MMP9 zymography

The MMP9 gelatinase activity was determined by zymography using a gelatin zymography assay kit (P1700, Applygen, Beijing, China) as described previously [30]. Briefly, the brains obtained above were homogenized in Lysis buffer on ice. Total protein concentrations were determined using the Bradford assay (Bio-Rad, California, USA). Equal amounts (50 μg) of total protein extract were prepared and MMP9 activity was measured by SDS-PAGE under non-reducing conditions. The gel contained 1% gelatin and 30% acrylamide. Electrophoresis was carried out at 4 °C. After washing with buffer A (containing 2% Triton X-100) from the kit, the gel was incubated at 37 °C with buffer B (containing the necessary metal ion: 5 mmol/CaCl₂, 1 μmol/L ZnCl₂). MMP activity was visualized by staining with Coommasie Blue R-250 (P1501, Applygen, Beijing, China). Signals were detected using the gray value of bands and quantified with Image Pro Plus 7 software. After obtaining the absolute gray value of each band, the relative gray value of SAH group was obtained by the absolute gray value of the SAH group / the absolute gray value of the Sham group.