Neuroprotective Effects Of NSTK On The AD Mouse Model And Its Mechanism About Regulating Energy Metabolism Disorder

Alzheimer’s disease（AD）,a neurodegenerative disorder,could be induced through complex multiple mechanisms.The scientific evaluation of candidate drugs used for the treatment of AD needs to adopt AD model mimicking the pathologic characteristics,especially the corresponding animal model.The energy metabolism disorder involving in multiple pathways（oxidative stress,inflammation etc.）was considered as an early signal of AD.Aiming at energy metabolism disorder could effectively improve the therapeutic effects of AD.In our previous researches,we focused on AEA analogs N-fatty acyl amino acids with intellectual property rights.We have found N-stearoyltyrosinate（NSTK）a neuroprotective candidate drug with improved solubility stability and good druggability against cerebral ischemia after system researches.NSTK acting as fatty acid amide hydrolase（FAAH）inhibitor intervened in multiple ways of cerebral ischemia through endocannabinoid system and showed good neuroprotective effects.Because of the similar intervention mechanism between cerebral ischemia and AD,we conducted a preliminary study on the activity and mechanism of NSTK in the treatment of AD at the cellular level and we found NSTK inhibited oxidative stress,excitotoxicity.To further evaluate the feasibility and mechanism of NSTK to the treatment of AD,we investigate the mechanism of NSTK in AD mouse model involved in inflammation,oxidative stress,glucose metabolism disorder.We explore the mechanism related to inflammation,oxidative stress of NSTK through intervening in energy metabolism disorder in U251cells.PART I:The neuroprotective activity of NSTK was evaluated on AD mouse modelWe chose APP/PS1/Tau and Tau transgenic mice to study the neuroprotective effect of NSTK on AD by the observation of animal’s behavior,histopathology or ELISA.The results showed that NSTK（30 mg/kg,45 mg/kg）improved the performance of transgenic mice in animal behaviors（spontaneous locomotor activity,low anxiety）,ameliorated spatial memory deficits.Because of Tau transgenic mouse not completely mimic behavioral impairment,we focused on the neuroprotective effects of NSTK on APP/PS1/Tau transgenic mice.Our biochemical analysis suggested that NSTK ameliorated Aβand Tau pathology(decreased Aβdeposits,the content of Aβ₄₂ and Aβ₄₀,inhibited NFTs)in cortex and hippocampus.Consistently,NSTK decreased the neuronal loss and inhibited the proliferation of reactive astrocytes in cortex and DG.These results suggested that NSTK showed good neuroprotective effects on AD mouse model and multiple ways might be involved.The forementioned results about the candidate drug NSTK with good druggability against cerebral ischemia provide the experimental date in the treatment of AD.PART II:The multiple intervention mechanisms of neuroprotetive effects of NSTK on APP/PS1/Tau transgenic mice were evaluatedThe pathogenesis of AD involved in inflammation,oxidative stress,glucose metabolic disorders is complex,some therapeutic agents aiming at the specific etiology and pathogenesis are only modestly efficacious in vivo.It is a better option to explore new agents intervening in multiple processes of AD.We evaluated the effects of NSTK on inflammation,oxidative stress and glucose metabolism related factors and the expression of protein in APP/PS1/Tau mouse model.The results showed that NSTK（30mg/kg,45 mg/kg）inhibited the content of inflammatory cytokines（IL-1β,TNF-α）and the expression of NF-κB,decreased the content of MDA and increased the activity of SOD,CAT,increased the content of glucose and the expression of glucose related protein（GLUTs,p-AMPK）.The results indicated that NSTK showed neuroprotective effects through intervening in multiple pathways involved in inflammation,oxidative stress,glucose metabolism disorder.These results support the conclusion“NSTK showed good neuroprotetive effects in AD mouse model”in the previous chapter from the mechanism and establish the experimental base of the whole animal model about the research“NSTK regulate energy disorder,inflammation,oxidative stress”in next chappter.PART III:NSTK regulated energy metabolism disorders in U251 cellsSince energy metabolism disorder is an early signal of the occurrence of AD,interfering in energy metabolism had great influence in the inflammation and oxidative stress during the pathogenesis of AD.Regulation on energy metabolism,which will improve inflammation and oxidative stress,is beneficial to the treatment of AD.Energy metabolism disorder is mainly showed by the decrease of glucose uptake.In this part,a hypoxic damage model of U251 cells was introduced to imitate energy metabolism disorder and evaluate the efficacy and mechanism of NSTK on glucose uptake.The decrease of glucose uptake induced by hypoxia was reversed by NSTK（1-5μmol/L）treatment for 48-96 h in a time-/dose-dependent manner.When U251 cells was treated with NSTK for 48 h,the decrease of glucose uptake induced by hypoxia was reversed,while the increase of the levels of IL-1β,TNF-αand MDA were not,suggesting NSTK reversed the decrease of glucose uptake before improving inflammation and oxidative stress.In hypoxic damage U251 cell model,the expression of GLUTs,which were closely related to glucose uptake,were significantly reduced and the reduction was reversed when U251 cell model was treated with NSTK for 96 h,suggesting both the energy metabolism disorder induced by hypoxia and the improvement produced by NSTK were closely related to GLUTs expression.When treated with AMPK not PI3K inhibitor,the upregulation of GLUTs induced by NSTK was reversed,suggesting the regulation GLUTs by NSTK was mediated by AMPK pathway and did not depend on the level of insulin.Moreover,we found that AMPK not PI3K inhibitor could reverse the effect of NSTK on glucose uptake,inflammation and oxidative stress of hypoxic damage U251 cell model,suggesting AMPK pathway was involved in the regulation NSTK on glucose uptake,inflammation and oxidative stress,which were insulin-independent.Finally,we found that the phosphoration of AMPK was upregulated when treated with NSTK,which could be blocked by AMPK inhibitor,suggesting the involvement of AMPK pathway in NSTK’s regulation of glucose uptake,inflammation and oxidative stress in hypoxic damage U251 cell model.In this chapter,we clarify the intervention of NSTK in the energy metabolism disorder in hypoxic damage cell model,the improvement of NSTK on inflammation and oxidative stress and the mechanisms involved.The results not only provided theoretical support for the application of NSTK in AD treatment,but also enrich the research of the action mechanism of the neuroprotective agents mediated by endocannabinoid system.This study identified the neuroprotective effect of NSTK on AD animal model and elucidated the action mode of NSTK in improving inflammation and oxidative stress via regulating energy metabolism for the first time.These results enriched the theoretical basis of the neuroprotective effect of NSTK and provided experimental data for NSTK as a leading compound.