Study On The Anti-Alzheimer's Disease Effects And Mechanisms Of Dendrobium Nobile Alkaloids And Dendrobium Alkaloid

Alzheimer’s disease（AD）is a neurodegenerative disease characterized by progressive dementia.The patients of AD are increasing year by year,and medical expenses remain high.Amyloid precursor protein（APP）is a key protein related to AD,which is a precursor to generate amyloidβ（Aβ）peptides through sequenced cleavage,and is also the upstream protein of tau hyperphosphorylation to form neurofibrillary tangles,involved in the pathological process of synaptic damage in AD.APP undergoes a variety of protein post-translational modifications.The ubiquitination-dependent process of APP affects the endocytosis,intracellular sorting,and finally degradation in lysosomes.Reducing APP expression and inhibiting the formation of cytotoxic peptide fragments could exert neuroprotective effects.At present,there is still a lack of effective anti-AD drugs to halt disease progression.The complex pathological mechanisms behind AD etiology mean that a single-target therapeutic strategy cannot achieve disease control.Traditional Chinese medicine has the characteristics of multiple pathways and multiple targets,which can play an overall regulatory role.It is significance to search for anti-AD drugs from the treasure-house of traditional Chinese medicine.Our previous research showed that Dendrobium nobile Lindl alkaloid（DNLA）exhibited neuroprotective,anti-aging,and alleviating effects on cognitive deficits in AD animal model.However,the mechanism involved is still unclear.DNLA contains several monomer components.Confirming the monomeric components of anti-AD efficacy in DNLA can be conducive to drug quality control.This study intends to explore the protective effects and possible mechanisms of DNLA and its major monomer component,Dendrobine（DDB）,in regulating APP protein and synaptic damage,so as to provide a potential agent of therapy for AD.In this study,an acute synaptic injury model was established by injecting Aβ_25-35（10μg）into the bilateral hippocampus of SD rats.DNLA（40 and 80 mg/kg/d）was intragastrically administrated 7 days prior to Aβinjection,and continued for 28 days.The spatial learning and memory,synaptic morphology,synapse-related proteins and Wnt signaling components GSK3βandβ-catenin phosphorylation were evaluated.Rat primary cortical neuron cultures and APP695-PC12 cells were used to evaluate axonal mitochondria distribution,ROS production,amyloidogenesis,and Wnt pathway in the protection.DNLA ameliorated Aβ-induced cognitive impairment,increased the number of synapses,elevated the postsynaptic density thickness and expression of synapsin and PSD95 in the hippocampus,and suppressed Aβ-mediated GSK3βactivity and theβ-catenin phosphorylation.In primary neurons and APP695-PC12cells,DNLA restored Aβ_25-35induced mitochondrial dysfunction,inhibited ROS production and amyloidogenesis.Furthermore,the Wnt/β-catenin pathway inhibitor Dkk-1 blocked the effect of DNLA on the expression of Aβ_1-42and PSD95.To confirm whether DDB is the main active component of DNLA exerting anti-AD effects.3×Tg-AD model mice were intragastrically administrated of DNLA（20mg/kg）and DDB（10,20 mg/kg）from 4 to 7 or 12 months.Cognitive deficits and pathological changes in mice brain were evaluated.We found that DDB reduced the levels of APP and APP fragments and increased the expression of synapse-related proteins PSD95,SYN,and NCAM in the brains of 7-month-old 3×Tg mice.DDB or DNLA treatment could improve episodic memory,spatial learning and memory ability,anxiety,and reduced amyloid plaques in cortex and hippocampus in 12-month-old 3×Tg-AD mice.Compared with the model group mice,DDB also decreased the levels of APP,s APPβ,CTFβ,AT8 and p-Ser396 tau.Moreover,RNA-sequencing analysis showed that synaptic cycling and synaptic development signalling pathways associated with the synaptic function were enriched after administration of DDB,indicating that alleviating synaptic damage may be a key target for DDB to exert its anti-AD effect.In summary,DDB may act as the main component of DNLA to regulate APP pathway proteins,improve Tau pathology and synaptic damage,and thereby alleviate behavioral disorders in AD mice.To further explore the possible mechanism of DDB on down-regulating APP protein expression in vitro.The results revealed that DDB could accelerate the degradation of APP protein in APP-HEK293 cells.And the expression of endogenous APP and its toxic metabolites s APPβand Aβ₄₂were reduced in DDB-treated cortical neurons of APP/PS1 mice.APP was rapidly degraded when the cells pretreated with DDB,and this degradation was partially attenuated by lysosomal inhibitor chloroquine.Mass spectrometry was used to detect APP-interacting differential proteins,and it was found that DDB can affect APP ubiquitination process.Here we show that DDB can stimulating APP lysine 63（K63）-linked polyubiquitination,which affects APP intracellular transport and thus the production of its metabolites.We also found that the expression of APP on the plasma membrane in DDB-treated cells was decreased.Furthermore,DDB decreased the colocalization of APP with Rab5 and increased the colocalization with Rab7.The expression of APP,s APPβand Aβ_1-42in early endosomes was also decreased.Taken together,these findings indicated that DNLA could promote the rescue of Aβ-mediated synaptic injury and suppress the amyloidogenic processing of APP in a manner requiring activation of Wnt/β-catenin signaling pathway.DDB could alleviate behavioral disorders and play a protective role in brain and synapse in 3×Tg-AD mice,which indicates that DDB is the active component of DNLA to exert anti-AD effect.The neuroprotective effect of DDB may be related to regulating APP K63-linked polyubiquitination.DDB limits amyloidogenic processing of APP and associated neuronal dysfunction by promoting the degradation of APP targeting the endosome-lysosomal pathway.