Research On The Mechanism Of BACE1 Expression And Interventtion By Di-3-N-Butylphthalide In Cell Model Of Oxygen-Glucose-Deprivation

As China has entered the aging society,the incidence of cerebrovascular accident is increasing yearly.Governmental statistics showed that the incidence of stroke rised at an annual rate of 8.7% in 2012.A foreign research showed that about 64% stroke survivors with varying degree of cognitive impairment,and roughly one-third of those would progress to definite dementia.This has become an urgent problem to solve.As ischemic stroke is about 80% in all types of strokes,it has become the focus of research in the field of cognitive impairment after stroke.Although the pathogenesis of cognitive impairment following ischemic stroke has not been clear,reports suggested that the interaction between cerebrovascular injuries and neurodegeneration might be the possible pathological mechanism,which is similar with Alzheimer’s diseases.And increasing studies have supported the notion that cognitive impairment was closely related to extracellular Aβ deposition in brain after ischemic stroke.Moreover,preventing Aβ production and(or)promoting Aβ clearance can improve cognitive decline in post-stoke patients.As BACE1 was identified to be the key rate-limiting enzyme in Aβ metabolism,it has been considerated as another promising therapeutic target for Aβ clearance.Many studies indicated that up-regulation of BACE1 at the transcriptional and protein level led to increased Aβ level,while the reversal results occurred due to the application of BACE1 inhibitors.So Aβ level may be affected by regulation of BACE1 activity.Therefore,the improvement of cognitive decline after stroke could be achieved indirectly through reduction of Aβ level by regulation of BACE1 activity.Autophagy is a kind of self-digestion process in cell in which misfolded proteins,damaged or aged organelles and invading microorganisms are transported to lysosome for degradation.Recent studies have shown that BACE1 metabolized via lysosome degradation pathway.Previous studies from our team showed that Aβ generation was prevented through up-regulation of autophagy in mice brain objected to cerebral ischemia.Domestic studies showed that dl-3-n-butylphthalide(NBP)could also regulate autophagy.NBP is a new drug initially extracted from a kind of Celery in south China,displaying multi-target role in the treatment of acute ischemic stroke.In order to investigate the possible mechanism involved with the regulation of BACE1 expression by autophagy or NBP medication,the cell model of cerebral ischemia in vitro was set up through oxygen-glucose-deprivation(OGD)in Neuro-2a/APP695 cells stably over-expressing wild-type human APP695 protein in our experiment.Expression of BACE1 and its regulation by NBP will be observed and analyzed by the mothod of MDC staining,GFP-LC3 localization,immunoblotting,confocal laser microscopy and transmission electron microscopy technology.Our experimental results showed that the expression of autophagy-specific protein,LC3 and Beclin-1,remarkable increased,while level of BACE1 decreased.This was observed in the groups of OGD and OGD plus Rapamycin compared to control group.The reversal results occurred due to the application of autophagy inhibitor 3-MA.Similarly,the enhancement of autophagic phenomenon was also observed in the above two groups with ultrastructural morphologic assessment.All of the results obtained above suggested that reduction in BACE1 expression could be achieveted by autophagy up-regulation,thereby might decreasing the level of Aβ,providing a new option for the clinical treatment of cognitive impairment.Next,the influence of NBP on BACE1 expression and its possible mechanisms were investigated.As compared to control group,pretreatment with 10μM NBP in the groups of OGD+NBP and OGD+NBP+Rapa could attenuate autophagic level,lower BACE1 expression,raise Bcl-2 level along with reducing Bax level and inhibiting the release of cytochrome C from mitochondria.Thus it protected cells from stress status induced by OGD.Meanwhile,the reverse effects were observed when the medication conbination with 3-MA in the group of OGD+NBP+3-MA.The phenomenon of co-localization of protein LC3 and Aβ,downstream metabolic product of the enzyme BACE1,was observed in the groups of OGD and OGD+NBP+Rapa under confocal laser microscopy.The ultrastructural morphologic changes in the cells pretreated with NBP in the group of OGD+NBP also supported that NBP could attenuate autophagic level and protect cells from stress insult induced through OGD.In summary,conclusion may be made from above results that reduced expression of BACE1 can be modulated through autophagy activation.Moreover,the expression of BACE1 can also be down-regulated with NBP medication of which the related mechanism may be through autophagy pathway and(or)anti-apoptotic pathway.This suggests that NBP may serve as a promising clinical option for the treatment of cognitive impairment in ischemic stroke survivors,providing a new clue for the clinical treatment of cognitive impairment.