Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway methods
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Materials and methods
Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical University. EDA was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%) at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) and ML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these three drugs were determined according to previous studies [ - ].
Elevated plus maze (EPM)
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and time spent in closed and open arms was calculated.
Immunofluorescence and image analysis
Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 2 h at room temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with the secondary antibodies for 2 h at room temperature. Primary antibodies include: Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat anti-GFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2 (1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin 30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43 (1:100, Thermo, 710700). The nuclei were counterstained with DAPI for 10 min. Fluorescent images were capt...
Measurement of MDA, SOD, GSH, GSH-PX and T-AOC
The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A001-3-2) and GSH activity assay kit (No. A006-2-1), GSH-PX activity assay kit (No. A005-1-2) and T-AOC assay kit (S0121, Beyotime, China). MDA, SOD, GSH and GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat the brain tissues before measuring the above assay kits. Briefly, the tissues were weighted and PBS (pH 7.2-7.4, 0.01 mol/L) was added at a 1:9 proportion (MDA, SOD, GSH and GSH-PX kits) and a 1:5 proportion for T-AOC. Next, the samples were treated with tissue lyser (Jingxin, China), followed...
Western blotting (WB)
The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-ra...
Enzyme-linked immunosorbent assay (ELISA)
The levels of TREM2, GFAP, IL-1β, IL-6 and TNF-α in the Hip and mPFC were measured by ELISA kits (Jianglai Biological, Shanghai, China). The tissues were weighed, and PBS was added at a 1:9 proportion and homogenized. Then, the homogenate was centrifuged at 5000 rpm for 15 min, and collecting the supernatant to measure. According to the instructions, 100 µl standard/sample was added and incubated for 1 h at 37 °C. Then, biotin antibody was added into each well and incubated for 1 h at 37 °C. The plate was then washed with washing buffer and streptavidin-HRP was added for 30 min at 37 °C. Next, TMB substrate was added to each well and kept away from light for 15 min at 37 °C after washing. Finally, stop solution was added and the optical density was measured at 450 nm.
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Protein expression by western blot
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Evidence quotes (6)
Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical University. EDA was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%) at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) and ML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these three drugs were determined according to previous studies [ - ].
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and time spent in closed and open arms was calculated.
Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 2 h at room temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with the secondary antibodies for 2 h at room temperature. Primary antibodies include: Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat anti-GFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2 (1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin 30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43 (1:100, Thermo, 710700). The nuclei were counterstained with DAPI for 10 min. Fluorescent images were captured with a fluorescence microscope (VS200; Olympus, Japan) and a confocal microscope (A1R, Nikon, Japan). The number of cells was counted manually containing the intact hippocampus (Hip) and medial prefrontal cortex (mPFC) from 3 mice in each group. Branch length and branch numb...
The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A001-3-2) and GSH activity assay kit (No. A006-2-1), GSH-PX activity assay kit (No. A005-1-2) and T-AOC assay kit (S0121, Beyotime, China). MDA, SOD, GSH and GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat the brain tissues before measuring the above assay kits. Briefly, the tissues were weighted and PBS (pH 7.2-7.4, 0.01 mol/L) was added at a 1:9 proportion (MDA, SOD, GSH and GSH-PX kits) and a 1:5 proportion for T-AOC. Next, the samples were treated with tissue lyser (Jingxin, China), followed by centrifuging the homogenate at 12,000 rpm for 10 min at 4 °C, and collecting the supernatant to measure.
The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-rad, 170-6515) were incubated for 2 h at room temperature. The signals were visualized using ECL chemiluminescence reagent (Beyotime; US Everbright Inc.).
The levels of TREM2, GFAP, IL-1β, IL-6 and TNF-α in the Hip and mPFC were measured by ELISA kits (Jianglai Biological, Shanghai, China). The tissues were weighed, and PBS was added at a 1:9 proportion and homogenized. Then, the homogenate was centrifuged at 5000 rpm for 15 min, and collecting the supernatant to measure. According to the instructions, 100 µl standard/sample was added and incubated for 1 h at 37 °C. Then, biotin antibody was added into each well and incubated for 1 h at 37 °C. The plate was then washed with washing buffer and streptavidin-HRP was added for 30 min at 37 °C. Next, TMB substrate was added to each well and kept away from light for 15 min at 37 °C after washing. Finally, stop solution was added and the optical density was measured at 450 nm.
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