Caspase-3 Activity Assay

The activity of caspase-3 was detected by the caspase-3 colorimetric assay kit. Briefly, 6 h after the last injection, the young rats were sacrificed by decapitation, and the brain tissue was quickly separated. Fresh frozen brain tissue was homogenized in lytic buffer to extract protein or extract protein directly from PC12 neural cell line. The protein concentration was then determined using the Bradford Protein Assay Kit. After that, 50 μL of the lysate supernatant containing 200 μg of protein, 50 μL of reaction buffer, and 5 μL of caspase-3 substrate was incubated in a 96-well plate at 37°C for 4 h with a microtiter plate reader (ThermoFisher, Shanghai, China). The absorbance was measured at a wavelength of 400 nm. The absorbance of the blank sample without the caspase-3 substrate was subtracted from these values. The results implied that the final caspase-3 activity was the ratio of the control group.