Dl-butylphthalide Inhibits Rotenone-induced Microglia Oxidative Stress Via Keap1-Nrf2-HO-1 Signaling Pathway

Objective:The herein study aim at evaluating the potential of dl-butylphthalide to inhibit rotenone-induced microglia oxidative stress and its molecular mechanism.Methods:1.CCK-8 detected the cytotoxicity of NBP and rotenone in BV2 cells.The measurement of BV2 cell viability under different concentrations of rotenone or NBP was using CCK-8.BV2 cells were seeded in 96-well plates at a density of 3000cells/well for 24 h,then the medium was replaced by DMEM with 10%FBS containing different doses of rotenone or NBP.Each concentration was replicated with3 wells.After 24 h,each well was added 10μL CCK-8 reagent.The absorbance was recorded within 4 h at 450 nm on Model 550 micro-plate reader（BIO-RAD company,America）.2.Detection of mitochondrial membrane potential（MMP）by JC-1BV2 cells were seeded in 6-well plates at a density of 4*10^6 cells/well for 24 h,then,treated by rotenone（0.05μM）with or without NBP（200μM）for 24 h,after that,BV2 Cells were harvested,resuspended in PBS and immediately stained with JC-1（1mg/m L in DMSO）,and incubated at 37°C for 30 minutes in darkness.After washing twice in ice-cold PBS,the samples were subject to a Cyto FLEX flow Cytometer（Beckman Coulter,America）.All data were collected from three independent experiments.3.Measurement intracellular reactive oxygen species（ROS）Intracellular reactive oxygen species（ROS）were examined by DCFH-DA.After treated by rotenone with or without NBP for 24 h,BV-2 cells were incubated with10μmol/L DCFH-DA for 30 min at 37℃under 5%CO2,washed triple with PBS.Then visualized using the fluorescent microscope.（Leica,German）Fluorescence images were captured through a 505–530 nm band pass filter（10X）.4.Nuclear extract preparationBV2 cells were seeded 4*10^6 in 6-wells plate for 24 h,then treated by rotenone（0.05μM）with or without NBP（200μM）for another 24 h.Then the cells were used for nuclear extract preparation.We used NE-PER Nuclear and Cytoplasmic Extraction Reagents（Thermo Scientific,Rockford,IL）for nuclear extraction according to the instructions.Then concentration of extracted protein was evaluated with BCA protein assay Kit.5.Western blotThe equal amount of protein in each group was separated by10%SDS-polyacrylamide gels followed by transfer to polyvinylidenedifluoride（PVDF）membrane（Milli-pore,Billerica,MA,USA）.Subsequently,the membranes were blocked with Tris Buffered Saline Tween-20（TBST）containing 5%non-fat milk for 1 h at room temperature.Keap1,Nrf2,and HO-1 were detected by immunoblotting using specific primary antibodies and secondary antibody.β-tubulin and Lamin B was employed as control for equal loading of cell lysates.All primary antibodies are from rabbit and the concentration used is 1:1000.The secondary antibody is anti-rabbit.The density analyzed by Image J and the data analyzed by one-way analysis of variance（ANOVA）.6.Statistical analysisAll images were analysis was performed with the Image J software.Statistical analysis was analyzed by one-way analysis of variance（ANOVA）and was performed with the Graph Pad Prism 7.0 software.All data were expressed as MEAN±SD for three independent experiments*P<0.05,**P<0.01,compared with the control group;^#P<0.05,^##P<0.01,compared with the Rot group.Results:1.The results of CCK-8 method showed that when the concentration of NBP was 300μmol/L,the cell viability of BV2 microglia decreased significantly（P<0.05）,but in the range of 0-200μmol/L,there was no significant effect on the activity of BV2microglia.Therefore,50μmol/L,100μmol/L and 200μmol/L were used in the follow-up experiments The results of CCK-8 showed that rotenone had no effect on the cell viability of BV2 microglia at the concentration of 0-0.05μM,but significantly inhibited the cell viability of BV2 microglia at the concentration of 0.25μmol/L（P<0.01）.2.Morphological observation showed that rotenone could significantly change the morphology of BV2 microglia（P<0.01）,NBP could alleviate the activation of BV2microglia induced by rotenone,and the effect was the best when NBP（200μmol/L）（P<0.01）,so the concentration of NBP（200μmol/L）was selected for the next experiment.3.JC-1 showed that rotenone（0.05μmol/L）could significantly reduce the mitochondrial membrane potential in BV2 cells（P<0.01）,while NBP（200μmol/L）could significantly alleviate the effect（P<0.01）,but NBP alone had no significant effect on the mitochondrial membrane potential.4.The detection of ROS kit showed that rotenone（0.05μmol/L）could significantly increase the level of ROS in BV2 cells（P<0.01）,while NBP（200μmol/L）could significantly alleviate the effect（P<0.01）,while NBP alone had no significant effect on mitochondrial membrane potential.5.The results of Western blot showed that rotenone could significantly reduce the level of HO-1 in the cytoplasm（P<0.01）,and NBP could increase the level of HO-1in the cytoplasm by reducing the level of Keap1 and Nrf2 protein in the cytoplasm,increasing the level of Nrf2 in the cytoplasm and nucleus.Conclusion:The study demonstrated that NBP inhibits rotenone-induced microglia oxidative stress via Keap1-Nrf2-HO-1 signaling pathway.Therefore,the current results supported the notion that NBP treatment could decrease oxidative stress and might have considerable value as a therapeutic agent against Parkinson’s disease.