Salvianolic Acid B Attenuates Mitochondrial Stress Against Ab Toxicity In Primary Cultured Mouse Neurons

BackgroundAlzheimer s disease(AD)is the most common type of dementia in the elderly population,and its clinical manifestations include memory loss,cognitive impairment and personality changes.The most typical pathology of AD is extracellular plaque deposition and intracellular neurofibrillary tangles;the former is consists of the depositions of amyloid β(Aβ),and the latter is caused by the spiraling hyperphosphorylated Tau protein.And it is generally considered that Aβ makes the main virulence factor of AD.The specific mechanism of Aβ-induced neuronal toxicity remains unknown.But oxidative stress mediated by Aβ has been repeatedly confirmed as a crucial pathological factor in the pathogenesis of AD.In recent years,the relationship between mitochondrial dysfunction and synaptic failure caused by oxidative stress and neuronal loss during the pathogenesis of AD has been emphasized.Studies have shown that Aβ will lead to excessive production of mitochondrial reactive oxygen species and enhanced oxidative stress in cells;those two factors strengthen each other to form a vicious circle,leading to severe oxidative damage and mitochondrial dysfunction.Salvianolic acid B(SalB)is a biologically active component extracted from the roots of Salvia miltiorrhiza.As traditional Chinese medicine,Salvia miltiorrhiza is widely used in the treatment of a variety of diseases including cardiovascular and cerebrovascular diseases,hepatitis and so on.Pharmacological assays have demostrated that salvianolic acid B has antioxidant activity as a potential active oxygen scavenger.A variety of active oxygen scavengers have been confirmed to have the ability to perotec neurons in AD-related environments.Among those active oxygen scavengers,plant-derived active oxygen scavengers are valued for their ease of extraction and safety.Therefore,in this study we will explore the protective effect of salvianolic acid B on reducing mitochondrial oxidative stress and mitochondrial function in AD environment.Objectives1.To detect the protective effect of salvianolic acid B on mitochondrial bioenergetics in Aβ insulted environment.2.To determine the effect of salvianolic acid B on alleviates mitochondrial oxidative stress in Aβ-treated neurons3.To exam the influence of salvianolic acid B on mitochondrial dynamics in AD related environment4.To mearsure the positive impact of salvianolic acid B on synaptic density of neurons in Aβ rich environments.Methology1.Primary neuron culture and the drug treatment.Animal expriments were approved by Shandong University Animal Use and Care Committee(IACUC).The procedures of animal operation were strictly comply with relevant regulations.Newborn C57BL/6j mouse puppies within twenty four hours fully sterilized were applied to primary neuron culture process.Mature cortical and hippocampal neurons at Div 12-16 days were treated with drugs.Aβ and salvianolic acid B were applied to treat neurons and scramble Aβ were designed to serve as strict control.2.Salvianolic acid B cytotoxicity assay:MTT assay for the viability of neuronal cells treated with different concentrations of salvianolic acid B3.Mitochondrial function detection:TMRM staining for mitochondrial membrane potential examnation,fluorescence detection of ATP production.4.Mitochondrial redox state detection:4-HNE Elisa to demonstrate lipid oxidation,MitoSOX Red staining mitochondrial reactive oxygen species(ROS)assay5.Mitochondrial dynamic detection:Western Blot to mitochondrial division/fusion protein expression level.6.Synaptic density analysis7.Statistical analysis:The results were exhibited in form of mean±s.e.m,one way anova were performed by SPSS software to compare the difference between multiple groups and student’s t test or Bonferroni post hoc analysis were applied to confirm the varience of two groups.P<0.05 were considered of statistical significance.Results:The MTT assay showed that 0-400 uM salvianolic acid B was non-cytotoxic and inhibited the formation of mitochondrial superoxide,thus preserved the redox balance in Aβ-treated neurons.Mitochondrial membrane potential,ATP assay a showed that salvianolic acid B protected mitochondrial bioenergetics function under Aβ interference.Detection of axon mitochondria length and mitochondrial division,fusion-related proteins expression level revealed that salvianolic acid B maintained mitochondrial dynamics.Synaptic density analysis showed that salvianolic acid B maintained synapse abundancy.ConclusionAs an antioxidant,Salvianolic acid B can maintain the mitochondrial redox homeostasis,reduce the mitochondrial dynamic abnormality caused by Aβ,protect the mitochondrial function as well as synaptic function in Aβ environment of primary cultured mouse neurons.