6’’’-Feruloylspinosin Activates Autophagy Via The AMPK/mTOR Signaling Pathway To Alleviate Beta-amyloid Induced Toxicity

Alzheimer’s disease（AD）is a progressive neurodegenerative disease.One of the typical pathological manifestations of AD is the deposition of large amounts of beta-amyloidβ（Aβ）in the cerebral cortex,hippocampus,certain subcortical nuclei and thalamus,thus forming senile plaques（SPs）with Aβas the core,triggering oxidative damage,causing neuroinflammation,etc.,which induces neuronal apoptosis and impairs the cognitive abilities of patients.Ziziphi Spinosae Semen（ZSS）is a traditional Chinese medicine that exhibits significant biological activity in the regulation of neurological functions.6′′′-Feruloylspinosin（6-FS）is one of the main flavonoid components of date palm.6′′′-Feruloylspinosin has been shown to extend the life span of nematodes in Alzheimer’s disease models and has potential therapeutic effects in Alzheimer’s disease,but systematic reports are still lacking.Therefore,the following investigations were conducted to investigate the mechanism of 6′′′-Feruloylspinosin intervention in Alzheimer’s disease:Aβ_1-42 was used to induce SH-SY5Y cell injury and 6′′′-Feruloylspinosin was administered for intervention.The effects of 6′′′-Feruloylspinosin on Aβ_1-42-induced damage in SH-SY5Y cells were investigated from multiple perspectives,including cell viability,cellular oxidative stress,cellular mitochondrial status and cellular autophagy.（1）Aβ_1-42 treatment of SH-SY5Y cells was used to construct the neural cells injury model.It was found that 5 and 10μmol/L Aβ_1-42 significantly reduced SH-SY5Y cells viability（p<0.001）and altered SH-SY5Y cell morphology.5,10,and 20μmol/L 6′′′-Feruloylspinosin inhibited Aβ_1-42-induced apoptosis and increased cell viability in a concentration-dependent manner（p<0.001）;improved oxidative stress in Aβ_1-42-induced damaged cells;regulated apoptosis-related protein expression,increased the expression of the anti-apoptotic protein B cell lymphoma/leukemia-2 protein,and decreased the expression of the pro-apoptotic protein Bcl-2-associated death promoter recombinant protein（p<0.01）.（2）5μmol/L Aβ_1-42 induced mitochondrial damage in SH-SY5Y cells,reduced intracellular ATP content（p<0.001）,decreased mitochondrial membrane potential（p<0.001）,and increased the expression of intracellular mitochondrial transport proteins:outer mitochondrial membrane receptor Tom20 and inner mitochondrial membrane receptor Tim23 in SH-SY5Y cells（p<0.001）.10μmol/L 6′′′-Feruloylspinosin inhibited Aβ_1-42-induced mitochondrial damage,increased the intracellular ATP level and mitochondrial membrane potential level in SH-SY5Y cells,and regulated the expression of mitochondrial membrane transport-related proteins Tom20 and Tim23.（3）Aβ_1-42 was used to construct a neural cell injury model,and cells were treated with6′′′-Feruloylspinosin,the cellular autophagy agonist Rapamycin（RAPA）and the cellular autophagy inhibitor 3-methyladenine（3-MA）.It was found that 5μmol/L Aβ_1-42 and 5mmol/L 3-MA treatment resulted in decreased cell viability（p<0.001）,increased intracellular ROS content（p<0.001）,decreased cellular mitochondrial membrane potential（p<0.001）and affected the expression of autophagy-related proteins.Administration of 6′′′-Feruloylspinosin and RAPA increased cell viability（p<0.001）,decreased intracellular ROS accumulation（p<0.01）,and increased cellular mitochondrial membrane potential levels（p<0.01,p<0.05）.It also upregulated the expression of yeast Atg6 homolog（Beclin1）,decreased the expression of nuclear pore glycoprotein（P62）and the ratio of microtubule-associated protein 1 light chain 3 II/I（LC3 II/I）,regulated cellular autophagy and alleviated cellular damage.（4）A nerve cell injury model was constructed using Aβ_1-42 and cells were treated with6′′′-Feruloylspinosin,AMPK agonist Phenformin and AMPK inhibitor Compound C.Aβ_1-42and Compound C treatment resulted in decreased cell viability（p<0.001）,increased intracellular ROS content（p<0.01,p<0.001）,and affected the expression of cellular AMPK/m TOR autophagy pathway-related proteins.6′′′-Feruloylspinosin and Phenformin treatment of cells increased cell viability（p<0.001）,improved cell morphology and reduced intracellular ROS accumulation（p<0.01,p<0.05）.Up-regulated the phosphorylation level of5′-adenosine monophosphate-activated protein kinase（AMPK）protein and decreased the phosphorylation level of Unc51-likekinase（ULK1）,mammalian target of rapamycin（m TOR）protein.This suggests that 6′′′-Feruloylspinosin may affect the expression of AMPK/m TOR autophagy pathway-related proteins to protect damaged mitochondria and alleviate cellular oxidative damage,thereby reducing Aβ_1-42-induced cellular damage.