| Objective:Parkinson's disease(PD)is the second most important age related neurodegenerative disorder following Alzheimer's disease.Arachidonic acid(AA)and their derivatives are important endogenous substances involved in inflammatory reactions,among them,the Epoxyeicosatrienoic acids(EETs)are mainly metabolized by CYP2Js and CYP2Cs of cytochrome P450.The research shows that the expression of CYP2Js in the brain is abundant,and the content of CYP2Cs is low.This paper intends to explore the possible protective effects and its mechanism of brain CYP2J in animal models of Parkinson's disease(PD)by studying in vivo and in vitro.Methods:U251 cells were given LPS(1 ?g/ml)for 24 h or given TLR4 inhibitor CLI-095(1 ?M)and MyD88 inhibitor ST 2825(20 ?M)pretreated 1 h before giving LPS 24 h,quantitative real-time PCR(qRT-PCR)detects the expression of CYP2J2 and transcription factor CREB,the amount of phosphorylated CREB in the nucleus was detected by imunofluorescence.Chromatin immunoprecipitation assay(ChIP)was used to detect the binding of CREB with CYP2J2 promoter.CYP2J2 overexpression plasmid transiently transfected U251 cells in 24 h and qRT-PCR detected SOD1,NRF2,KEAP1,CAT,GPX1,MANF,PERK,IRE la and ATF6 mRNA expression.U251 cells were given AUDA(1 ?M)and 14,15-EET(1 ?M)in 24 h respectively,or after pretreatment AUDA 1 h,then giving 14,15-EET to play with 24 h,qRT-PCR detection related antioxidant stress gene expression.CYP2J3 overexpression plasmid was transfected into the substantia nigra neurons of the Wistar male rats by lentivirus packaging,the animals were killed 3 days,12 days and 21 days respectively after injection,and the transfection efficiency was observed by immunofluorescence method.Male Wistar rats were injected with LPS or 6-OHDA into the right SNc as PD model(n = 9).Animals were given apomorphine(0.5 mg/kg)after LPS injection 12 days and 21 days,to rotational behavioral testing.The animals were killed after LPS injection 21 days,and qRT-PCR was used to detect the expression of CYP2J2,CREB,and the related antioxidant stress genes;the right substantia nigra of the brain was used to immunohistochemical staining of TH.In the LPS induced PD animal model,rats were randomly divided into control group,single group LPS(10 ?g),LPS and CYP2J3 overexpression group,LPS and empty plasmid group.In the 6-OHDA induced PD animal model,Rats were randomly divided into control group,single group 6-OHDA(8 ?g),6-OHDA and CYP2J3 overexpression group,6-OHDA and empty plasmid group.CYP2J3 overexpression group was injected with CYP2J3 overexpression plasmid(2 ?L)in SNc,after 3 days later,LPS or 6-OHDA was injected;empty plasmid group was given empty pHAGE plasmid,after 3 days later,LPS or 6-OHDA was injected;the animals in the control group were injected with 2 ?L sterile PBS or sterile saline containing 0.2%vitamin C at the same site at the same time points.Results:In vitro experiments,compared with the control group,LPS significantly inhibited the expression of CYP2J2 and CREB,the decrease rate was 67.43%(p<0.001)and 34.8%(p<0.01)respectively.Compared with the LPS group,the TLR4 inhibitor CLI-095 pretreated cells 1 h and then treated with LPS,CYP2J2 and CREB mRNA levels increased 2.5 times(p<0.001)and 1.43 times(p<0.001),respectively.In comparison with the single LPS group,the MyD88 inhibitor ST 2825 pretreated cells 1 h and then treated with LPS,CYP2J2 and CREB mRNA levels increased 2 times(p<0.001)and 1.3times(p<0.01),respectively.There was no statistical difference between the ST2825 group or CLI-095 group with control group.Transfection of CYP2J2 expression plasmid or administration of exogenous 14,15-EET,oxidative stress genes SOD1,CAT,GPX1,NRF2 and KEAP1 significantly increased(p<0.001),but there was no obvious change of MANF,PERK,IRE1 alpha,ATF6 expression(p>0.05).By immunofluorescence analysis,LPS significantly reduced the amount of p-CREB protein in the nucleus(p<0.01).In ChIP analysis,LPS inhibited the binding of CREB to the CYP2J2 promoter(p<0.01).In the LPS induced rat PD model,animals were rotated at 115 rotations/0.5 h and 233 rotations/0.5 h on 12 and 21 days respectively,whereas the control group did not rotate.Compared with the LPS group,the number of rotations in the LPS + CYP2J3 overexpression group decreased by 42.6%(p<0.01)at 12 days.On 21 days rotation decreased by 12.7%(p<0.05).LPS + plasmid control group and LPS group had no difference.Immunohistochemical data showed that after 21 days of LPS injection,the dopaminergic neurons in the right substantia nigra decreased 58%compared with the uninjured side(p<0.01).In the LPS + CYP2J3 overexpression group,the dopaminergic neurons in the right substantia nigra decreased 43%compared with the uninjured side(p<0.01),LPS + empty plasmid group and LPS group animals were not different.Compared with the control group,LPS inhibited CYP2J3 and antioxidant genes such as SOD1,CAT,GPX1,NRF2 and KEAP1 expression(p<0.001);compared with LPS group,after transfected CYP2J3 overexpression plasmid,the expression of antioxidant genes such as CAT,GPX1,SOD1,NRF2 and KEAP1 were significantly increased(p<0.001).In the PD model induced by 6-OHDA,animals were rotated at 118 rotations/0.5 h and 241 rotations/0.5 h on 12 and 21 days respectively,whereas the control group did not rotate.Compared with the 6-OHDA group,the number of rotations in the 6-OHDA + CYP2J3 overexpression group decreased by 60.7%(p<0.01)at 12 days.On 21 days rotation decreased by 21.4%(p<0.05).6-OHDA + plasmid control group and 6-OHDA group had no difference.Immunohistochemical data showed that after 21 days of 6-OHDA injection,the dopaminergic neurons in the right substantia nigra decreased 73.65%compared with the uninjured side(p<0.001).In the 6-OHDA + CYP2J3 overexpression group,the dopaminergic neurons in the right substantia nigra decreased 57.8%compared with the uninjured side(p<0.01),while 6-OHDA + empty plasmid group and 6-OHDA group animals were not different.Compared with the control group,the CYP2J3 and antioxidant stress genes were significantly downregulated in the 6-OHDA group(p<0.001);Compared with the 6-OHDA group,the expression of antioxidant stress genes such as SOD1,CAT,GPX1,NRF2 and KEAP1 significantly increased after transfection of CYP2J3 expressing plasmid(p<0.001).Conclusions:TLR4-MyD88 pathway participates in the process of LPS regulate CYP2J.LPS inhibits the expression of CYP2J2 at the transcriptional level by inhibiting the binding of CREB to the CYP2J2 promoter site.The data show that transfection of CYP2J can upregulate the expression of antioxidant genes like NRF2 and alter the oxidative stress status of neurons.Treated exogenous substances 14,15-EET can up regulate the expression of mitochondrial antioxidant genes,suggesting that CYP2J may play a role in protecting neurons from injury through metabolite EETs.In the PD animal model of inflammation and oxidative stress,we can find CYP2J of the substantia nigra has the function of protecting dopaminergic neurons and can delay the process of disease.Exogenous administration of CYP2J metabolite EETs is expected to combat the loss of dopaminergic neurons in the early stages triggered by inflammation and oxidative stimuli. |
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