| Major depressive disorder(MDD)afflicts approximatily 10%men and 20%women of the population all over the world and is one of the leading causes of disability and economic burden,core symptoms of which include depressed mood,anhedonia(reduced ability to experience pleasure from natural rewards),irritability,difficulties in concentrating,and abnormalities in appetite and sleep(neurovegetative symptoms).In addition,MDD is often a complication to many chronic diseases,including cardiovascular disease,chronic liver disease and type 2 diebetes.Therefore,the prevention of MDD is a major medical issue for us.There are many hypothesises for depression,including the monoamine-deficiency hypothesis,the Hypothalamic-Pituitary-Cortisol system,synaptogenic model,adult neurogenesis model,neuroinflammation and glutamate imbalance.All of these hypothesises are implicated to the dysfunction of neural system.And the antidepressants which are designed base on the hypothesis are low efficacy and slow response.So we should change our mind to investigate the mechanism of depression in another way?Whether peripheral system plays a role in the pathologic of depression?Not only depression has a high morbidity in normal people,but also it usually accompany with many chronic diseases,for example chronic liver disease,diabetes and cardiovascular disease.Recently studies report that approximately 20%?60%patients with Chronic Hepatitis C(CHC)suffer from mood disorder.And about 15%?49.5%untreated CHC patients display depressive symptom but most of them are not diagnosed as depression before they infected hepatitis C virus.For the patients with cirrhosis,56.7%patients are diagnosed as mild depression,40.7%patients are moderate depression and 6%?10%are severe and extreme depression.At least 25%patients among the patients with non-alcoholic fatty liver disease(NAFLD)are diagnosed as depression.Therefore,all these evidences suggest that the liver may play a role in depression.There are kinds of metabolisms in the liver,including lipid metabolism,protein metabolism,and carbohydrate metabolism.All of these metabolisms are main dependent on the physiology of liver.And metabolism disorder is usually associated with many diseases.Previous studies have found that metabolic abnormalities are often associated with depression,such as insulin resistance and abdominal obesity,which increase glucocorticoid concentrations in serum.Excess glucocorticoids can induce hyperactivity of hypothalamic-pituitary-Adrenal axis(HPA-axis).However,hyperactivity of HPA-axis is a vital pathological symptom to depression.In addition,fatty acid metabolism disorder has been consistently reported to major depressive disorder.Previous studies show lower concentrations of ?3 long chain polyunsaturated fatty acids,for example eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),and decrease in overall FA unsaturation,chain length and peroxidizability both in acutely depressed and remitted patients.Meanwhile another paper implicates the ?6/?3 ratio in the body will change according to acute stress.All of these evidences suggest that there may be a relationship exists between FA-metabolism disorder and depression.Many kinds of fatty acids exist in our body,including arachidonic acid(AA),EPA and DHA.Fatty acids play a key role in structure and function of nervous and cardiovascular system.The cell membranes contain a lot of FAs,whose unsaturation and chain length will determine membrane fluidity and influence functioning of membrane bound proteins.In addition,the peroxidizability of membrane FAs has impact on the membrane susceptibility to oxidative stress.EPA,DHA and AA play an important role in inflammatory regulation and maintenance of brain cytoarchitecture.Arachidonic acid(AA)belongs to ?6 long chain polyunsaturated fatty acid.It is an important component of cytomembrane and a precursor for many bioactive compounds.AA is one of the essential fatty acids for human,and it is found in common meats.Mammals not only can obtain the AA from food,but also can synthesize AA from the linoleic acid in the liver.There are three key pathways in the arachidonic acid metabolism,including the cyclooxygenase(COX),lipoxygenase(LOX)and cytochrome P450(CYP)pathways.The AA-COX pathway is involved in pain,inflammation,blood clotting and pulmonary hypertension.And the metabolite leukotriene C4 in the AA-LOX pathway will cause asthma and allergies.The CYP450 pathway is major and important for AA metabolism.The epoxygenase CYP enzymes convert arachidonic acid into epoxyeicosatrienoic acids(EETs),by catalyzing the epoxidation of arachidonic acid olefin bonds,resulting in the production of four regioisomeric EETs:5,6-EET,8,9-EET,11,12-EET and 14,15-EET.EETs are endothelium-derived hyperpolarizing factors(EDHFs)that protect from ischemic injury,anti-inflammatory actions,antiplatelet aggregation,vasodilation,antidiabetics and cell proliferation.The four regioisomeric EETs distribute in different organs.14,15-EET is generated mainly in the liver,and 11,12-EET is the primary product in the kidney.Both 5,6-EET and 8,9-EET are observed in the heart.Soluble epoxide hydrolase(sEH)can decrease EETs levels and thereby diminishing their beneficial properties by converting EETs to their corresponding diols(dihydroxyeicosatrienoic acids;DHETs).The DHETs accumulates in the body will result in inflammation,pain,diabetes,kidney disease and cardiovascular disease.sEH is an enzyme which belongs to epoxide hydrolase family.It can change the epoxide chemical property into inactive substrate by hydrolyzing.Human sEH are encoded by EPHX2 gene which locates in the Chromosome 8p21-p12.The mammalian sEH is a homodimer with monomers arranged in an anti-parallel form.Each monomer is composed of two domains:the carboxy-terminal domain,which contains ?/?-hydrolase fold structure and has epoxide hydrolase activity,and the amino-terminal domain,which also contains ?/(?)fold topology belonging to the haloacid dehalogenase enzyme superfamily and hydrolyses phosphates on lipophilic backbones.These two domains are linked by a proline-rich peptide segment.The amino-terminal domain has a critical role in maintaining the stabilization of sEH homodimer.And the homodimer form is the key structure for keeping sEH acivity.So far,sEH is found to broadly distribute in vertebrate tissues.However sEH activity is various in different tissues.The specific activity of sEH is highest in the liver,followed by the kidney,with lower levels in extra hepatic tissues including brain,lung,heart,spleen and so on.Growing evidences have proved that the EPHX2 gene shows polymorphism,which reflectes in gene mutation.The gene mutation will change the amino acids that encod the sEH protein,and then affects the activity of sEH.For example,the Lys55Arg and Glu470Gly mutations increase the sEH activity,and then decrease the EETs level in the blood.These two mutations result in a high risk to get atherosclerosis and stroke.On the contrary,the Arg287Gln mutation decreases the sEH activity,which reduces the hydrolysis of EETs.This mutation will lower the risk to get a stroke and has a protection for the nervous system.To this end,we ask whether the liver had a relationship to the depression.What happen within liver in depression model?In order to answer this question,we developed an animal model of depression following a CMS(Chronic Mild Stress)paradigm.CMS paradigm is a well-established behavioral model of depression.In briefly,the mice were subject to series of stress,including restraint,forced swim in ice-cold water(5 min),food and water deprivation(24 h),cage tilting(45°),reversal of the light/dark cycle,strobe light,and pairing with another stressed animal,in a schedule that lasts for 3 weeks and was repeated thereafter.And the control mice were subjected to normal conditions.After 3 weeks of CMS,mice showed sucrose preference and coat state deterioration,which could be reversed by chronic antidepressant treatments.At the end of CMS,we made a quantitative analysis to the liver of mice by iTRAQ(isobaric Tags for Relative and Absolute Quantitation).We found that AA metabolism was significantly different in the liver of mice which displayed a depressive-like behavior when compared to the normal mice.This result suggested that there may be a relationship between AA metabolism disorder and depression.Furthermore,we detected the expression of enzymes that involved in the AA metabolism after CMS by quantitative RT-PCR.The data showed that the mRNA of enzymes which involved in AA-COX,AA-LOX and AA-CYP pathway did not change in the liver after CMS.However,the mRNA of sEH,an enzyme that involved in the downstream of AA-CYP pathway,was increased in the liver after CMS.Our data suggested that the change of sEH in the liver might be the reason for the AA metabolism disorder after CMS.sEH is an enzyme which can convert the EETs into DHETs.To this end we wanted to know how the expression of sEH changes in the liver during CMS process.Then we detected the expression of liver's sEH at different time points after CMS paradigm.Our result showed that the expression of sEH had a time dependent increased in the mice liver during CMS.But no alteration to the expression of sEH was observed in the other extra hepatic tissues including kidney,heart and pre-frontal cortex(PFC).This was suggesting that the liver sEH may play a role in the pathologic mechanism of depression.Then we asked whether antidepressants could decrease the expression of sEH in the liver.In order to answer this question,we treated the C57BL/6J mice with fluoxetine,imipramine and haloperidol(an antianxiety agent)and found that the expression of sEH was significantly decreased in the liver after chronic treatment with antidepressants.But no changes were observed in the other extra hepatic tissues.Meanwhile,the expression of sEH in the liver was normalized by chronic fluoxetine and imipramine treatment after CMS model.All these results suggested that the liver sEH likely involved in the antidepressant-like effects.Now we asked whether deletion of sEH would produce antidepressant-like effects.So,we first tested the possibility that sEH knock-out was involved in antidepressive-like behaviors with a battery of behavioral assays,including force swimming test(FST),tail suspension test(TST),open field test(OPT),sucrose preference and the state of mouse's fur.In the FST and TST,sEH-/-.mice decreased the immobile time.And no difference was observed on open field test.In addition,sEH-/-mice displayed an increase in their preference for sucrose,with no differences in total fluid intake(data not shown).And the physical state of the animal's coat also was improved in sEH-/-mice.Together,these results suggested that deletion of sEH poruduces antidepressive-like behaviors in mice.However,we did not know which tissue contributed to the antidepressant-like effects of sEH knock-out mice..Many studies had indicated that sEH had a highest expression and specific activity in the liver,so we infered that the antidepressant-like effects observed in sEH knock-out mice was mainly caused by liver sEH deletion.In order to verify our hypothesis,we first needed to build a method to specifically decrease sEH expression in the liver but did not affect the expression of sEH in the other tissues.Recombinant adenoviruses encoding green fluorescence protein(Control-shRNA)and optimal sEH shRNA specific sequences(ephx2-shRNA)were constructed and amplified.The expression of sEH was significantly decreased at the 14 days after the injection of adenovirus via the tail vein.And no alterations were observed to the expression of sEH in other extra hepatic tissues including hippocampus,PFC,kidney,heart,spleen and pancreas.Also no any toxic reaction was observed in the liver after the infecton.Then in order to detect the antidepressant-like effects of the Ephx2-shRNA,we used CMS paradigm.Mice were randomly divided into seven groups(control,control-shRNA,Ephx2-shRNA1,Ephx2-shRNA2,Fluoxetine,Imipramine and saline)and subjected to CMS,meanwhile the control mice were subjected to normal conditions.The state of each mouse's fur and sucrose preference were evaluated at each week.After 3 weeks of CMS,mice showed sucrose preference and coat state deterioration.Strikingly,after 4 weeks treatment,Ephx2-shRNA1,Ephx2-shRNA2,Fluoxetine and Imipramine significantly improved sucrose preference and the physical state of the fur,suggesting that decrease the expression of sEH in the liver produced antidepressant-like effects.Our results showed that genetic deletion of sEH produced antidepressant-like effects.So we wondered whether sEH inhibitors also could produce antidepressant-like effects.TPPU(N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoro-methoxy)phenyl)-urea)and TUCB(Trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid)were two effective sEHIs,which could inhibit sEH activity effectively in vivo by intraperitoneal injection.The CMS result showed that TPPU,TUCB and Imipramine significantly improved sucrose preference and the physical state of the fur after 4 weeks treatment.Strikingly,we found that TPPU produced a fast-onset antidepressant-like effects(7 days)compared to imipramine.All these results suggested that sEH inhibitors could produce fast antidepressant-like effects.We provided here evidence that the expression of sEH was increased specifically in liver after depression and was decreased after the treatment wiht antidepressants.Genetic deletion of sEH produced antidepressant-like effects.And the sEH inhibitors produced fast antidepressant-like effects.Thus our results suggest that the liver may play an important role in the pathologic of depression via the liver sEH.The liver sEH would be a novol target for fast antidepressant. |
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