Cell experiment

The Jurkat T-cell line was used as the T-cell model to examine the regulation of T-cell proliferation and apoptosis through CB2Rs. The CCK-8 assay was carried out according to the manufacturer’s instructions for detection of cytoactivity. The Jurkat cells were inoculated in 96-well plates at a density of 2000 cells per well. The cells were treated with GW 0, 5, 10, 20, or 40 μg/mL. The cytoactive detection was conducted at 3, 6, 9, 12, 16, 20, or 24 h after GW treatment. The apoptosis of Jurkat cells was measured in the GW and GW plus AM630-treated groups using flow cytometry analysis. Jurkat cells were collected 24 h after the treatment with GW 10 μg/mL + AM630 1 μg/mL, GW 20 μg/mL + AM630 2 μg/mL, or GW 40 μg/mL + AM630 4 μg/mL. Cells were then stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). The percentage of Annexin V-FITC/PI-positive Jurkat T cells was determined by flow cytometry to evaluate the degree of apoptosis. For Western blot detection of Parp, the treatments of the Jurkat T cells used different concentrations of GW (0, 5, 10, or 20 μg/mL), different concentrations of AM630 (0, 5, 10, or 20 μg/mL), or GW (20 μg/mL) plus AM630 (2 μg/mL). The L02 liver cells were cocultured with Jurkat cells using a Transwell coculture system [20] to research the immune injury of the hepatocytes with 10% fetal bovine serum in 1640 medium for the coculture. Con A at 5 μg/mL was used to stimulate the Jurkat cells to release cytotoxic cytokines. GW 5 μg/mL or 10 μg/mL were used to reveal whether activation of CB2R could protect the L02 cells against cell toxicity from the Jurkat T cells. Furthermore, the AM630 was added to test whether the GW’s effects could be reversed, to confirm the critical roles played by the CB2Rs.