Ablation Of Soluble Epoxide Hydrolase Produces Antidepressive-like Behaviors In Adult Mice

Depression is a common mood disorder with 12-17%lifetime prevalence and is anticipated to be the first leading cause of disability worldwide in 2020.Due to the high prevalence,a high reoccurrence rate and a tendency of suicide,depression has become the most major public health problem.However,the neurobiological mechanisms of depression is still unclear.Although great progress has been made in the area of antidepressant pharmacotherapy,i.e.selective serotonin reuptake inhibitors(SSRIs)and serotonin-norepinephrine reuptake inhibitors(SNRIs)are clearly superior to the older TCAs and MAO inhibitors in terms of efficacy and tissue tolerability,there are three major areas need to be improved.The first is slow response.The vast majority of patients do not respond until 3-6 weeks after the initiation of treatment.It usually takes 6-8 weeks for patients treated with standard dose of antidepressants to return to premorbid levels of functioning.Second is the efficacy.After 6-8 weeks,only 35-45%of patients treated with standard doses of antidepressants return to premorbid levels of functioning.Third is the side effects,including sexual functioning,sleepiness,and weight gain.Therefore,unraveling the neurobiological mechanism of depression,developing new agents with a faster response,a longer duration and less side-effects are in urgent demand.Epoxyeicosatrienoic acids(EETs)is a major product of cytochrome P450-catalyzed arachidonic acid metabolism.EETs exhibit a wide variety of biological functions in anti-inflammation,prevention of platelet aggregation,neuroprotection after ischemia,promotion of angiogenesis and regulation of kidney tubules.The metabolism and biological functions of EETs is mainly regulated by soluble epoxide hydrolase(Ephx2,sEH).sEH is a bifunctional enzyme with COOH-terminal hydrolase and NH2-terminal phosphatase activities,widely expressed in vessels,liver,kidney,heart and brain of mammal animals.It was firstly characterized in early 1973 by Gill whose study focused on Juvenoid 1-(4'-Ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans2-octene.sEH can rapidly convert epoxyeicosatrienoic acids(EETs)to dihydroxyeicosatrienoic acids(DHETs)which has quite low biological activities.Based on the biological functions of EETs and the role of sEH,sEH inhibitors can decrease the degradation of EETs,increase the biological activities of EETs.Therefore,disruption of sEH activity is becoming an attractive therapeutic target for inflammatory diseases,cardiovascular diseases and ischemia.It has been reported that EETs has anti-inflammatory effects.Combined administrations of non-steroidal anti-inflammatory drugs(NSAIDS)and EETs can effectively alleviate pain and inflammation.The anti-inflammatory actions that are attributed to EETs include decreased aggregation of human polymorphonuclear leukocytes and decreased leukocyte adhesion to endothelial cells.The potential role of EETs in hypertension and cardiac remodelling has been determined using the selective sEH inhibitor,N-adamantyl-N'-dodecylurea(ADU),in deoxycorticosterone acetate(DOCA)-salt hypertensive rats.Administration of ADU prevented the further increase in systolic blood pressure and left-ventricular wet weight and normalized endothelial function,EETs can also up-regulate the expression and activity of endothelial nitric-oxide synthase that attribute to prevention of increase in blood pressure.Although the roles sEH played in the pathophysiology of inflammation,cardiovascular and brain injuries have been intensively reported,little is known about the potential effects sEH exerts on depression.In our study,we firstly utilized sEH knock out mice to identify the potential role sEH played in depression.We performed both forced swim test(FST)and tail suspension test(TST)on sEH knock out mice and found sEH knock out mice displayed a much shorter period.of immobility time than wide type mice(t-test,n>10,P<0.05).This results demonstrated that sEH knock out exhibit anti-depressive like behavior.We then conducted open field test(OPT)to investigate locomotor activities of sEH knock out mice and no differences were observed between sEH knock out mice and wide type mice(t-test,n=10 or 15,P>0.05).In elevated plus maze test(EPM)and novelty suppressed feeding test(NSF),which are anxiety-related,there were no differences between sEH knock out mice and wide type mice in the time spent in the open/closed arm,latency or homecage food intake(t-test,n=19 or 20 for EPM,n=12 or 15 for NSF,P>0.05).The inhibition of sEH can promote EETs levels,among 4 kinds of EETs(5,6-EET?8,9-EET?11,12-EET?14,15-EET),14,15-EET possesses the most high biological activity.We gave administration of 14,15-EET(i.c.v.)to C57BL/6J mice to mimic the effects of sEH inhibition and evaluated depressive-like behavior by utilizing forced swim test.C57BL/6J mice which received 0.25ug or 4ug 14,15-EET displayed a significant shorter period time of immobility when compared with control mice which received ACSF injection(P<0.01).14,15-EET(i.c.v.)did not exert influence on the locomotor activity of C57BL/6J mice.This data implied that 14,15-EET injections can induce anti-depressive like behavior in adult mice.There were no differences of total distance and number of rearing between control mice and 14,15-EET treated mice(ANOVA,n=11 or 12,P>0.05).14,15-EE-5(Z)E is an antagonist of EETs.It can effectively inhibit the biological activity of EETs.To further confirm the anti-depressive like behavior of sEH-/-mice was induced by the increased level of EETs,we then gave sEH-/-mice administration of 14,15-EE-5(Z)E(i.c.v.)to reduce the increased EETs level in the brain.Forced swim test was performed to evaluate depressive-like behavior.The data showed 14,15-EE-5(Z)E administration led to a longer period of immobility(t-test,n= 9,P=0.006).14,15-EE-5(Z)E(i.c.v.)can reverse the anti-depressive like behavior of sEH-/-mice in FST.To sum up,our study implicated that EETs-sEH system plays an important role in producing anti-depressant effects.Some studies which based on the cardioprotective effects of sEH postulated that there may be an EET receptor(EETR)within the mitochondria;As we know,mitochondria is the most important source of ATP.Our previous study demonstrated that astrocyte-derived ATP serves as a regulator of depressive-like behavior in mice and 7-day treatment with ATP is enough to stably reverse the avoidance behavior of depressed mice,indicating that ATP could be a fast antidepressant drug.Therefore,here we raise our hypothesis that whether ATP release from astrocytes is associated with the anti-depressant like behavior of sEH knockouts,and through which mechanisms sEH regulate ATP release from astrocytes.Firstly,we utilized immunological skills to investigate the expression of sEH in the astrocytes and found sEH was widely exist in cytoplasm.Then,we incubated brain slices of prefrontal cortex(PFC)and hippocampus from both sEH knock out mice and wide type mice to evaluate the released ATP level.Notably,ATP level was increased in PFC slices,but not hippocampus of sEH knock out mice when compared with wide type mice(t-test,n=15,P<0.001).To investigate whether astrocytes contribute to the increase of ATP,we cultured cortex astrocytes both from sEH knockout mice and wide type mice in vitro,and measured the released ATP level.ATP level was increased in cultured astrocytes of sEH knockout mice compared with wide type ones(t-test,n=5,P<0.001).To further characterize the effects of sEH exerts on ATP release from astrocytes,we treated the cultured astrocytes with AUDA,a widely used sEH inhibitor,at different concentrations for 30min and the astrocytes given vehicle treatment for 30min was used as control.We found ATP level was significantly increased in the 5uM AUDA,not luM treated astrocytes when compared with control ones(ANOVA,n=15 or 16,P<0.01).Then we considered whether AUDA could promote ATP release in a time-dependent manner,we gave 5uM AUDA administration to cultured astrocytes for 5min,10min and 30min.Only 30min treatment of AUDA at 5uM can significantly increase ATP release from astrocytes when compared with the control group which treated with vehicle(ANOVA,n?13,P<0.05).To further confirm the regulatory role of sEH played in ATP release from astrocytes,we treated cultured astrocytes with 14,15-EET at luM for 30min and then examined ATP level.14,15-EET can also significantly promote ATP release from astrocytes when compared with control group(ANOVA,n?8,P<0.05).In summary,these results show that sEH knock out mice display anti-depressant like behavior which can be reversed by 14,15-EE-5(Z)E administration(i.c.v.).14,15-EET injections(i.c.v.)can induce anti-depressive like behavior in adult mice.ATP level is increased in PFC slices and cultured astrocytes of sEH knock out mice.Both AUDA,a kind of sEH inhibitor and 14,15-EET,a substrate of sEH can promote ATP release from astrocytes.Our study indicates that EETs-sEH system plays an important role in producing anti-depressant effects.