| Cytochrome P450(cytochrome P450,CYP)superfamily is involved in the metabolism,inactivation and activation of endogenous and exogenous substances.CYP superfamily is mainly expressed in the liver,but recent studies have shown that CYPs are also expressed in extrahepatic organs,such as the brain.Studies have shown that brain CYPs can metabolize or activate exogenous drugs and poisons within the brain.In the brain,there are significant differences in the expression levels of different CYP isoforms.The expressed CYP isoforms were different in various brain regions and neuronal cell types.According to mRNA level analysis,the major CYP isoforms expressed in the brain include CYP46A1,CYP2D6,CYP2E1,CYP2J2,CYP2U1 and CYP1B1.CYP2A1,CYP2J1 and CYP2U1 were expressed in neurons and astrocytes,and CYP1B1 was mainly expressed in microglia.Cell membrane phospholipids can be metabolized to arachidonic acid(AA)by phospholipase.AA can be further metabolized to more than 100 kinds of derivatives.These derivatives are the material basis for the cells to complete some important biological function.AA mainly through three ways to metabolize:(1)cyclooxygenase(COX)metabolic pathway;(2)lipid oxidase(LOX)metabolic pathway;(3)CYPs metabolic pathway.The metabolic reactions of arachidonic acid catalyzed by CYPs can be divided into:(1)propylene oxidation reaction,the main metabolite of hydroxyeicosatetraenoic acid(HETE),including 5-,8-,9-,11-,12-,15-HETE;(2)?and ?-1 hydroxylation reaction,including the main metabolites 19-,20-HETE;(3)epoxidation,including the main metabolites of eneecosatrienoic acid(EET),including 5,6-,8,9-,11,12-,14,15-EET.EETs are hydrolyzed to soluble biologically stable metabolites by soluble epoxide hydrolase(sHE).In the peripheral,CYP isoforms involved in AA metabolism include CYP1A,CYP1B,CYP2B,CYP2C,CYP2E,CYP2J,CYP2U,CYP4A,CYP4F.However,little is known about the biological behavior of brain CYPs in the AA metabolic network.Glutamate is an important excitatory neurotransmitter in the human central nervous system,and participates in the regulation of various physiological functions of central nervous system.Astrocytes are the largest number of neuronal cells in the brain,and paly an important role in glutamic acid-glutamate cycle.The recent study found the presence of glutamate receptors on astrocytes,suggesting that glutamate may affect the neurophysiological function of astrocytes.In this paper,we used in vitro and in vivo experiments to study the regulation of brain CYPs with metabolic function of AA by glutamate using molecular biology and analytical chemistry techniques.The results of the study will provide an important data base for further understanding of the regulation of brain CYPs-mediated AA metabolism by neurotransmitter.Part I:Glutamate regulates brain CYP2J and its mediated arachidonic acid epoxide metabolitesObjective:Glutamate is the main excitatory neurotransmitter in the brain,and glutamate excitotoxicity is involved in the pathophysiological processes of a variety of neurodegenerative diseases.It is known that mGluR3 and mGluR5 are the major glutamate receptors expressed in the astrocytes,and mGluR5 may play an important role in maintaining glutamate homeostasis.CYP2J is an important extracellular expression of the CYP isoform,which has been shown to play an important role in the metabolism of AA epoxide EETs.EETs can act as autocrine or paracrine factors,and play a role in the regulation of inflammation,vascular tension and so on.The aim of this part is to investigate the effect of glutamate on brain CYP2J and CYP2J-mediated EETs metabolites.Methods:Human glioma cell U251 cell lines were treated with different concentrations(10-50 ?M)of glutamate for 24 h,and mGluR5 and CYP2J2 mRNA levels were detected by quantitative RT-PCR.The U251 cell line was pretreated with the specific inhibitors of mGluR5 and MAPK(ERK,JNK,p38)for 30 min,then treated with glutamate(50 ?M)for 24 h.The mRNA level of CYP2J2 was detected by quantitative RT-PCR.After pretreatment with the specific inhibitors of MAPK molecules for 30 min,glutamate(50 ?M)was administered for 24 h,and the binding ability of CREB to CYP2J promoter was detected by chromatin immunoprecipitation(ChIP).The CREB expression plasmid and the recombinant plasmid containing the CYP2J2 promoter were co-transfected in U251 cell line to detect the regulation of CREB on CYP2J2 promoter.Wistar rats were mated,and the newborn rats were randomly divided into control group and glutamate group.The newborn rats in the glutamate group were subcutaneously injected with glutamate(4 mg/g)at 1,3,5 and 7 days.At 10 weeks after birth,the animals were sacrificed.The levels of mGluR5 and CYP2J2 mRNA in different brain regions,as well as the levels of EETs and DHETs,were measured in different brain regions(hippocampus,cerebellum,frontal cortex and brainstem).DMEM derivatization was used to treat EETs and DHETs,and the respective standards of different metabolites were prepared.The levels of EETs and DHETs in different brain regions were detected by LC-MS/MS,and the differences of metabolites in different brain regions were analyzed by PLS-DA analysis.Results:In vitro experiment,glutamate regulated CYP2J2 mRNA levels in a dose-and dose-dependent manner,compared with the control group.The pretreatment of MPEP could attenuate the induction of CYP2J2 by glutamate,suggesting that mGluR5 may mediate the regulation of CYP2J2 by glutamate.Compared with the control group,glutamate treatment upregulated ERK1/2,p38 and JNK phosphorylated protein levels.In U251 cell lines respectively pretreated with ERK1/2(U0026),p38(SB202190)and JNK(SP600125)for 30 min,the induction of CYP2J2 mRNA by glutamate were significantly inhibited.The data from chromosome immunoprecipitation experiments showed that glutamate(50 ?M)increased the binding of CREB to the CYP2J2 promoter,which was 1.78 times higher than that of the control group.However,the administration of specific inhibitors for ERK1/2(U0126),p38(SB202190),JNK(SP600125)inhibited the binding of CREB to the CYP2J2 promoter.In the rats treated with glutamate,the mGluR5 level in the frontal cortex,hippocampus,cerebellum and brainstem were increased by 1.49-fold,3.82-fold,1.46-fold and 1.41-fold,respectively,compared with the controls.The results suggest that high doses of glutamate exposure during childhood can lead to mGluR5 up-regulation,which causes sustained activation of the glutamate system.At the same time,the expression level of CYP2J3 in glutamate-treated animals was respectively increased in the frontal cortex,hippocampus,cerebellum and brainstem by 2.21-fold,8.27-fold,1.5-fold and 3.53-fold.The levels of EETs and DHETs metabolites in the hippocampus and cerebellum,frontal cortex and brainstem were significantly different by PLS-DA analysis.The EETs and DHETs were 1.16-fold,1.18-fold,1.18-fold and 1.19-fold higher in the frontal cortex,hippocampus,cerebellum,brainstem and brainstem of adult animals,compared with the controls.Conclusion:Glutamate can induce the expression of brain CYP2J in a time-and dose-dependent manner,and affect the formation of EETs within the brain.Glutamate activates MAPK signaling pathway(ERK1/2,p38 and JNK)molecules through the receptor mGluR5 on astrocytes,thereby increasing the binding of CREB to the CYP2J2 promoter and up-regulating CYP2J.Part ?:Glutamate regulates brain CYP1B1 and CYP2U1 and its mediated arachidonic acid hydroxy metabolitesObjective:Morphological data showed that astrocytes were an important component of functional neurovascular units(NVU).Vascular endothelial cells and astrocytes together constitute the basement membrane barrier,and endothelial cells,peripheral cells and end-feet of astrocytes closely linked.Previous studies have shown that CYP1B1 and CYP2U1 are highly expressed in astrocytes.In addition,CYP1B1 and CYP2U1 are the only CYP isoforms that can be detected in freshly isolated human brain microvessels.The in vitro experiments showed that CYP1B1 could metabolize AA to the midchain of hydroxy dioctatetraenoic acid(HETEs)and terminal chain HETEs.At the same time,CYP2U1 can metabolize AA to 19-HETE and 20-HETE.In view of a various metabolites from AA with vascular regulation activity,the aim of this part is to further study the regulation of brain CYP1B1 and CYP2U1 and HETEs metabolic levels by glutamate based on Part ?.Methods:Human glioma cell U251 cell line and immortalized human brain microvascular endothelial cell line hCMEC/D3 were treated with different concentrations(10-50 ?M)glutamate for 24 h.CYP1B1 and CYP2U1 were detected by quantitative RT-PCR MRNA levels.U251 cell line was pretreated with the specific inhibitors for mGluR5 and MAPK(ERK,JNK,p38)for 30 min,then treated with glutamate(50 ?M)for 24 h.The levels of CYP1B1 and CYP2U1 mRNA were detected by quantitative RT-PCR to detect whether MAPK is involved in the regulation of glutamate on CYP1B1 and CYP2U1 mRNA.After pretreatment with the specific inhibitors of MAPK molecules for 30 min,glutamate(50 ?M)was administered for 24 h,and the binding ability of CREB to CYP1B1 and CYP2U1 promoter was detected by chromatin immunoprecipitation(ChIP)in U251 cells.The CREB expression plasmids and the recombinant plasmids containing CYP1B1 and CYP2U1 promoters were co-transfected in U251 cell lines to detect the regulation of CYP1B1 and CYP2U1 promoters by CREB.Wistar rats were mated,and the newborn rats were randomly divided into control group and glutamate group.The newborn rats in the glutamate group were subcutaneously injected with glutamate(4 mg/g)at 1,3,5 and 7 days.At 10 weeks after birth,the animals were sacrificed.The levels of CYP1B1 and CYP2U1 mRNA and HETEs were measured.DMEM was used to derivatize HETEs to prepare the respective standards for different metabolites.The levels of HETEs in different brain regions were measured by LC-MS/MS,and the differences of metabolites in different brain regions were analyzed by PLS-DA analysis.Results:In vitro experiments showed that glutamate(10,25,50 ?M)could upregulate the expression of CYP1B1 and CYP2U1 mRNA in U251 and hCMEC/D3 cell lines in a dose-dependent manner.In the U251 cell line,the mRNA levels of CYP1B1 and CYP2U1 increased by 1.68-fold and 1.80-fold,respectively,compared with the control group.At the same time,the mRNA levels of CYP1B1 and CYP2U1 were increased by 1.79-fold and 1.34-fold in hCMEC/D3 cell lines.In the U251 cell line,MAPK specific inhibitors(U0126,SB202190,SP600125)and mGluR5 specific inhibitor,MPEP,could significantly inhibit the induction of CYP1B1 and CYP2U1 mRNA by glutamate.Compared with the control group,ERK,p38 and JNK phosphorylated protein levels were up-regulated in U251 cells treated with glutamate(50 ?M)for 15 min.However,the activation of MAPK pathways by glutamate can be attenuate by mGluR5 specific inhibitor(MPEP).The results of immunofluorescence showed that CREB protein was mainly present in the nucleus,and glutamate could up-regulate the phosphorylated CREB level in the nucleus.The administration of mGluR5-specific inhibitor MPEP significantly inhibited the phosphorylation of CREB protein induced by glutamate.At the same time,ChIP experiments confirmed that glutamate could induce the binding of CREB to CYP1B1 and CYP2U1 promoters,and MPEP reduced the binding of CREB to the promoters.Compared with the control group,the levels of CYP1B1 mRNA in the frontal cortex,hippocampus,cerebellum and brainstem were respectively increased by 1.65-fold,1.53-fold,1.58-fold and 1.54-fold after subcutaneous injection of monosodium glutamate;CYP2U1 mRNA levels were increased by 1.31-fold,1.82-fold,1.47-fold and 1.56-fold.The HETEs levels of metabolites in the hippocampus and cerebellum,frontal cortex and brainstem were significantly different by PLS-DA analysis.Compared with the control group,the total amount of 5-,8-,11-,12-,and 15-HETE in the glutamate treated group increased by 1.15-fold,1.12-fold,1.13-fold and 1.10-fold in the frontal cortex,hippocampus,cerebellum and brainstem respectively.The 5-HETE production in the glutamate treatment group was increased significantly in the frontal cortex,hippocampus,cerebellum and brainstem.Compared with the control group,the total amount of 19-and 20-HETE generated in the frontal cortex,hippocampus,cerebellum and brainstem from glutamate-treated animals were increased by 1.12-fold,1.21-fold,1.17-fold and 1.10-fold respectively.Although the amount of 19-HETE production did not change,the generation of 20-HETE was significantly increased in all tested brain regions.Conclusion:The results suggest that low concentration of glutamate can change the metabolism of AA via the regulation of brain CYPIB1 and CYP2U1.Glutamate up-regulated CYPIB1 and CYP2U1 expression through MAPK-CREB signaling pathway coupled with mGluR5 in astrocytes.Combined with the experimental data from Part I,these data indicate that glutamate may affect AA metabolism within the brain via brain CYPs.Although it still need to further study whether neurons regulate cerebral blood flow through astrocytes,the data from this paper suggest that CYPs in astrocytes can be an important pathway for AA metabolism in the central nervous system,and that is neurotransmitter glutamate can regulate brain CYPs. |
PDF Full Text Request