| At least 10%of the world's population is affected by a wide range of mental disorders;as many as 700 million people get mental disorder in 2010.The global economic costs on mental disorders were estimated $2.5 trillion in 2010 and are projected to reach $6.0 trillion by 2030.40.5%of the years lived with disability(YLDs)among mental illness is depression;The next are anxiety disorders,substance by drug abuse,alcohol addiction,and schizophrenia,bipolar disorder.Depression has a high incidence and high morbidity.It brings a heavy burden to the society and affects the people's mental health.First of all,depression has a high incidence:around 20%to 25%of women and 7%-12%of the men is likely to suffer depression.With the increase of the accelerating rhythm of life and social pressure,that figure is likely to further be increased;Second,depression cause a high number of disabilty:Major depressive disorder is the second leading cause of YLDs globally and ranks among the four largest contributors to YLDs in each of the socially diverse regions spanning the six continents assessed in the Global Burden of Disease Study 2010 the age of developing depression is 18 to 44,which is young adults,and the suicide,disability caused by depression will take a heavy burden to the society;In addition,depression will further exacerbated inflammatory disease,cardiovascular disease,which will increase the loss of social wealth.Therefore,at present,depression research and treatment has become a important scientific problems need to be solved.The pathogenesis of depression is complex.Researchers thought that the occurrence of depression is closely related with neural circuits,synaptic activity,inflammation factors,and the hypothalamus-pituitary-adrenal axis.However,the clinical diagnosis and treatment is still relatively limited.The pathogenesis of depression still need to be further explored.The diagnosis of depression is mostly by the depression scale after the patients get the disease.Early intervention can get good result,but early diagnosis is not easy:there is few biological indicators can help;In labortory examination,although the 5-HT level can be reduced in urine and cerebrospinal fluid,but it is not feasible becouse it usually need to exclude the depressive mood caused by mental disorder and somatopathy.In terms of treatment,the hospitals still use monoamine antidepressants now:targeting on neurotransmitters,assisting by electric shock,psychotherapy,etc.But becouse of individual differences,antidepressants are still low efficiency(about 35%patients can not be cured by it),need long time(usually several weeks and months is needed)to work,and disgusting side effects.In the brain,more than 90%cells are glial cells.The astrocyte is largest size,and the most widely distributed glial cells.Astrocytes distribut between neurons,giving nutrition and support them;besides,astrocytes involved in many aspects such as neuronal information transmission,neural stem cells regeneration,participation in the role of the learning and memory,antioxidant and so on.The astrocyte activity is abnormal in cerebrovascular disease,epilepsy,Parkinson's disease,Alzheimer's disease,and nerve demyelinating disease.In recent years,researches have shown that the decline of astrocytic ATP release is important for the development of depression,after supplement of ATP;depressive symptoms can be significantly improved in mice.Astrocytes have variety of metabolic pathways,including glutamate-glutamate cycle,sugar-lactic acid metabolism,arachidonic acid metabolism pathways.Most previous studies focused on glutamate-glutamic acid circulation pathway,but in fact,arachidonic acid metabolism pathway is also very important.Keeping a stable injection pressure is very important for the neuron;and arachidonic acid has this function.When neurons activity increases,there is more blood flow in the brain,and arachidonic acid level will increase;when neuron activity is reduced,cerebral blood flow decreases,arachidonic acid production also declines.There are three pathways in arachidonic acid metabolism:cyclo-oxygenase,lipoxygenase,cytochrome CYP450.cyclo-oxygenase metabolic pathway is associated with the occurrence of cardiovascular disease,lipoxygenase metabolic pathway is closely associated with inflammation,and cytochrome CYP450 way was thought to be associated with the regulation of cerebral blood flow in the past.But it is not clear the relationship between cytochrome CYP450 pathway and neuronal diseases such as emotion,learning,memory now.SEH(soluble epoxide hydrolase)is encoded by EPHX2 gene,with a molecular weight of 62kD.Its distribution and biological activity is different in different organizations or individuals.It is mainly expressed in mammalian livers,kidneys,and the brain tissues.In brain tissue,sEH is mainly expressed in astrocytes,oligodendrocytes,and vascular endothelial cells.There is also some researches consider that sEH expresses in neurons in amygdala.Under the action of the cytochrome P450,arachidonic acid is degraded into EETs and HETEs.EET has four isomers:5,6-EET,8,9-EET,11,12-EET and 14,15-EET.A competitive inhibitor of EET is EEZE,which can block EET biological activity.EET can act on the surrounding cells through autocrine and paracrine,EET has anti-inflammatory,anti-apoptotic,mitogenic,promoting angiogenesis,analgesic and anti-oxidation effect.In the neuroscience aspect,EET may regulate cerebral blood flow;analgesia;releaseing of neurohormone,promoting the growth of axons.Under the action of sEH,EETs can be rapidly degraded into inactive DHETs;sEH inhibitors can inhibit the enzyme activity of sEH.Therefore,arachidonic acid-EET pathway can regulate neuro activity;the abnormity of neuro acvity is related to depression.We hypothesize that:inhibition of sEH or increase EET level can produce antidepressant effect.First of all,we need to verify whether sEH inhibitors can have antidepressant effect.Through lateral ventricle injection of sEH inhibitors TUCB and TPPU in C57 mice,the acute forced swimming experiments were done 15 minutes later to determine antidepressant effect.Compared with the control group,the sEH inhibitors TUCB group of 0.2 u M,1 u M,and TPPU group 1 u M,5 u M and 10 u M the immobility time was significantly shortened,The open field test confirmed that this is no physical change in mice.After 10 days social defeat model,we keep pumping the lateral ventricle 1 u M TPPU for 7 days and observed a obviously increase of the contact time in the middle area,which shows the antidepressant effect of sEH inhibitors.So acute and chronic experiment shows that giving sEH inhibitors to the lateral ventricle can produce antidepressant effect.In order to further verify delivery way and time of the sEH inhibitors,we used social defeat model,after defeating for ten days,we intraperitoneal injectioned the sEH inhibitors(1 mg/kg)for 7 days and tested its antidepressant effect,founding that compared with the control group,mice of TUCB group spend more time in the middle area,but there is no statistically significant.Mice of TPPU group spend more time in the middle area compared with controls.Imipramine group as positive contrast,because it needs to 28 days to work on this disease,so it cannot produce antidepressant effect temporarily.Traditional antidepressants usually take a long time to react,in social defeat model,they may need 28 days.But we give sEH inhibitors for seven days and observed significant antidepressant-like effects.This showed us that,compared with traditional antidepressants;sEH inhibitors display a rapid antidepressant effect.The substrate of sEH is four kinds of EET,inhibition of sEH will raise EET levels.In order to detect the role of EETs in depression,we give different EETs on mice lateral ventricle and made forced swimming test.The results suggest that only 14,15-EET has obvious antidepressant effect.Other EET display no function like this.Open field test results show us that the EET will not affect physical much on mice.After making sure of the antidepressant effect of EET,then we tested their levels on mouse model and depression patients.After 10 days social defeatl,we divided the mice into susceptible and unsusceptible groups.These mice plasma were made into samples by solid-phase extraction and detected the EETs level by Lc Ms-ms,finding that the mice of susceptible group the four kinds of EETs levels were significantly lower than control.To the mice of unsusceptible group,except 14,15-EET level decreased,other EET levels has no significant change(P>0.05).The depression patients' blood samples supported our preliminary findings,that the four kinds of EET levels are lower than control,but the sample has no statistical difference.So we get a preliminary conclusion that,in depression,EET levels dropped significantly.Above the pharmacology experiment results indicate that sEH inhibitors and 14,15--EET can produce rapid antidepressant effect;And in depression,each EET levels declines.This reminds us that in depression,sEH become hyperactivity,which may lead to all sorts of EET get hydrolysis and the EETs level decreased;Among them,14,15-EET,which is the most important in the development of depression,decreased most significantly.But after sEHI or 14,15-EET treatment,the depressive state will be changed.In order to further validate the conclusion we used the sEH knockout mice.The researches about the mice reported that its EET level,particularly 14,15-EET level is obviously higher than the wild type mice.Through the forced swimming test and tail suspension test,we see the immobility time of sEH knockout mice was significantly reduced when compared with wild type mice.This showed that the the sEH knock out mice is anti-depressant;And through injecting to the lateral ventricle EEZE,blocking the activity of EET,the immobility time has no statistical difference with normal mice and the anti-depressant effect disappeared.This further supports the results above,which is,14,15-EET play an important role in the antidepressant response;after the sEH gene knockout,although other epoxide hydrolase such as mEH may take part in the metabolism of EETs,EET level,especially the 14,15-EET level increased and which will produce anti-depressant effect.We proved by pharmacology and knockout mice that inhibit or knock out sEH can produce rapid antidepressant effect above;Next we will further discussed their mechanisms.First by using immunofluorescence histochemistry,we found that in hippocampus and cortex,there are more than 80%sEH-positive cells colocalized with GFAP-positive cells,but there is no sEH-positive cells colocalize with NEUN-positive cells,which means that in cortex and hippocampus,sEH expresses in astrocytes,rather than the neuron.Further we found sEH distribut granularly in astrocytes.By immunofluorescencel and confocal microscope we found that sEH colocalized with lysosome marker lysotracker,but not with mitochondria marker mitotracker,that is to say,sEH distribute on the lysosome in glial cells.Our previous work has found that ATP released by astrocytes lysosome produce antidepressant effect:after lysosome intaking of ATP,it was transferred to the cell membranes,the membrane fused and the ATP was released.Therefore,the speed of the ATP releas depends on:the speed of the lysosome intaking ATP and the speed of lysosomes fusing with the cell membrane.SEH expresses in the lysosome in glial cells,this suggests that sEH inhibitors may generate antidepressant effect by acting on ATP.So we tested ATP levels in the sEH knockout mouse brain.After 12 minutes incubation,the solution was centrifuged and supernatant were used to detect ATP levels.We found that in prefrontal cortex,ATP levels of sEH knockout mice were significantly higher than that of wild-type mice,but it didn't change much in hippocampus.In order to know where did the increased ATP from,we cultured cortical astrocytes of the transgenic mice,founding that the ATP levels increased significantly compared to control.This shows that ATP levels was increased in the sEH knockout mouse,and it is likely to be caused by increased astrocytic ATP.In order to further explore the role of sEH inhibitors on astrocytes ATP release,we tried different concentrations of TUCB and TPPU in cultured astrocytes,discovering that the group of giving TUCB 20 u M,50 u M,100 u M,200 u M,ATP release significantly increased;Giving TPPU 50 u M,100 u M,200 u M group cells,ATP release significantly increased.After the experiments above,we tried 14,15-EET in astrocytes,and find that it can also produce similar effect.But other EET does effect much on ATP release.The above results show that,14,15-EET or sEH inhibitors can cause significant increase in astrocyte ATP release.Calcium imaging experiments found that 1 u M 14,15-EET can promote calcium level,which may increase ATP levels and produce antidepressant effect.Directly injecte sEH inhibitors or 14,15-EET to prefrontal cortex(TUCB,0.1 u M,TPPU,1 u M,14,15-EET,1 u g),and in forced swimming test we can see mice will get significantly shorter immobility time.Open field test shows that mice activity did not change much.This account for that in the brain,sEH inhibitors act on the prefrontal cortex.Above all,we think that in the prefrontal cortex actrocyte,sEH inhibitors can inhibit lysosome sEH,by increasing 14,15-EET levels and improving calcium levels,finally it will increase the ATP release,thus make rapid antidepressant effects. |
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