Western Blotting
Six rats were randomly chosen for western blot analysis. After sacrifice, the white matter was removed on an ice plate and homogenized in ice-cold radioimmunoprecipitation assay buffer (Solarbio; cat. no. R0010) according to the manufacturer’s protocol, followed by centrifugation (4°C; 12,000× g; 10 min) to obtain total protein. For quantification of NF-κB, nucleoproteins were extracted with the Nuclear Protein Extraction Kit (Solarbio; cat. no. R0050) according to the manufacturer’s protocol. Protein concentration was measured with the BCA Protein Assay kit (Solarbio; cat. no. PC0020), and 40 μg per sample was separated by 10% sodium dodecyl sulfate-polyacrylamide electrophoresis and electro transferred to a polyvinylidene difluoride membrane (Millipore, Beijing, China) that was blocked in 5% fat-free milk for 1 h before overnight incubation at 4°C with primary antibodies against MBP (1:1,000), Olig2 (1:500), p-STAT3 (1:1,000), NF-κB (1:1,000), SIRT1 (1:500), p-AMPK (1:1,000), and TNF-α (1:1,000) as well as β-actin (1:4,000; loading control). The following day, the membrane was washed three times with Tris-buffered saline with 0.1% Tween-20 and then incubated for 45 min at 37°C with HRP-conjugated goat anti-rabbit IgG H&L (1:5,000; Abcam; cat. no. ab6721). Protein bands were visualized with an enhanced chemiluminescence kit (Wanlei Biotechnology Company; cat. no. WLA003) and quantified with Gel-Pro-Analyzer software (Media Cybernetics). The relative intensity of each band was normalized to that of β-actin.