The Effect And Mechanism Of Quercetin On Apoptosis Of PC12 Mitochondria Induced By Aβ_25-35 Through JNK Signaling Pathway

Alzheimer’s disease（AD）is characterized by progressive decline of cognitive and memory functions.Clinical studies have shown that the decrease of estrogen level in women after menopause leads to an increased risk of oxidative stress and accelerates the process of neurodegenerative diseases.It is speculated that estrogen level is closely related to the occurrence of AD.However,the clinical use of estrogen is controversial.Studies have shown that it can take up to 10 years for estrogen to improve cognitive impairment in postmenopausal women,and long-term use of estrogen significantly increases the risk of breast,endometrial and cervical cancers.Quercetin is a natural phytoestrogens with similar chemical structure and estrogen effect.Quercetin（Que）widely exists in fruits and vegetables and medicinal plants,and has no obvious toxic and side effects.Previous studies of our group found that quercetin had a weak estrogen-like effect on MCF-7 and T47D cells,and could promote synaptic plasticity and growth of rats’cerebral cortex and hippocampus through estrogen receptorα（ERα）.The neuroprotective effect of quercetin on tau hyperphosphorylation through PI3K/Akt/GSK3βsignaling pathway was studied by exploring the relationship between ER and phosphorylation regulation pathways and pathological changes of AD.The MAPK/ERK signaling pathway was affected by ERαmediating,and apoptosis was regulated.Previous studies have shown that quercetin has an estrogen-like neuroprotective effect,but its mechanism is complex and involves the interaction of multiple intervention pathways.Among them,the ER-mediated JNK signaling pathway is related to the mitochondrial apoptosis pathway.This study will start with the protective effect of quercetin on Aβ_25-35-induced apoptosis of PC12.Using estrogen receptor antagonist and JNK signaling pathway inhibitor,we explored the neuroprotective effect of quercetin through the association between JNK signaling pathway and ER.In this study,PC12 cells were incubated with Aβ_25-35for 24h to construct AD damage model,and the molecular mechanism and target of the neuroprotective effect of quercetin on the cellular level were further discussed.The experimental groups were normal control group,AΒ_25-35model group(20μmol·L^-1),positive control group(0.1μmol·L^-1)with17β-E₂and Gen(50μmol·L^-1),low,middle and high Que groups(40,60,80μmol·L^-1).Cell Counting Kit-8（CCK8）method was used to detect the activity of PC12 cells under estrogen depletion condition after24h co-incubation with Aβ_25-35.Fluo-4 AM dye fluorescence was used to detect the change of calcium ion in PC12 cells.Mito-Tracker Red CMXRos fluorescence probe was used to observe the morphological changes of mitochondria and Rhodamine 123 staining was used to detect the changes of mitochondrial membrane potential.The early apoptosis rate was evaluated.Caspase-3 activity detector was employed to detect Caspase-3 activity.The expression of ERa,ERβ,p-JNK and Cytochrome C（Cyt C）proteins were detected by immunofluorescence staining combined with confocal laser scanning.The expression of ERα,ERβ,p-JNK,JNK,Bcl-W,Bim and Cyt C proteins were detected by Western blot.In addition,estrogen receptorα/βinhibitor MPP/PHTPP and JNK specific inhibitor SP600125 were used to investigate the neuroprotective mechanism of quercetin.The results of growth curve showed that PC12 cells were best treated 24-72h after inoculation.The data after AΒ_25-35stable membrane-forming drug administration showed that,compared with normal control group,Aβ_25-35significantly inhibited the viability of PC12cells（P<0.001）,promoted the Ca²⁺outflow（P<0.001）,changed the morphology of mitochondria（P<0.001）and decreased the mitochondrial membrane potential（P<0.001）,and then caused mitochondrial damage.Low,middle and high dose of quercetin could increase the viability of injured cells（P<0.001）,improve the morphology of mitochondria,maintain the normal structure of mitochondria,and increase the mitochondrial membrane potential（P<0.001）.The concentration of Ca²⁺was decreased（P<0.01）.Compared with the model group,quercetin reversed the decreased expression of ERαinduced by Aβ（P<0.01）,but increased the expression of ERβ（P>0.05）.When the binding of quercetin to ERαwas inhibited by the addition of MPP（P<0.05）,the expression of Caspase-3 increased.Quercetin inhibited the phosphorylation of JNK（P<0.001）,decreased the expression of Bim（P<0.01）,decreased the release of Cyt C（P<0.01）,and promoted the expression of Bcl-W（P<0.001）,the protective mechanism of SP600125 inhibitor on Aβ-induced mitochondrial apoptosis in PC12 cells is the same as that of SP600125 inhibitor.Quercetin can inhibit-induced mitochondrial apoptosis of PC12 cells induced by Aβ_25-35and exert the neuroprotective effect of estrogen.The mechanism may be related to the activation of JNK signaling pathway,and then exert the neuroprotective effect.