Active pro caspase 3

The protein expression of Aβ_1-40, COX-2, iNOS, TNF-α, IL-1β, Bax, Bcl-2, and caspase-3 was analyzed using Western blot. Hippocampus were removed from the brain hemispheres and homogenized in radioimmunoassay lysis buffer containing protease inhibitors. The homogenized samples were centrifuged for 15 min at 12,000 g and 4°C. Protein concentrations from each sample were determined using the BCA protein assay kit (Biocolor Biotechnology, China). 50 μg protein of each sample was heated at 100°C for 5 min, and then separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk in PBS buffer for 2 h, and then incubated with primary antibodies against Aβ_1-40 (1:1,000, Abcam, USA), IL-1β (1:1,000, Abcam, USA), COX-2 (1:1,000, Abcam, USA), TNF-α (1:1,000, Abcam, USA), iNOS (1:1,000, Abcam, USA), Bax (1:1,000, Abcam, USA), Bcl-2 (1:1,000, Abcam, USA), active + pro caspase-3 (1:1,000, Abcam, USA) and β-actin (1:2,000, Beyotime, China) at 4°C overnight, followed by horseradish peroxidase conjugated secondary antibodies (1:2,000, Beyotime, China) incubation. Immunoblots were visualized with chemiluminescence reagent BeyoECL Plus (Beyotime, China) and quantified with Quantity One software v4.52 (Bio-Rad).

Protein samples (10 to 40 μg) were subjected to SDS-PAGE and transferred to Immobilon P membranes (Millipore Corp., Billerica, MA). After blocking, membranes were probed with primary antibodies against active + pro caspase 3 (1:1000; Abcam), α SMA (1:1000; Abcam), phosphorylated NF-κB-p65 (1:1000; Cell Signaling, MA, USA), total NF-κB-p65 (1:1000; Delta Biolabs), Cytochrome c (1:1000; Santa Cruz Biotechnology, CA, USA), GAPDH (1:2000; Santa Cruz Biotechnology, CA, USA), CD10 (1:1000; R&D System, MN, USA), iNOS (1:1000; Abcam), HO-1 (1:1000; Assay designs, NY, USA), or CYGB (1:3000; our laboratory). Membranes were then incubated with horseradish peroxidase conjugated secondary antibodies at 1:2000 dilutions. Immunoreactive bands were visualized using the electrochemiluminescence detecting reagent (GE Healthcare UK Ltd, Buckinghamshire), and documented with the Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan) coupled with image analysis software (Multi Gauge version 3.1; Fujifilm, Tokyo, Japan).