Inhibition Of Soluble Epoxide Hydrolase Ameliorates Pathology In An Alzheimer’s Mouse Model

Alzheimer’s disease(AD)is a common chronic senile dementia with the highest prevalence,and its main symptom is progressive loss of cognitive funtion.The etiology of AD is complex and its pathogenesis is unclear.The main neurological pathological features of AD are β-amyloid(Aβ)plaques,Neuronal tangels,accompanied by inflammation and gliosis,loss of neurons and synapses,and vascular lesions.In addition to these hallmark features,excessive inflammatory responses and innate immune responses such as glial hyperplasia and abnormal changes in inflammatory factors have also been observed in patients with AD.Although the pathological features are clear,and in recent years AD biomarkers and clinical imaging studies have brought hope for early diagnosis,there are still more than 50%of AD patients can not be diagnosed and received assistance.In addition,there is no safe and effective drug that can reverse the pathological process of AD.Therefore,elucidating the pathogenesis of AD and finding a safe and effective drug for reversing the pathological process of AD and improving the cognitive impairment of AD is a key scientific issue in the current research on prevention and treatment of AD.More and more studies have found that unsaturated fatty acids(PUFAs)and their metabolites are closely related to AD.Arachidonic acid(ARA)in unsaturated fatty acids is closely related to the development of inflammation.Therefore,the enzymes,receptors,and metabolites of the ARA-COX,ARA-LOX metabolic pathways are widely used as targets for the development of anti-inflammatory drugs,including various COX inhibitors,LOX inhibitors,etc,Epoxyecosatrienoic acid(EET),the metabolite of ARA-CYP metabolites,has a wide range of biological activities,such as anti-hypertensive,anti-inflammatory,cardiovascular and renal protection and so on.Soluble epoxide hydrolase(sEH)is a key enzyme for the hydrolysis of EET and plays an important role in the EET signaling pathway.EETs can be rapidly hydrolyzed by sEH to the corresponding diols to lose their activity and even cause toxicity.Inhibition of sEH enzyme activity can increase EETs level.However,it is unclear whether sEH-mediated EET signaling pathway is involved in the pathophysiology of AD.The goal of this study was to reveal the role and mechanism of sEH in the pathogenesis of AD.Firstly,we used high-performance liquid chromatography and mass spectrometry detection techniques and found that the EETs levels of plasma,hippocampus and cortex in C57 mice showed a decreasing trend with age;EETs,especially 14,15-EET levels were significantly reduced in the cortex,hippocampus,and plasma of 5xFAD mice,suggesting that EETs levels change during aging and in AD pathology.Secondly,we hybridized 5xFAD mice with Ephx2 gene(sEH-encoding gene)knockout mice to obtain 5xFAD;Ephx2-/-and littermate control mice.We then used thioflavine staining,immunofluorescence staining,and ELISA techniques to find that knocking out the Ephx2 gene in 5xFAD mice significantly decreased A(3 levels in the brain of 5xFAD mice and decreased Aβ deposition.Behavioral experiments revealed that Ephx2 knockout significantly improved learning and memory in 5xFAD mice.Thirdly,we found that intraperitoneal inj ection of the TPPU,a sEH inhibitor,in 5xFAD mice significantly decreased brain Aβ levels,Aβdeposition,and improved learning and memory.Fourthly,we used western blotting and qPCR techniques and found that knockout of Ephx2 gene does not affect the levels of APP and its cleavage fragments such as APP-CTF and APP-NTF protein,and the mRNA level of APPase BACE1,suggesting Ephx2 gene Knockout does not affect the production of Aβ.Using high performance liquid chromatography coupled with mass spectrometry detection,we found that knockout of Ephx2 gene or 5xFAD mice treated with sEH inhibitor TPPU can significantly increase the level of plasma,hippocampus and cortex EETs in mice,especially 14,15-EET level.It is suggested that EETs,especially 14,15-EET,may play an important role in reducing the Aβ level and Aβ deposition.Fifth,the in vitro results showed that 14,15-EET can inhibit the polymerization of Aβ and can depolymerize the polymerized Aβ in a concentration-dependent manner.Finally,using immunofluorescence and real-time fluorescence qPCR,we found that knocking down Ephx2 gene significantly inhibited the excessive activation of microglia,reduced the number of microglia,and increased microglial phagocytosis abilityin the brain of 5xFAD mice.The above results indicate that sEH is involved in the pathological process of AD;knocking out of sEH gene(Ephx2)or inhibiting sEH enzyme activity can improve the pathological symptoms of AD,and its mechanism may be achieved by inhibiting Aβ aggregation and increasing the phagocytosis ability of microglia.