The Protective Effect And Mechanism Of Quercetin-3-O-Glucuronide On SH-SY5Y Cells Induced By Aβ_1-42

Alzheimer’s disease（AD）is a progressive neurodegenerative disease,which is characterized by extracellular senile plaque deposits,intracellular neurofibrillary tangles,and neuronal loss.Amyloid-β（Aβ）plays a critical role on the progress of AD.Previous studies have found that Aβcould induce neuronal synaptic damage by down-regulating brain-derived neurotrophic factor（BDNF）-related signal pathways,which could cause neuronal cell apoptosis and death,and ultimately lead to AD.Although there were treatments proposed for this hypothesis,no effective drugs were available for AD so far.In addition,these drugs had a series of problems,such as:short duration of action,obvious toxicity and side effects,difficulty in origin and difficulty in penetrating the blood-brain barrier.Quercetin-3-O-glucuronide（Q3G）,as a flavonoid compound with good food safety,has several pharmacological activities such as scavenging free radicals,anti-oxidation,anti-aging and inhibiting tumor cell growth.At present,studies have shown that Q3G exerted its neuroprotective effect through anti-oxidation and inhibiting the formation of Aβ,however,whether Q3G could improve AD by regulating BDNF-related signaling pathways has not been reported.In our study,we first treatment the SH-SY5Y with Aβto design cell injury model,and then,Q3G was used to explore its protective effects and specific mechanism.This study aimed to identify the protective of Q3G on Aβ-induced damage with associated pathways for AD treatment.Thus,the main content of this study was divided into the following two sections:Part 1 The protective effect of Q3G on damage of SH-SY5Y cells induced by Aβ_1-42Objective:Aβ_1-42 were added to SH-SY5Y cells to establish a cell injury model in vitro,and to explore the effect of Q3G on the cell damage.Methods:（1）SH-SY5Y cells injury model induced by Aβ_1-42SH-SY5Y cells were divided into control group and different doses（5,10,20,40and 50μM）of Aβ_1-42group,and were incubated for four different time periods（12,24,36 and 48 h）,respectively.The cell viability was detected by cell counting kit-8（CCK-8）.The optimal incubation dose and time of Aβ_1-42were selected according to the results of cell viability.（2）The protective effect of Q3G on SH-SY5Y cells induced by Aβ_1-42SH-SY5Y cells were treated with different doses of Q3G（0,1,2,5,10,20,40 and50μg/mL）for 1 h before 20μM or 0μMAβ_1-42 incubation,respectively.The cell viability was measured by CCK-8.The optimal incubation dose of Q3G was selected according to the results of cell viability.（3）The effect of Q3G on apoptosis of SH-SY5Y cells induced by Aβ_1-42SH-SY5Y cells were divided into four groups.Control group was treated with incomplete cell medium,Aβgroup was treated with 20μMAβ_1-42,Q3G+Aβgroup was treated with 10μg/mL Q3G for 1 h before 20μMAβ_1-42incubation,Q3G group was treated with 10μg/mL Q3G.After 24 h,Hoechst 33342/PI staining and Annexin V-FITC/PI dual-label flow cytometry were used to measure the cell apoptosis.Results:（1）According to the CCK-8,exposure to Aβ_1-42 could cause different degrees of cell viability reduction.Compared with control group（96.29±0.65%）,the survival rate of cells was significantly decreased to 79.40±1.59%after treatment with 20μMAβ_1-42for 24 h,and the difference was statistically significant（P<0.05）.Thus,20μM was selected as the intervention dose of Aβ_1-42and 24 h was chosen as the intervention time.（2）According to the CCK-8,Q3G could inhibit the cell death induced by Aβ_1-42and the cell survival rate was also gradually increased with an increase in Q3G concentration.When the dose of Q3G was 10μg/mL,the cell survival rate was the highest,reaching 88.16±1.11%,and the difference with the 0μg/mL Q3G（78.95±1.61%）was statistically significant（P<0.05）;The 20,40 and 50μg/mL Q3G treatment could also protect cells from death,although not significant.Thus,the dose of 10μg/mL Q3G was chosen for the successive test.（3）Hoechst 33342/PI staining results showed that the cell apoptosis were obviously increased with the incubation of Aβ,while Q3G reversed the toxicity of Aβ.Annexin V-FITC/PI dual-label flow cytometry results found that compared with control group,the rates of early cell apoptosis（13.57±0.62%vs.7.25±1.00%,P<0.01）,the rates of late cell apoptosis（2.39±0.34%vs.0.57±0.19%,P<0.01）and the rates of total cell apoptosis（15.96±0.84%vs.7.82±1.19%,P<0.01）were increased in Aβgroup,while the rates of early cell apoptosis（9.90±1.68%vs.13.57±0.62%,P<0.05）,the rates of cell late apoptosis（1.41±0.16%vs.2.39±0.34%,P<0.05）and the rates of total cell apoptosis（11.31±1.59%vs.15.96±0.84%,P<0.05）were decreased in Q3G+Aβgroup.Besides,compared with Aβgroup,the rates of early cell apoptosis（6.62±0.65%vs.13.57±0.62%,P<0.01）,the rates of cell late apoptosis（1.20±0.43%vs.2.39±0.34%,P<0.05）and the rates of total cell apoptosis（7.82±0.23%vs.15.96±0.84%,P<0.01）were also decreased in Q3G group.Conclusion:Exposure to Aβ_1-42could lead to a decrease in survival rate,and promote the occurrence of apoptosis in SH-SY5Y cells,while Q3G could improve the cell damage caused by Aβ_1-42.Part 2 The effect of Q3G on BDNF signal pathway in SH-SY5Y cells induced by Aβ_1-42Objective:This study aimed to identify the protective effect of Q3G on cyclic-AMP response-element-binding protein（CREB）/BDNF expression in SH-SY5Y cells through the activation of protein kinase B（AKT/PKB）and extracellular signal-regulated kinase（ERK）signal pathways.Methods:（1）Effect of Q3G on the expression of BDNF signaling pathway-related proteins in SH-SY5Y cells induced by Aβ_1-42SH-SY5Y cells were divided into four groups.Control group,Aβgroup,Q3G+Aβgroup and Q3G group.Control group was treated with incomplete cell medium,Aβgroup was treated with 20μMAβ_1-42,Q3G+Aβgroup was treated with 10μg/mL Q3G for 1 h before 20μMAβ_1-42incubation and Q3G group was treated with 10μg/mL Q3G.After 24 h,western blot（WB）was used to measure the expression level of BDNF signaling pathway-related proteins（2）Effect of Q3G on the expression of BDNF signaling pathway-related proteins in SH-SY5Y cells induced by Aβ_1-42 after inhibitor treatmentSH-SY5Y cells were divided into six groups.Control group,Aβgroup,Q3G+Aβgroup,Q3G group,Q3G+Aβ+LY294002（an inhibitor of the PI3K/AKT signaling pathway）group and Q3G+Aβ+PD98059（an inhibitor of the ERK signaling pathway）.Q3G+Aβ+LY294002 group was treated with 10μg/mL Q3G for 30 min before 10μM LY294002 incubation,and after 30 min,the 20μMAβ_1-42 was added.Q3G+Aβ+PD98059 group was treated with 10μg/mL Q3G for 30 min before 30μM PD98059incubation,and after 30 min,the 20μMAβ_1-42 was added.After 24 h,WB was also used to measure the expression level of BDNF signaling pathway-related proteins.Results:（1）the protein expression levels of BDNF,p-CREB/CREB,p-AKT/AKT and p-ERK1/2/ERK1/2 in each group.Compared with the control group,the protein expression level of BDNF in the Aβgroup was significantly reduced（P<0.05）and the phosphorylation expression level of its gene regulatory protein CREB（p-CREB/CREB）was correspondingly reduced（P<0.05）.Besides,its upstream key proteins,including the phosphorylation expression levels of AKT（p-AKT/AKT）and ERK（p-ERK/ERK）were also significantly decreased（P<0.05）.Compared with the Aβgroup,the protein expression levels of BDNF,p-CREB/CREB,p-AKT/AKT and p-ERK/ERK were significantly increased in Q3G+Aβgroup and Q3G group（P<0.05）.（2）the protein expression levels of BDNF,p-CREB/CREB,p-AKT/AKT and p-ERK1/2/ERK1/2 in each group after inhibitor treatmentCompared with the Aβgroup,the protein expression levels of BDNF,p-CREB/CREB and p-AKT/AKT were significantly increased in Q3G+LY294002+Aβgroup（P<0.05）.However,compared with the Q3G+Aβgroup,the protein expression levels of BDNF,p-CREB/CREB and p-AKT/AKT were significantly decreased in Q3G+LY294002+Aβgroup（P<0.05）.Compared with the Aβgroup,the protein expression levels of BDNF,p-CREB/CREB and p-ERK/ERK were significantly up-regulated in Q3G+PD98059+Aβgroup（P<0.05）.However,compared with the Q3G+Aβgroup,the protein expression levels of BDNF,p-CREB/CREB and p-ERK/ERK were significantly down-regulated in Q3G+PD98059+Aβgroup（P<0.05）.Conclusion:（1）Aβ_1-42 could significantly down-regulate the protein expression of BDNF,CREB,p-CREB,AKT,p-AKT,ERK1/2 and p-ERK1/2,while Q3G could up-regulate the expression levels of these proteins.（2）Taken together,our research elucidated that Q3G could alleviate damage and reduce apoptosis of SH-SY5Y cells induced by Aβ_1-42through regulating the PI3K/AKT/CREB/BDNF and ERK/CREB/BDNF signaling pathways.