The Effect Of Sulforaphane On Anti-oxidative Stress Related Mechanisms In N2a/APP Cells

Objective:Based on the theory of "protein cascade hypothesis",this study aimed to explore the protective effect of sulforaphane(SFN)in the oxidative damage of N2a/APP cells in vitro and its molecular mechanism.Methods:The in vitro cell models of AD were generated using N2a cells stably transfected with APP gene.To determined the dose,the relative suvival rate of N2a/APP cells treated with different concentrations of SFN for 24h was tested by CCK-8.The preliminary experiments were divided into five groups:control group(N2a/WT),AD group(N2a/APP),high dose SFN group(3.75μM),medium dose SFN group(7.5μM)and low dose SFN group(15μM).Subsequent experiments were divided into four groups:control group(N2a),AD group(N2a/APP),SFN group(15μM)and siNrf2+SFN group.The ELISA was used to determine the content of Aβ and Trx in cells.ROS fluorescence probe was used to determine the positive rate of reactive oxygen species(ROS).Trace method was used to detect the content of reduced glutathione(GSH)and the enzymes changes in activity levels of thioeredoxin reductase(TrxR),glutathione peroxidase(GSH-Px),glutathione S-transferase(GSTs)and glutamate-cysteineligase(GCL).RT-PCR was used to detect changes in mRNA levels of BACE1.The protein change in Nrf2 was observed with Western Blot.After silence Nrf2 gene by liposome transfection,Wes tern Blot was used to detect the efficiency.The Aβ protein levels was observed by ELISA.ROS fluorescence probe was used to detect the relative levels of H2O2 and NO.Results:1.Within a certain concentration range,SFN had a protective effect on N2a/APP cells.When the concentration was greater than 17.486μmol/L,SFN had a obviously inhibitory effect on the growth of N2a/APP cells,and showed a dose-effect relationship.The IC50 of SFN was 17.486μmol/L.2.Compared with the control group,the Aβ protein content,ROS positive rate and the BACE1 mRNA in the model group were significantly higher;the protein content of Trx and GSH was decreased;the enzyme activities of TrxR,GSH-Px,GSTs and GCL went down clearly;there was no significant difference in the expression of Nrf2.3.Compared with the model group,the Aβ protein content,ROS positive rate and the BACE1 mRNA in the SFN group showed a downward trend;the protein level of Trx and GSH was increased in varying degrees;the enzyme activities of TrxR,GSH-Px,GSTs and GCL showed an evident increasing;the expression of Nrf2 was up-regulated,and the effect of the high dose group was significant.4.Compared with SFN group,the protein level of Aβ and the positive rate of ROS in the siNrf2+SFN group were increased remarkable.Conclusion:1.SFN has protective effects on AD model cells,and can reduce the expression of Aβ and BACE1.2.SFN can increase the potein level of Trx and GSH,rise the activity of anti-oxidation enzymes,eliminates ROS accumulation in AD model cells and relieve the oxidative damage.3.SFN can up-regulate the expression of Nrf2.4.The antioxidation and neuroprotective effects of SFN are partly achieved by activating the Nrf2/ARE pathway.