Study On The Effect And Molecular Mechanism Of Butyrolactone Derivative On The Alzheimer Disease Cell Model

Background and objectiveAlzheimer Disease (AD) is the most common progressive degenerative disease in nervous system, which is the etiological factor of dementia. AD patients have increased everyyear. It has become the forth cause of death in elderly people in developed countries. The pathogenesis of AD isn't clear up to date, and there isn't specific treatment for AD patients. So to make clear of the pathogenesis of AD and find new treatments for different aspect will have great value.There are obvious atrophy in temporal lobe, parietal lobe and prefrontal lobe. The pathology of AD is senile plaques (SPs), neurofibrillary tangle, neurons loss in temporal lobe and hippocampus, granulovacuolar degeneration and vascular amyloidosis. Senile plaques are the extracellular deposit of β-amyloid protein (A(3), presenilin1, presenilin2, Anti-chymotrypsin al, apolipoprotein E, macroglobulin a2and ubiquitin. Neurofibrillary tangles are the intracellular deposit of phosphorylated tau proteins. SPs, NFTs and neuron loss are characteristic pathological changes. On the basis of previous researches Aβ hypothesis was proposed that the extra deposit of Aβ was the common pathway in pathogenesis of AD. To make clear of the mechanism of Aβ cytotoxicity and to prevent or eliminate Aβ deposit is the key target in Alzheimer desease researches.Autophagy is the generally existed degradation-recirculation system in eucaryotic cells. There are three types of autophagy:macroautophagy, microautophagy and chaperon-mediated autophagy. Macroautophagy is the common type. Recent researches had found that autophagy-lysosome system was involved in Aβ deposit. At the early stage of brains of AD patients and8weeks APP/PS1transgenic mice autophagosome were found abundantly existed.Our previous research had found a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) could inhibit autophagy in HUVECs, and protects cells from apoptosis and senescence induced by deprivation of serum and growth factor. Could it have the same effect in neural cells especially in AD cell models? Could it protects cells against Aβ-induced cytotoxicity and reduces Aβ deposit for AD treatment?Base on the backgrounds mentioned above, the objective of this study was to investigate the effect of autophagy involved in AD pathogenesis and to find the key factor or protein of modulating autophagy by using small molecule3BD0. This research was help to make clear of the mechanism of AD pathogenesis and to find new therapeutic targets for new drugs.Contents1. To investigate wheather extra-Aβ25-35induced cytotoxicity in PC12cells and induced autophagy.2. To investigate whether3BDO protects cells against Aβ cytotoxicity and to find its mechanisms.3. To investigate whether3BD0could decrease the level of extracellular Aβ formation in20E2cells (APP transgenic cells) and to find its mechanisms.4. To investigate the molecule mechanism of3BD0inhibits Aβ-induced autophagy.Methods1. The building and culture of Alzheimer Disease cell model:PC12cell or SH-SY5Y cell treated with oligomer Aβ25-35are one of the AD cell model;20E2cell (HEK293/APP) was another AD cell model.2. The detection of cell viability:cell viability was measured by SRB assay.3. Measurement of apoptosis:determination of apoptosis by Hoechst33258staining.4. Measurement of autophagy:4.1The quantity and distribution of acidic vesicle were analyzed by acridine orange (AO) staining under fluorescent microscope.4.2the level of LC3-Ⅱ was analyzed to estimate whether autophagy was induced.5. The detection of ROS level:ROS level was measured by fluorescent probe (DCHF).6. Measurement of Na+K+-ATPase:the Na+K+-ATPase assay was performed according to the Na+K+-ATPase detection kit.7. Measurement of mitochondrial membrane potential (MMP):MMP was measured by fluorescence probe (JC-1) under confocal laser scanning microscope.8. Detection of extracellular concentration of Aβ1-40and Aβ1-42:extracellular concentration of Aβ1-40and Aβ1-42were measured according to ELISA kit.9. Expression analyses of FLJ11812:The mRNA FLJ11812was analyzed by semi-quantitative RT-PCR and agarose gel electrophoresis.10. Analysis of expression of proteins:The protein levels of LC3-II, p62, p70s6k, p-p70s6k, p62, APP, IDE, NEP were analyzed by western blot assay.1. The effect and its mechanism of3BDO on Aβ25-35cytotoxicity in PC12cells1.1Aβ25-35could reduce cell viability of PC12cells in a dose-and time-dependent manner.1.23BDO could protect cells against Aβ25-35cytotoxicity by pretreatment for1h. Pretreatment with60and120μM3BDO for1h then Aβ25-35inhibited the decrease in cell viability induced by10μM Aβ.1.33BDO could reduce the increase of ROS level induced by Aβ25-35. ROS level were increased after Aβ treatment for2h and4h compared with control cells, and3BDO, after pretreatment for1h, could reduce the increase of ROS level induced by Aβ25-35.1.43BDO could restore the Aβ25-35-reduced Na+, K+-ATPase activity. The activity of Na+, K+-ATPase was reduced compared with control cells after10μM treatment for4h, and3BDO could restore the impairment of Na+, K+-ATPase activity.1.53BDO had no influence on Aβ-disturbed MMP. When PC12cells were treated with10μM Aβ for4h, MMP was higher compared with control cells. While with3BDO pretreatment for1h followed by Aβ25-35-treatment for another4h, MMP slightly decreased but not reached to the significant level compared with Aβ25 35-treatment alone.1.6Study on the mechanism of3BDO protecting effect.1.6.1Both10μM Aβ25-35and120μM3BDO had no effect on cell apoptosis after48h treatment, the number of apoptotic nuclei did not differ between experimental groups. This implied that the protective effect of3BDO against Aβ25-35-induced cytotoxicity might not be through its anti-apoptosis effect. Acidic compartments of PC12cells markedly increased after Aβ25-35-treatment for4h, and120μM3BDO inhibited the increase.1.6.2Because the level of LC3-Ⅱ is directly associated with number of autophagosomes and is generally used to monitor autophagy, we used western blot analysis to examine the protein level of LC3-Ⅱ. Aβ25-35and the autophagy-inducer rapamycin greatly elevated the level of LC3-Ⅱ, which could be inhibited by60and120μM3BDO.2. The effect and its mechanism of3BDO on Aβ genesis in20E2cells.2.1We used ELISA assay to examine the concentration of Aβ1-40. The results showed that120μM3BDO could inhibit the genesis of Aβ1-40in both SH-SY5Y cells and20E2cells, but it had no significant influence in HEK293cells.2.2We used ELISA assay to examine the concentration of Aβ1-42.3BDO could decrease the extracellular level of Aβ1-42in20E2cells, but it had no significant influence in HEK293cells and SH-SY5Y cells.2.3We used western blot analysis to evaluate the level of APP protein after specialized treatments. The level of APP protein in20E2cells was obviously higher than that in HEK293cells, the APP level didn't change in20E2cells and SH-SY5Y cells after120μM3BDO treatment for24h.This implied that3BDO could reduce the extracellular level of Aβ1-40and Aβ1-42in20E2cells not through reducing the level of APP.2.43BDO could decrease the level of LC3-Ⅱ protein in HEK293cells, SH-SY5Y cells and20E2cells. We used Western blot to examine the level of LC3-Ⅱ protein which was the special marker of autophagy and the level of p62whose level could reflex the integrity of autophagy flux. LC3-Ⅱ proteins were decreased in all three cell lines after120μM3BD0treatment for24h and the p62levels increased after3BD0treatment both in20E2cells and SH-SY5Y cells.2.53BD0increased Aβ degradating by upregulating degradating-enzyme IDE and NEP. We used western blot to examine the levels of NEP and IDE in20E2cells. After120μM3BD0for24h the levels of NEP and IDE were significantly increased in20E2cells. This implied that3BD0could increase Aβ degradation.3. The molecular mechanism of3BDO inhibiting autophagy.3.1mTOR-dependent pathway is the classical signaling pathway involved in autophagy process, mTOR is the key modulating fator. We measured the phosphorylated level of p70S6K, which is the substrate of mTOR. Meanwhile we treat cells with rapamycin as positive control. The result showed that both rapamycin and AP induced autophagy through inhibiting the activity of mTOR, and3BD0could inhibit autophagy through activating activity of mTOR.3.2The effect of FLJ118112during the process of3BD0modulating autophagy. FLJ11812is the differential expression gene in pre-and after-treatment with3BDO by using genechip detection. When SH-SY5Y cells were treated with10μM Aβ25-35, the expression of FLJ11812is decreased, and3BDO could up-regulate the expression of FLJ11812.3.3When over expressing FLJ11812, the autophagy level was inhibited. Meanwhile3BDO inhibited the level of LC3-Ⅱ and upregulate p62level in both non ang overexpressing FLJ11812groups.3.4When we used siRNA to downregulate the expression of FLJ11812, LC3-Ⅱ has no significant change between control and3BDO groups.Conclusion1. Aβ induced cytotoxicity and reduced cell viability in a time-and dose-dependent manner.2.3BD0protected cells against Aβ-induced cytotoxicity through inhibiting the increase of intracellular ROS level and restored the Aβ25-35-reduced Na+K+-ATPase activity. However3BDO had no significant influence on Aβ-disturbed MMP.3.3BDO protected cells against Aβ25-35-induced cytotoxicity might not be through its anti-apoptosis effect but through inhibiting autophagy.4.3BD0decreased Aβ1-40and Aβ11-42genesis in20E2cells through inhibiting autophagy level and inhibiting expression of Aβ-degrading enzyme (IDE, NEP).5.3BDO might inhibit autophagy through up-regulation activity of mTOR signaling pathway, meanwhile we found that FLJ11812might have important effect in3BDO-modulating autophagy, which was increased expression induced by3BDO.