Caspase 9

Curcumin (CUR) and THC were obtained from Wuhan reagent company (Wuhan, China). Dimethylsulfoxide (DMSO), 5-fluoro-2,4 (1 h,3 h) pyrimidinedione (5-FU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), rhodamine 123 (Rh123), ethylenediaminetetraacetic acid (EDTA), RNAse-A and propidium iodide (PI), phenylmethyl-sulfonyl fluoride (PMSF), 4′,6-diamidino-2-phenylindole (DAPI), electro-chemi-luminescence (ECL), dichlorofluorescein diacetate (DCFH-DA), and tris-HCl and glycine were obtained from Sigma-Aldrich (St, Louis, MO, USA). Assay kits of BCA, caspase-3, caspase-9, and lactate dehydrogenase (LDH) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phosphate buffer solution (PBS), 0.1% tween-20 in phosphate buffer solution (PBST), and sodium dodecyl sulfonate (SDS) were purchased from Zhengjiang Wanbang Pharmaceutical Co. (Wenling, China). The primary antibodies against Bax, Bcl-2, poly (ADP-ribose) polymerase (PARP), cytochrome c, and the horseradish peroxidase (HPR)-conjugated goat anti-mouse secondary antibody were provided by BioVision, Inc., (BioVisio, Palo Alto, CA, USA). All the other cell culture reagents were purchased from Sinopharm (Beijing, China), and all the other chemicals were of analytical grade.

The activities of 8-hydroxy-2′-deoxyguanosine (8-OHdG), caspase-3, caspase-8, caspase-9 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), aspartate aminotransferase transaminase (AST) and alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyltransferase (GGT) (Shanghai Yuanye Bioengineering Institute, Shanghai, China) were determined using commercially available enzyme-linked immune sorbent assay (ELISA) kits. Briefly, liver tissues were homogenized in 0.9% of saline solution and then centrifuged at 12,000 ×g for 15 min, to release the enzymes into the solution. Then, the microplates were coated of with anti-8-OHdG, caspase-3, caspase-8, caspase-9, AST, ALT, GGT, and ALP, followed by detection with a horseradish peroxidase-labeled substrate after incubation for 10 minutes at 37°C. Absorbance values were then read in a spectrophotometer at 450 nm.