Western blotting assays

Following the behavioral tests, the mice were sacrificed with an overdose of pentobarbital sodium (150 mg/kg). Lumbar spinal cord samples were obtained and were then homogenized in RIPA lysis buffer that contained 1% protease inhibitors (Sigma-Aldrich, USA), and centrifugated at 12,000g, 4°C for 20 min for supernatant collection. The protein concentration in each sample was quantified using a BCA analysis kit (Beyotime, Shanghai, China). ²⁴ Proteins were then collected, separated on SDS-PAGE gel, and transferred to a PVDF membrane. Membranes were blocked with QuickBlockTM Blocking Buffer for 1 h, incubated with the appropriate primary antibodies overnight at 4°C, and then incubated with HRP-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Protein bands were probed using an ECL detection reagent, visualized using an iBright 1500 instrument (Invitrogen, USA), and analyzed using ImageJ. β-actin (1:5000) was used as a loading control. The following primary antibodies were used at the dilution as 1:1000: anti-IL-1β, anti-GFAP, anti-Nrf2, anti-AMPK, anti-phospho-AMPK, anti-Drp1, Anti-phospho-DRPp1 (Ser616), anti-phospho-Drp1 (Ser637), anti-NDUFB11, anti-DHODH, anti-Cyt-C, anti-NLRP3, anti-cleaved-Caspase-1.