Study On The Protective Effect And Mechanism Of Quercetin On β-amyloid-induced Apoptosis InPC12 Cells Mitochondria

Alzheimer’s disease（AD）is a major public health problem,with progressive neurological loss,cognitive impairment and executive impairment as the main clinical manifestations.Epidemiological studies have shown that the incidence of women is significantly higher than that of men.It is speculated that the occurrence of AD may be related to the level of estrogen in women.The etiology and pathogenesis of AD are very complicated.Estrogen replacement therapy（ERT）is commonly used in clinical practice.It can significantly alleviate the symptoms of menopause in women,but the clinical trial data of postmenopausal women show that there are adverse reactions to estrogen therapy,which makes its application in clinical treatment limited.Phytoestrogens have attracted widespread attention.Studies have found that when phytoestrogens with a chemical structure similar to estrogen are used in the treatment of neurodegenerative diseases,phytoestrogens not only have an estrogen-like neuroprotective effect but also reduce the occurrence of adverse reactions.Therefore they are expected to become drugs for the prevention and treatment of AD.Quercetin（Que）is a natural flavonoid phytoestrogens derived from a variety of fruits and vegetables.It has a unique neuroprotective effect without obvious toxicity and adverse reactions.In this study,Aβ_25-35induced PC12 cell injury to establish an in vitro model of AD,and explore the neuroprotective effect of quercetin in the process of mitochondrial apoptosis and its molecular mechanism.After treating PC12 cells with Aβ_25-35 for 24h,the positive control drugs were added with 0.1μmol·L^-117β-E2,50μmol·L^-1 Gen and 40,60,80μmol·L^-1 Que and continued to incubate for 24h.MTT method was used to detect the effect of quercetin on the activity of PC12 cell AD injury model under the condition of estrogen depletion;Mito Tracker Red mitochondrial fluorescence probe was used to observe the morphological changes of mitochondria;2,6-DPIP reduction method was used to determine the activity of succinate dehydrogenase;spectrophotometry Na⁺-K⁺-ATP enzyme activity was measured by Rhodamine 123 fluorescent dye;mitochondrial transmembrane potential was measured by Rhodamine 123;ELISA double antibody sandwich method was used to measure intracellular cytochrome C concentration;Caspase-3 activity was measured by spectrophotometry,and quercetin was measured by immunofluorescence staining.Aβ_25-35 damages the expression of the two subtypes of estrogen receptor ERα,ERβand p-AMPK in PC12 cells.Western blot detects ERαand ERβin damaged PC12 cells.The mitochondrial pathway apoptosis protein Bcl-2 mediated by AMPK signaling pathway,Bax,Caspase-3,Cyt-C,p-AMPK,t-AMPK protein expression changes.And use estrogen receptor antagonist ICI182,780,AMPK specific inhibitor Compound C to explore the molecular mechanism of quercetin exerting neurop.The results showed that Aβ_25-35 at 20μmol·L^-1 could significantly reduce the survival rate of PC12 cells after 24h（P<0.01）.The positive control drugs 17β-E2,Gen and Que groups are comparable to the model control group.Ratio can significantly increase the survival rate of cells（P<0.05）.Que can improve mitochondrial morphology;increase mitochondrial succinate dehydrogenase（SDH）activity and mitochondrial Na⁺-K⁺-ATPase activity;stabilize Aβ_25-35 induced mitochondrial transmembrane potential of PC12 cells;inhibit Caspase-3 Activity;reduce the release of Cytochrome C.The results show that Que can inhibit PC12cell damage and mitochondrial dysfunction induced by Aβby improving the morphology of PC12 cell mitochondria,stabilizing mitochondrial membrane potential and membrane permeability,and increasing cell mitochondrial viability.The results of immunofluorescence staining and Western blot showed that compared with the model group,quercetin increased the expression of ERαand p-AMPK protein（P<0.05）.Although there was an increase in the expression of ERβprotein,there was no significant difference（P>0.05）.And in the Western blot experiment,the expression ratio of apoptosis-related protein Bcl-2/Bax increased and the expression of Caspase-3 protein decreased in the experimental group（P<0.05）.When the quercetin estrogen receptor was inhibited by ICI182,780,the expression of p-AMPK protein decreased（P<0.05）,the ratio of Bcl-2/Bax decreased and the expression of Caspase-3 and Cytochrome C protein increased（P<0.05）.When Compound C acts on quercetin,the ratio of Bcl-2/Bax decreases and the expression of Caspase-3and Cytochrome C protein increases（P<0.05）.Quercetin inhibits Aβ_25-35induced PC12 cell mitochondrial pathway apoptosis through signaling pathways to play a neuroprotective effect,and binding with ERαcan mediate the activation of AMPK signaling pathway,and then exert neuroprotective effects.