The Role Of Soluble Epoxide Hydrolase In Neuronal Loss In MPTP-induced Parkinson's Disease Model

Parkinson's disease(PD)is the second most common neurodegenerative disease and is widely thought to primarily affect the dopamine(DA)neurons of the substantia nigra pars compacta(SNpc).Loss of SNpc dopamine neurons results in a concomitant depletion of dopamine(DA)in the striatum,impairing the nigrostriatal system to prevent the execution of coordinated movements.Clinically PD is characterized by motor abnormalities including resting tremor,bradykinesia,muscular rigidity and postural instability.According to the epidemiological survey,the incidence of disease in the general population increases with age fromg 1-2%at 50 years to approximately 5%by the age of 85.By the time a patient first presents with symptoms of Parkinson's disease at the clinic,a significant proportion(50-70%)of the cells in the substantia nigra par compacta(SNc)has already been destroyed.At present there is no cure to rescue the loss of dopaminergic neurons.Since its introduction in 1817,levodopa has remained the most efficacious treatment of PD.Unfortunately,its use is associated with motor complications such as wearing off,dyskinesias,and 'on-off' phenomenon.These complications occur in about 50%of levodopa-treated patients who have received the drug for more than 5 years,in 80%of patients treated for 10 years,and in nearly all patients with young-onset disease.Because PD effects so many old people in the world,the scientists devote themselves to study it.But the etiological factors involved in the development of PD are still elusive,but there is considerable evidence that a combination of genetic susceptibilities and environmental factors plays a critical role in disease pathogenesis.Sixteen PD-related genes have been found.Compare with the other genes,we know more about a-Synuclein,Parkin,LRRK2,Pink-1,UCHL-1.Actually,about 5-10%of all parkinsonian cases are familial,most of the parkinsonian cases are sporadic.The study of environmental factor begins from the discovery of MPTP,a drug with neurotoxicity.MPTP induced-Parkinsonism was first reported in a group of drug addicts,that developed PD after the injection of a synthetic heroin in the 1980s.Later studies found that after administration,MPTP can pass the blood-brain barrier and be metabolized by monoamine oxidase to MPP+.MPP+,which can be selectively uptaken by dopamine transporter,inhibits complex I in the mitochondria and induces degeneration of DAergic neurons.Because of this found,people pay more attentions to the chemicals from environment.Later the scientists found people who always exposed to pesticide and herbicide will get the higher rate of PD.As the research goes on,we know that PD patients have protein aggregation in their brain which calls"lewis body".In 1997,the scientists found the mutant of a-Synuclein is the main component of "lewis body".And now mitochondrial dysfunction,oxidative stress and inflammation are considered to be the major mechanisms for Parkinson's Disease.The study of Parkinson's disease is never stopped but now the etiology is still unclear.For this reason there is no guidance for clinic.So we study sporadic PD to find an underlying mechanism which can reduce dopaminergic neurons' susceptibility to environment stimulators.Epoxide hydrolases catalyse the hydrolysis of electrophilic,and therefore potentially genotoxic epoxides to the corresponding less reactive vicinal diols,which explains the classification of epoxide hydrolases as typical detoxifying enzymes.Microsomal epoxide hydrolase and soluble epoxide hydrolase are the main two kinds of epoxide in our body,one exists in microsoma,named microsomal epoxide hydrolase and the other exists in cytoplasm,named soluble epoxide hydrolase.Soluble epoxide hydrolase(sEH)is the major enzyme responsible for the metabolism and inactivation of epoxyeicosatrienoic acids(EETs).EETs are produced by the cytochrome P450(CYP450)epoxygenase pathway of arachidonic acid(AA)metabolism and tend to be anti-hypertensive,anti-inflammatory and protective against ischemic injury in periphery system.In the central nervous system,EETs are thought to play a role in the regulation of local blood flow,protection from ischemic injury,inhibition of inflammation,the release of peptide hormones and modulation of fever.EETs have been found to activate GPCR,TRPV4,PPAR-gamma and dopamine D3 receptor.However,little is known about the relationship between soluble epoxide hydrolase and Parkinson's disease.Since the metabolism of EETs by sEH reduces or eliminates their bioactivity,inhibition of sEH has become a therapeutic strategy for clinical use.In this study we use the soluble epoxide hydrolase gene deletion mice mating with the C57BL/6 background.We use sEH(-/-)as the experiment group and the littermate sEH(+/+)asa control to establish the PD animal model.Our purpose is to find wether the changes in sEH are involved in the pathogenesis of PD by modulating the vulnerability of dopaminergic neurons to environmental stress,and explor the mechanism underlying.We first establish the PD model in C57BL/6 wild type mice.We employed acute PD model.Eight to ten week-old male mice received one i.p.injection of MPTP · HCl per day(40 mg/kg per day of free base)for two consecutive days and were killed at 14 days after the last injection.Controls received saline injections only.At the end of the experimental period,mice were anesthetized with 10%chloralic hydras and perfused transcardially with ice-cold normal saline and then perfused transcardially with buffered paraformaldehyde(4%w/v)and the brain was dissected out and processed for immunohistochemistry.The number of dopaminergic neurons in SNc in control group and MPTP-treated group is 137±3 and 73±6 respectively.Dopaminergic neurons in the MPTP-treated group is significant fewer than in the control group.Next,we confirmed the genotype of mice.We used polymerase chain reaction(PCR)to identify the genotype,but also immunocytochemistry and western blotting to confirm the deletion of protein expression.All the data illustrate that sEH is not expressed in the gene knockout mice.After confirming the genotype of mice,PD model was established in sEH(-/-)and sEH(+/+)mice.Twelve sEH(-/-)or sEH(+/+)mice were divided into two groups respectively,with one group receiving MPTP and the other group receiving the same volumn of saline.The dose and the scheme of MPTP were used as described above.The number of dopaminergic neurons in SNc is 141 ±5 in sEH(+/+)/Saline group,and 139±8 in sEH(-/-)/Saline group((P>0.05).The number of dopaminergic neurons in SNc is 65±9 in sEH(+/+)/MPTP group,98±4 in sEH(-/-)/MPTP group((P<0.01).All the results indicated that sEH are involved in the pathogenesis of PD by modulating the vulnerability of dopaminergic neurons to the neurotoxicity induced by MPTP.To confirm that the neuroprotective effect in sEH knockout mice is mediated by sEH deletion,we inhibited sEH in wild type mice to see if sEH inhibitor protected dopaminergic neurons from MPTP induced neurotoxicity.Two days before MPTP injection,5mg/kg AUDA was poured into mice stomach,then the following two days AUDA is administered together with MPTP.The number of dopaminergic neurons is 133±4 in Saline group,135±4 in AUDA group,81 ±7 in MPTP group,120±5 in MPTP+AUDA group.Difference between MPTP group and MPTP +AUDA group had statistical significance(P<0.01).Some studies had demonstrated that sEH inhibition can increase the level of EETs and 14,15-EET has the most important biological function.Because EETs have multiple biological functions,we hypothesize that the different susceptibilities between sEH(-/-)and sEH(+/+)groups is due to the different level of EETs.To verify our hypothesis EET inhibitor was administered into sEH(-/-)mice.1.5?g 14,15-EEZE(an inhibitor of 14,15-EET)was injected into lateral ventricle 30min before MPTP injection for two concessive days.The number of dopaminergic neuron is 100±6 in MPTP group,63±9 in MPTP+14,15-EEZE group,144±12 in control group.14,15-EEZE reversed the neuronal protecting effect in sEH(-/-)mice(P<0.05).To further prove that the neuroprotection in sEH(-/-)mice is mediated by increase of EETs,sEH(+/+)mice and sEH(-/-)were given 2?g 14,15-EET or 11,12-EET in lateral ventricle 30min before MPTP injection for two concessive days.Mice were killed at 14 days after the last injection.Controls received saline injections.For sEH(+/+)mice,the number of dopaminergic neuros in MPTP group is 65±9,in MPTP+14,15-EETs group is 93±3,in control group is 140±5.MPTP+11,12-EET group is 69±8.14,15-EET can protect dopaminergic neurons from MPTP-induced neurotoxicity in sEH(+/+)mice(P<0.05).However,for sEH(-/-)mice,the number of dopaminergic neurons in MPTP group is 99±4,in MPTP+14,15-EETs group is 89±7,in control group is 139±8,MPTP+11,12-EET group is 90±8.14,15-EETs or 11,12-EET group didn't protect dopaminergic neurons in sEH-/-mice(P>0.05).These data indicated that the increased EETs in sEH(-/-)mice protected dopaminergic neurons from MPTP-induced neurotoxicity.It is reported that EETs can activate PI3K/AKT signal,which is one of the most potent intracellular mechanisms to promote cell survival.So we want to know if EETs is involved in the PI3K/AKT pathway to protect dopaminergic neurons from damage induced by MPTP.In the beginning we established the PD cell model in cortical neurons dissected from sEH(+/+)or sEH(-/-)new born mice.We treated the neuron for 24h,or 48h,or72h with 20?M or 40?M MPP+(an active form of MPTP)to confirm that sEH gene deletion can rescue the neurons from MPP+ damage.After 20?M MPP+ treatment for 24h,the rate of survival in sEH(+/+)and sEH(-/-)are73%±7.46%and 86%±7.54%respectively(P>0.05).After 48h,the survival rate in sEH(+/+)and sEH(-/-)are48%±4.88%and65%±8.93%respectively(P<0.05).After 72h,the rate of survival in sEH(+/+)and sEH(-/-)are39%±4.12%and55%±11.06%respectively(P<0.05).After 40?M MPP+ treatment for 24h,the rate of survival in sEH(+/+)and sEH(-/-)are 68%±15.7%and76%±13.1%respectively(P>0.05).After 48h,the rate of survival in sEH(+/+)and sEH(-/-)are47%±6.5%and63%±10.2%respectively(P<0.05).After 72h,the rate of survival in sEH(+/+)and sEH(-/-)are35%±6.2%and50%±9%respectively(P<0.05).In conclusion,sEH deletion mice can protect dopaminergic neurons from neurotoxicity induced by MPTP in both PD animal model and cell model.And this phenomenon was mimiced by inhibiting sEH by its inhibitor in wild type mice.Together with MPTP treatment,exogenous EETs reduce the loss of dopaminergic neurons in sEH(+/+)mice but not in sEH(-/-)mice.On the contrary,inhibiting EETs abolishing the protecting role of EETs in sEH(-/-)mice.