To Compare The Characteristics Of Three Zebrafish Models Of Alzheimer’s Disease (AD) And Explore The Mechanism By Which DPA Attenuated AD-Like Chnages Induced By Aluminum

Alzheimer’s disease（AD）is a progressive neurodegenerative disease characterized with extracellular plaques and intracellular neuronal fibrillary tangles,accompanied with cognitive impairment and memory decline.The current clinical treatments cannot effectively improve AD symptoms and reverse AD progress.It is urgent to explore new pathogenesis of AD and develop effective medicines.In recent years,the neuroinflammatory hypothesis of AD has provided a new direction for the development of new treatmets for AD.Due to the complicated pathogeneses of AD,it is difficult to establish an accurate animal model with most neuropathological characteristics and clinical symptoms of AD,and the long establishment cycle of rodent model is not satisfied for drug discoveries.Therefore,the present study used highly humanized zebrafish to set up and compare three AD models induced by aluminum trichloride,intraventricular injection of Aβ_1-42 and diabetes mellitus.Previous studies have shown that omega（n）-3 unsaturated fatty acids（PUFAs）exert anti-inflammatory and neuroprotective effects,among which the new n-3PUFA docosapentaenoic acid（DPA）seems have stronger anti-inflammatory effects.Therefore,this study explored the mechanism by which DPA attenuated aluminum induced AD-like changes.The findings may provide a theoretical basis for DPA development in the treatment of AD.Method:1.To establish the zebrafish model induced by neurotoxic aluminum ion The adult zebrafish was exposured to 100μg/L aluminum trichloride solution at pH6.0±0.2.After 6 weeks,the behavioral changes of new object recognition and reward memory were detected.2.To detect the activation of microglia by aluminum ions transgenic zebrafish Tg（coro1α:eGFP）larva with fish fluorescent labeled microglia,were treated in 40mg/L and 45 mg/L aluminum solution with pH 6.0±0.2 for 4 days after screened the toxic concentration.The fluorescence intensity in the brain of the larva was photographed by confocal microscope.3.To establish AD model by intraventricular injection of Aβ_1-42 in zebrafish:1μL 20μM of Aβ_1-42 was injected into the brain ventricle of zebrafish,and the behavioral tests of new object recognition and reward memory were carried out after7 days of recovery.4.To establish of AD model of zebrafish diabetes induced by high glucose and high fat:diabetic model was established by high glucose environment（containing 2%glucose solution）and high-fat diet（containing 10%cholesterol）for 6 weeks.Then behavioral changes of new object recognition and reward memory were tested.5.Fluorescence quantitative PCR detected amyloidβ-precursor protein b（Appb）,microtubule associated protein tau a（mapta）,microtubule associated protein tau b（maptb）,acetylcholinesterase（AChE）,brain derived neurotrophic factor（BDNF）,cluster differentiation antigen 11B（CD11b）,interleukin（IL）-1β,mRNA levels of voltage dependent anion channel 1（VDAC1）and caspase-3 in the brain of the above three zebrafish models and their control groups.6.The AD model induced by aluminum ion was intervened with 1%DPA,and then the behavioral changes of new object recognition and reward memory were detected.After DPA intervention,the brain of zebrafish were detected by fluorescence quantitative PCR:β-site amyloid precursor protein cleaving enzyme 1（bace1）,BDNF,CD11b,glial fibrillary acidic protein（GFAP）,cyclin dependent kinase 5（CDK5）and glycogen synthase kinase 3β（GSK3β）were assayed in the fish brains by fluorescence quantitative PCR.ELISA kit was used to detect the expression of Aβ_1-42 and IL-6 in zebrafish brains.Results:1.AD model verification:New object recognition and reward memory behavior was investigated in three zebrafish models of AD induced by aluminum,Aβventricular injection and the diabete.Above models showed cognitive and memory impairments when compared to their control groups.Compared with the vehicle group,the mRNA levels of aluminum group of Appb,mapta,maptb,AChE,CD11b,IL-1βand VDAC1 were increased significantly,while the mRNA level of BDNF was decreased significantly.Compared with the vehicle group,the mRNA expression of caspase-3 increased.In a certain range,compared with control group,the activation of microglia was increased significantly with the increase of aluminum trichloride concentration.Compared with the sham group,Aβinjection group increased significantly the mRNA level of mapta,maptb,AChE,CD11b,IL-1β,VDAC1 and caspase-3.Compared with the sham group,the mRNA level of BDNF came down significantly,and the mRNA level of Appb increased.Continuous high glucose and high fat feeding significantly increased the body mass index and blood glucose in zebrafish.Compared with the control group,the mRNA levels of mapta,maptb,AChE and caspase-3 in diabetic model were significantly increased.Compared with the control group,the mRNA levels of Appb and CD11b rose up,and BDNF decreased.Compared with the Al Cl₃ group,the mRNA level of diabetic group of IL-1βincreased significantly.Compared with the Al Cl₃ group,the mRNA level of VDAC1 in diabetic model group increased significantly.Compared with the Aβgroup,the mRNA level of VDAC1 in diabetic model group increased much significantly.2.The effect of DPA on AD-like changes in zebrafish modelDPA reversed the cognitive and memory impairment of zebrafish caused by aluminum,and reversed the increase mRNA level of bace1,thereby reducing Aβdeposition.Moreover,DPA attenuated down-regulation of BDNF expression induced by aluminum,and inhibited the enhancement of microglia and astrocyte markers in the model group,thereby reducing the concentration of central IL-6 protein.In correlation,DPA inhibited CDK5 and GSK3βmRNA levels.Conclusion:1.Memory impairment induced by aluminum trichloride may result from increased AChE,Aβaggregation,microglia activation,release of proinflammatory factor IL-1β,damaging mitochondrial function,reducing BDNF and further cause neuronal apoptosis.2.Oligomeric state Aβcaused AD-like pathological changes may accelerate the formation of p-tau,increase nerve fibers and AChE,activate microglia,release a large number of pro-inflammatory factors,change the function of brain-derived neurotrophic factor,promote neuronal apoptosis,and finally lead to cognitive and memory impairment.3.The cognitive and memory impairments in diabetic models may be due to peripheral inflammation caused by metabolic disorders.Proinflammatory factors enter the central nervous system,leading to mitochondrial dysfunction and apoptosis,and promote p-tau formation and increase AChE activity.4.DPA can restore the function of microglia and astrocytes alleviate neuroinflammation,CDK5 and GSK3βexpression improve the level of BDNF and reduce Aβprotein deposition and finally improve the cognitive and memory impairment in zebrafish.