Protective Effect And Mechanism Of L-3-n-butylphthalide On Neuronal Damage Induced By A?_1-42 Oligomer

BackgroundAlzheimer's disease(AD)is a progressive neurodegenerative disease characterized by cognition and memory impairment,accounting for 60%-80%of patients with dementia.The clinical features of AD are progressively deterioration of memory,cognition,and executive dysfunction,resulting in a complete loss of social ability and self-care ability.The pathological hallmarks of AD are brain atrophy,massive loss of cortical and hippocampal neurons and synapses,extracellular amyloid-? plaques and intracellular neurofibrillary tangles.Alzheimer's disease is currently considered as a genetically heterogeneous disease,and its pathogenesis is still unclear.A variety of hypotheses and conjectures have been proposed for the pathogenesis of AD,including the cholinergic hypothesis,the A? protein cascade hypothesis,the Tau protein hyperphosphorylation hypothesis,the mitochondrial cascade hypothesis,and the neuroinflammation hypothesis and so on.The A? protein cascade hypothesis is the current dominant doctrine.First described by Hardy and Higgins in 1992,the amyloid cascade hypothesis pinpointed the overproduction of A?,the major component of amyloid plaques,as the initiating event in AD pathology,occurring upstream to the development of neuronal death,synaptic loss,tau tangles and declining cognitive function.A? is a polypeptide produced by proteolysis of ?-amyloid precursor protein(APP),and can exist in the form of monomers,oligomers,and fibrils in the brain.Early in the discovery of A?toxicity in AD,it was thought that the fibrillar form found within amyloid plaques was the most detrimental to neurons and synapses.However,various evidences suggested that amyloid plaque levels are not significantly associated with the levels and distribution of neurodegenerative and cognitive decline weakness.Later,researchers such as Selkoe proposed that the neurotoxicity of A? mainly comes from soluble A?oligomers,while the toxicity of A? monomers and fibrils is weak,and this view is widely supported by many clinical studies.Studies have found that A? oligomers could cause pathophysiological changes such as apoptosis,synaptic damage,and tau phosphorylation both in vitro and in vivo.First,A? oligomers could induce typical apoptotic changes such as cell shrinkage,cytoplasmic compaction,nuclear disintegration,chromatin condensation,apoptotic bodies and DNA ladder formation.Secondly,it was found that very low concentrations of A? oligomers could induce synaptic plasticity dysfunction and synaptic loss.Finally,it was found that A? can induce tau phosphorylation,disrupt microtubule stability,and cause neuronal death by activating glycogen synthase kinase 3?.However,the mechanism by which A? oligomers cause the aforementioned neurotoxicity is very complicated and has not yet been clarified.Studies have shown that A? could induce neuronal apoptosis and synaptic damage by inducing mitochondrial dysfunction,interfering with cell signal transduction pathways,producing excessive oxidative stress,inducing calcium dysregulation,and activating glial inflammatory responses.And the above cascade reactions were not independent,but intertwined,and the interaction of the above various cascade reactions eventually lead to the development of AD.Mitochondrial dysfunction played a very important role in A? neurotoxicity.As an energy-generating organelle necessary for cells,mitochondrial dysfunction is closely related to oxidative stress,apoptosis,and synaptic damage.Accumulating studies have found that mitochondria were particularly susceptible to A(3.A? could significantly inhibit the activity of cytochrome c oxidase and alpha ketoglutarate dehydrogenase,destroy mitochondrial energy production,and aggravate oxidative stress.In addition,A? could also directly act on multiple mitochondrial proteins,leading to mitochondrial permeability transition pores opened,calcium influx,mitochondrial membrane potential decreased.Dysfuncted mitochondria further exacerbated oxidative stress and calcium homeostasis imbalance,forming a vicious circle,eventually leading to neuronal apoptosis and synaptic damage.In addition,studies have found that mitochondrial dysfunction affects tau phosphorylation and neuroinflammatory responses.We believe that a drug which can significantly attenuate A?-induced mitochondrial dysfunction will bring new prospects for the treatment of AD.Studies have found that A? could affect mitogen-activated protein kinase(MAPK),phosphatide linositol-3-kinase/serine threonine kinase(PI3K/AKT),nuclear factor-KB(NF-?B),Wnt and other signaling pathways,then lead to neuronal apoptosis and synaptic loss.The PI3K/AKT pathway is a classical anti-apoptotic pathway which plays an important role in cell proliferation and differentiation,apoptosis and cell metabolism.It was found that A? could inhibit AKT phosphorylation by binding to a7nAChR and NMDA receptors or insulin receptors,induce abnormal expression of apoptotic proteins such as Bax,Bad,p53,increase permeability of mitochondria and endoplasmic reticulum,and promote neuronal apoptosis.However,activation of the PI3K/AKT signaling pathway could effectively attenuate neuronal apoptosis and synaptic loss induced by A?.Extracellular regulated protein kinase(ERK)signaling pathway is one of the important members of the MAPK signaling pathway family.It could transduce extracellular stimuli to the nucleus and is involved in the regulation of cell survival,proliferation and differentiation.A large number of studies have found that ERK signaling pathway was activated during the pathogenesis of AD,and that ERK activation was closely related to neuronal apoptosis and synaptic plasticity.As the population aging is aggravated,the incidence of AD is gradually increasing,but recent clinical trials of AD treatment drugs have all failed,so the development of effective treatment drugs is imminent.3-n-butylphathilide(NBP)is a ischemic stroke treatment drug developed in China.It is a synthetic racemate containing both L-form and D-form isomers.L-3-n-butylphthalide(L-NBP),which was originally extracted from the seeds of Apium graveolens Linn(Chinese celery),was the major pharmacological component.Previous studies have found that L-NBP could protect against ischemic stroke through multiple mechanisms,such as inhibiting platelet aggregation and thrombosis,protecting vascular endothelial cells,improving microcirculation in arterioles,decreasing oxidative damage and neuronal apoptosis,improving mitochondrial function and inhibiting infammatory response.Recent studies suggested that L-NBP could significantly improve the cognitive function of AD rats,reduce A? plaque deposition,reduce oxidative stress,inhibit glial activation,reduce neuronal apoptosis,improve synaptic function in the brain of AD rats.However,the mechanism by which L-NBP inhibited neuronal apoptosis and synaptic damage caused by A?1-42 oligomers is still unclear,and the role of PI3K/AKT and ERK signaling pathways in this process is unclear.Based on the previous researches,we designed this experiment to further investigate the protective effects and possible mechanisms of L-NBP.In vitro,we added A?1-42 oligomers to primary cultured neurons to create an ideal AD cell model,experiment groups were pre-incubated with L-NBP,then the effects of L-NBP on cytotoxicity,mitochondrial membrane potential decrease,neuronal apoptosis,synaptic loss and tau phosphorylation induced by A?1-42 oligomers were examined.In vivo,we prepared an animal model of AD by perfusion of A?1-42 oligomers into the lateral ventricle of SD rats.After administration of L-NBP,we examined the learning and memory ability,cortical cell apoptosis and tau phosphorylation in SD rats.We further studied the protective effect of L-NBP on neuronal damage induced by A? oligomers,and explored the changes of mitochondrial membrane potential,PI3K/AKT and ERK signaling pathways in this process,as to elucidate the possible molecular mechanisms by which L-NBP played the protective role.CHAPTER ? L-NBP could attenuate A?1-42-induced neuronal damageSECTION ? L-NBP could attenuate A?1-42-induced neuronal damage in vitroOBJECTIVEBy investigating the effects of L-NBP incubation on neuronal cytotoxicity,mitochondrial membrane potential,neuronal apoptosis,synaptic loss and tau phosphorylation induced by A?1-42 oligomers,we studied whether L-NBP has protective effects on neuronal damage induced by A?1-42 oligomers in vitro.METHODSThe AD cell model was prepared by adding A?1-42 oligomers into the primary cultured neurons,and pre-incubated with different concentrations of L-NBP for 4 hours.The cultured cells were divided into 5 groups:(1)control group,without any treatment;(2)A?1-42 treatment group(10?mol/L A?1-42 incubation for 24 hours);(3)A?1-42+0.1?mol/L L-NBP pretreatment group;(4)A?1-42+1?mol/L L-NBP pretreatment group;(5)A?1-42+10?mol/L L-NBP pretreatment group.After the treatment,the cell viability was detected by MTT assay,apoptosis was detected by Hoechst 33342 staining,synaptophysin expression was detected by immunofluorescence staining,and mitochondrial membrane potential was detected by JC-1 staining flow cytometry.The expression level of p-tau,total-tau,pro-caspase-3,cleaved caspase-3,and synaptophysin was detected by Western blot analysis.RESULTS1.Compared with the control group,the A?1-42 treatment group showed decreased cell viability,increased apoptotic neurons percentage,increased number of cells with mitochondrial membrane potential collapse,increased expression of p-tau and cleaved caspase-3,decreased expression of synaptophysin and pro-caspase-3,and the differences were statistically significant(P<0.01).2.Compared with the A?1-42 treatment group,the L-NBP pretreatment group showed increased cell viability,decreased apoptotic neurons percentage,decreased number of cells with mitochondrial membrane potential collapse,decreased expression of p-tau and cleaved caspase-3,and increased expression of synaptophysin and pro-caspase-3,and the differences were statistically significant(P<0.05).The protective effect of L-NBP was dose-dependent.CONCLUSIONL-NBP could significantly reduce neuronal cytotoxicity,mitochondrial membrane potential collapse,tau phosphorylation,neuronal apoptosis and synaptic loss induced by A?1-42 oligomers,which confirming L-NBP attenuated the neuronal damage induced by A?1-42 oligomers in vitro.SECTION ? L-NBP could attenuate A?1-42-induced neuronal damage in vivoOBJECTIVEBy detecting the effects of L-NBP on learning and memory ability,cortical apoptosis and tau phosphorylation in SD rats treated with A?1-42 oligomers,we investigated whether L-NBP could inhibit the neuronal damage induced by A?1-42 oligomers in SD rats.METHODSThe animal model of AD was prepared by injecting A?1-42 oligomer into the lateral ventricle of SD rats,and the sham group was injected with the same amount of physiological saline.L-NBP or vegetable oil was then administered to the stomach.After treatment,the passive avoidance test was applied to detect the learning and memory ability of the rats.TUNEL staining was performed to detect apoptosis of the cortex.The cortical protein was extracted and the expression of p-tau protein was detected by Western blot.RESULTS1.Compared with the sham group,the A?1-42 group showed shortened latency of entering the darkroom,increased numbers of mistakes entering the darkroom within 5 minutes,increased apoptotic cells and p-tau expression in cortex,and the differences were statistically significant(P<0.01).2.Compared with the A?1-42 group,the L-NBP group showed increased latency of entering the darkroom,decreased numbers of mistakes entering the darkroom within 5 minutes,decreased apoptotic cells and p-tau expression in cortex,and the differences were statistically significant(P<0.05).CONCLUSIONL-NBP could significantly improve the learning and memory ability,inhibit apoptosis and tau hyperphosphorylation in cortex of SD rats with A?1-42 oligomers infusion,which confirming L-NBP attenuated neuronal damage induced by A?1-42 oligomers in vivo.CHAPTER ? L-NBP could attenuate A?1-42-induced neuronal damage possibly via modulating both PI3K/AKT and ERK signaling pathwaysOBJECTIVEThe aim was to investigate the role of PI3K/AKT and ERK signaling pathways in A?1-42 oligomers leading to neuronal damage,and to further explore the molecular mechanism by which L-NBP protects by regulating PI3K/AKT and ERK signaling pathways.METHODS1.The AD cell model was prepared by adding A?1-42 oligomers in primary cultured neurons,and pre-incubated with different concentrations of L-NBP for 4 hours.The cultured cells were divided into 5 groups:(1)control group,without any treatment;(2)A?1-42 treatment group(10?mol/L A?1-42 incubation for 24 hours);(3)A?1-42+0.1?mol/L L-NBP pretreatment group;(4)A?1-42+1?mol/L L-NBP pretreatment group;(5)A?1-42+10?mol/L L-NBP pretreatment group.After treatment,the expression level of p-ERK,total ERK,p-AKT and total AKT was detected by Western blot.2.The AD cell model was prepared by adding A?1-42 oligomers in primary cultured cortical neurons.The cells were pre-incubated with different concentrations of LY294002 for 1 hour before L-NBP was added.The cultured cells were divided into six groups:(1)control group,without any treatment;(2)A?1-42 treatment group(10?mol/L A?1-42 incubation for 24 hours);(3)A?1-42+10?mol/L L-NBP pretreatment group;(4)A?1-42+10?mol/L L-NBP+20?mol/L LY294002 treatment Group;(5)A?1-42+10?mol/L L-NBP+40?mol/L LY294002 treatment group;(6)A?1-42+10?mol/L L-NBP+60?mol/L LY294002 treatment group.After treatment,the cell viability of each treatment group was examined by the MTT.3.The AD cell model was prepared by adding A?1-42 oligomer in primary cultured cortical neurons.The cells were pre-incubated with LY294002 and U0126 for 1 hour before L-NBP added.The cultured cells were divided into four groups:(1)control group,without any treatment;(2)A?1-42 treatment group(10?mol/L A?1-42 incubation for 24 hours);(3)A?1-42+10?mol/L L-NBP treatment group;(4)A?1-42+60?mol/L LY294002+10?mol/L L-NBP treatment group or A?1-42+10?mol/L U0126+10?mol/L L-NBP treatment group.After treatment,the mitochondrial membrane potential collaspe was detected by JC-1 staining flow cytometry,and the expression level of p-tau,total-tau,cleaved caspase-3,synaptophysin,p-AKT,total AKT,p-ERK,total ERK and ?-catenin was detected by Western blotRESULTS1.Compared with the control group,the A?1-42 treatment group showed decreased expression of p-AKT and increased expression of p-ERK,and the differences were statistically significant(P<0.01).Compared with the A?1-42 treatment group,the L-NBP pretreatment group showed increased expression of p-AKT and decreased expression of p-ERK,and the differences were statistically significant(P<0.05).And there was no significant difference in the expression levels of total AKT and total ERK between all the treatment groups.The results suggested that L-NBP could play a protective role by promoting AKT phosphorylation and inhibiting ERK phosphorylation.2.Compared with the control group,the cell viability of the A?1-42 treatment group was significantly decreased(P<0.01).Compared with the A?1-42 treatment group,the cell viability of the L-NBP pretreatment group was significantly increased.The differences were statistically significant(P<0.05).Compared with the L-NBP pretreatment group,the cell viability of A?1-42+L-NBP+40?mol/L LY294002 treatment group and A?1-42+L-NBP+60?mol/L LY294002 treatment group were significantly decreased,the differences were statistically signifiant(P<0.05).The results suggested that inhibiting AKT phosphorylation by LY294002 could block the protective effect of L-NBP on A?1-42-induced cytotoxicity3.Compared with the control group,the A?1-42 treatment group showed increased number of cells with mitochondrial membrane potential collapse,decreased expression of p-AKT,?-catenin and synaptophysin,increased expression of p-tau protein and cleaved caspase-3,and the differences were statistically significant(P<0.01).Compared with the A?1-42 treatment group,the L-NBP pretreatment group showed decreased number of cells with mitochondrial membrane potential collapse,increased expression of p-AKT,?-catenin and synaptophysin,decreased expression of p-tau and cleaved caspase-3,and the differences were statistically significant(P<0.05).Compared with the L-NBP pretreatment group,the A?1-42+L-NBP+LY294002 treatment group showed increased number of cells with mitochondrial membrane potential collapse,decreased expression of p-AKT,?-catenin,and synaptophysin,increased expression of p-tau protein and cleaved caspase-3,and the differences were statistically significant(P<0.05).The results showed that inhibiting AKT phosphorylation by LY294002 could block the protective effect of L-NBP on inhibiting mitochondrial membrane potential collapse,tau phosphorylation,neuronal apoptosis and synaptic loss.4.Compared with the control group,the expression level of p-ERK and cleaved caspase-3 in A?1-42 treatment group was increased significantly,and the differences were statistically significant(P<0.01).Compared with A?1-42 treatment group,the expression level of p-ERK and cleaved caspase-3 in L-NBP pre-incubation group was significantly decreased(P<0.05).Compared with the L-NBP pretreatment group,the expression level of p-ERK and cleaved caspase-3 in the A?1-42+L-NBP+U0 126 treatment group was significantly decreased(P<0.05).The results suggested that inhibiting ERK phosphorylation by U0126 had the synergistic effect with L-NBP on inhibiting neuronal apoptosis induced by A?1-42CONCLUSIONPretreatment with L-NBP effectively inhibited A?1-42 induced cytotoxicity,apoptosis,mitochondrial dysfunction,and synaptic loss in primary cultured cortical neurons in a dose-dependent way.The protective effects of L-NBP were accompanied by increased phosphorylation of AKT and decreased phosphorylation of the ERK.The neuroprotective effects could be abolished by LY294002,an inhibitor of PI3K-dependent protein kinase B.Furthermore,ERK pathway inhibitor U0126 enhanced the beneficial effects of L-NBP in inhibiting A?-induced apoptosis.So we conclude L-NBP has protective effects on A? induced neuronal damage through regulating both PI3K/AKT and ERK signaling pathways.