated with 10 ? mol/L FS for 24h),FS+Prdx2i group(PC12 cells with stable expression of Prdx2 shRNA were treated with 10 ?mol/L FS for 24h).4.Detection on the intracellular ferrous ion concentration:We used the Ferrozine assay to measure the ferrous ion concentration within the cells from each group,so as to determine that whether Prdx2 knockdown could affect the ferrous ion content in FS-treated PC12 cells.5.Detection of cell injury and apoptosis:The injury and apoptosis of PC12 cells were detected with LDH release assay and flow cytometry,respectively.6.Detection of apoptosis-related protien:The levels of apoptosis-related protein,Bcl-2,Bax,Cytochorme C and Cleaved Caspase-3 in PC12 cells were detected using Western Blot.7.Detection of OS-related markers:The extent of protein oxidation was evaluated by detecting 3'-Nitrotyrosine using immunofluorescence.The measurement of intracellular MDA content was applied to assess lipid peroxidation.The degree of DNA oxidative injury was evaluated by detecting the expression of ?-Histone H2A.X using Western Blot.8.Detection of inflammation-related markers:The levels of NF-?B p65 in nuclear protein and COX-2 in total protein were detected by Western Blot.The contents of TNF-? and IL-1 ? in supernatant were measured using ELISA.Results1.FS induced the transcription and expression of Prdx2 in PC12 cells:After FS stimulation,the levels of Prdx2 protein and mRNA increased.2.In PC12 cells with stable expression of Prdx2 shRNA,the transcription and expression of Prdx2 were significantly inhibited:In the Prdx2 shRNA stable cell line,almost all of the cells expressed GFP,and the levels of Prdx2 mRNA and protein were significantly decreased.3.Prdx2 knockdown had no effects on the ferrous ion concentration within FS-stimulated PC12 cells:The results of Ferrozine assay showed that FS stimulation increased the ferrous ion concentration in PC12 cells,while Prdx2 knockdown had no influence on that effect.4.Prdx2 knockdown intensified the FS-induced cell injury and apoptosis in PC12 cells:Prdx2 knockdown further promoted the FS-induced LDH release;The results of flow cytometry showed that FS increased the apoptosis rate of PC12 cells from 0.81%to 11.68%,while Prdx2 knockdown could further raise that rate to 19.21%.5.Prdx2 knockdown further promoted the FS-induced increase in the expressions of apoptosis-related protein in PC12 cells:FS increased the expression ratio of Bax/Bcl-2,and the expressions of Cytochrome C and Cleaved Caspase-3,while Prdx2 knockdown could further promote those effects.6.Prdx2 knockdown intensified the FS-induced OS in PC12 cells:The levels of 3'-Nitrotyrosine,MDA and ?-Histone H2A.X in PC12 cells were significantly increased after FS treatment,indicating FS could induce protein oxidation,lipid peroxidation and DNA oxidative injury in PC12 cells.Prdx2 knockdown could further promote the increases of these 3 markers.7.Prdx2 knockdown intensified the FS-induce inflammatory response in PC12 cells:Prdx2 knockdown could further promote the FS-induced NF-?B p65 nuclear translocation and the expressions of COX-2,TNF-a and IL-1 ?.Conclusion:1.Ferrrous ion stimulation could induce the transcription and expression of Prdx2 in PC12 cells.2.Prdx2 knockdown aggravates the ferrous ion-induced cell injury and apoptosis by intensifying OS and inflammatory response in PC12 cells,indicating that Prdx2 plays a vital role in the protection against the the ferrous ion-induced neurological injury. |
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