Western blot analysis was used to detect protein expression in the ischemic tissue, as described by us in previous work21 (link),22 (link). Upon completion of electrophoresis, proteins were transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies (rabbit polyclonal anti-Bcl-2, rabbit polyclonal anti-BAX antibody, rabbit polyclonal anti-TN-Fα antibody, or rabbit polyclonal anti- IL-1β antibody; Abcam, Cambridge, MA, USA) for 24 h at 4°C. Next, membranes were washed with PBS three times for 6 min per wash, and then re-incubated with a secondary antibody (goat anti-rabbit IgG, Santa Cruz, Dallas, TX, USA; goat anti-mouse IgG, Abcam) for 1 h at room temperature. Finally, an enhanced chemiluminescence system was used to detect immunoreactive bands. Western blot images for each antibody, including β-actin, were analyzed using an image analysis program (Image J 1.42, NIH, Bethesda, MD, USA) to represent protein expression in terms of relative image density; mean protein expression from the control group was assigned a value of 1 to serve as a reference.