Caspase 3

AZD7762 (S1532) was purchased from Selleck (Shanghai, China); DDP was purchased from MCE (Shanghai, China). This study used antibodies against caspase-3 (Servicebio, Wuhan, China), cleaved caspase-3 (Servicebio, Wuhan, China), and Ki67 (Servicebio, Wuhan, China).

Proteins extracted from liver tissues of each group were used in Western blotting to validate the network toxicology results. The liver tissues were lysed using lysis buffer containing a protease inhibitor cocktail to extract total liver protein of rats in each group. The protein concentrations of samples were determined using the BCA method, and proteins were denatured via boiling for 5 min. Next, 40 μg of total protein in each group was loaded and separated using sodium dodecyl sulfate-polyacrylamide (baiqiandu) and transferred to methanol-activated polyvinylidene fluoride membranes (Millipore). Next, the membrane was blocked in TBST containing 5% skimmed milk for 1 h at room temperature, and incubated with primary antibody (HMOX1, 1:2000, Boster; IL2, 1:2000, Bioss; Caspase-3, 1:1000, Servicebio; GAPDH, 1:6000, Abcam) overnight, followed by incubation with the secondary antibodies (HRP-Goat anti rabbit, HRP-Goat anti mouse, 1:50,000, Searcare) at room temperature for another 30 min. ECL was used for protein imaging and development.

For immunohistochemistry (Kusakawa et al., 2015 (link)), the slides were incubated with antibodies against caspase-3 (1:750, rabbit source, Servicebio, Wuhan, China), TNF-α (1:500, rabbit source, Servicebio, Wuhan, China), and IL-6 (1:1000, rabbit source, Servicebio, Wuhan, China) overnight at 4°C. Subsequently, paraffin sections of the tissues were washed with PBS, and the secondary antibody was added and incubated for 1 h.