The Role Of STAT3 In The Inflammatory Response And Oxidative Stress Of Microglia Induced By ?-amyloid Peptide

Objective:The pathogenesis of Alzheimer's disease(AD)is not yet fully understood,and the neurotoxicity of ?-Amyloid peptide(A?)is a key part in the pathogenesis of AD.Therefore,in this study,we investigated the expression and the effect of signal transducer and activator of transcription 3(STAT3)in the inflammatory response and oxidative stress of microglia induced by A?.Methods:.In the study,BV2 microglia was treated by A? to construct a cellular model of AD,and the expression of STAT3 was successfully silenced using siRNA method in microglia.The experiments were divided randomly into control group,A? group,control siRNA+A? group and siRNA STAT3+A? group.The optimum concentration and the incubated time of A? oligomer(A?O)in microglia cells were determined by CCK-8 assay with measuring the cell survival rate.The expression of STAT3 at protein level in BV2 microglial cells induced by A?O was measured by Western blotting.The expressions of IL1-? and TNF-? at mRNA and protein levels were detected with real-time quantitative PCR and enzyme linked immunosorbent assay(ELISA),respectively.The level of intracellular reactive oxygen species(ROS)was detected with ROS detection kit;biochemical methods were used for detecting the content of nitric oxide(NO),the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA);the level of cell apoptosis was determined by using flow cytometry;immunofluorescence method was used for determination of the phagocytic clearance of intracellular A? in B V2 cells and the level of A? was detected by Western blotting in the supernatant of BV2 cells.The experimental datas were analyzed by using SPSS 19.0 software.Results:1.The establishment of cellular model of AD and the treatments with siRNA:Different concentrations of A? induced cell death in a dose-dependent manner.When employing 0.5 ?mol/L of A?O,its cytotoxic effect was obvious and with 1.0 ?mol/L of A?O,nearly 50%of BV2 cells survived.Based on this result,1.0 pmol/L of A?O was selected as the optimal concentration for the treatment of experiment.siRNA method was used to silence the expression of STAT3 mRNA.The results showed that as compared with the control group,the levels of?-STAT3 and STAT3 protein were significantly decreased,indicating that this method can successfully silence the expression of STAT3 in BV2 cells.2.Effects of A?O for the expression of STAT3 protein.As compared with the control group,the expression of p-STAT3 was significantly increased in BV2 cells treated with ?1.0 ?mol/L of A?O.The levels of p-STAT3 varied in BV2 cells stimulated with 1.0 ?mol/L of A?O at different incubation times,in which the level of p-STAT3 in BV2 cells treated with 4 h was slightly higher than that with other incubation times.However,the expression of total STAT3 did not show any obvious changes.3.The levels of IL-1? and TNF-?in BV2 cells.The expressions of IL-1? and TNF-? at mRNA and protein levels were significantly increased in A? group compared to controls,while after suppression of STAT3,the expressions of IL-1? and TNF-? were significantly decreased as compared to A? group.4.Changes of oxidative stress in microglia.BV2 cells stimulated with A? led to a gradual increase in the contents of ROS,NO and MDA as well as decrease in the activity of SOD.Whereas,pre-treatment with siSTAT3 significantly inhibited the A?-induced production of ROS,NO and MDA,and raised the activity of SOD as compared with A? group.5.Apoptosis rate in microglia.The treatment with A?O significantly increased the percentage of apoptotic cells compared to controls.While pre-treatment with siSTAT3 resulted in the decreased apoptosis of the cells from 29.67%to 15.28%.6.Phagocytosis of A? by microglia.The results obtained by the determination of cell immunofluorescence showed that the fluorescence intensity was significantly decreased in A?-stimulated BV2 cells pre-treament with siSTAT3 compared to the A? group.Meanwhile,in Western Blotting we also found that A? in siSTAT3+A? group was lower than the A? group in culture medium.Conclusion:1.A?O has the significant neurotoxic effects on microglia and can produce obvious inflammatory reaction and high level of oxidative stress in microglial cells.2.BV2 microglia processing with A?O can cause the activation of STAT3.3.Supprssion of STAT3 by using siRNA can reduce the levels of inflammatory reaction and oxidative stress in microglial cells induced by A?O.4.Supprssion of STAT3 by using siRNA also reduces the apoptosis of microglia and enhances their ability of phagocytosis to A?O.In summary,STAT3 may involve the regulation in mechanism of inflammatory reaction,oxidative stress and cell apoptosis in BV2 microglia induced by A?.