Fig. 3

Pyroptosis-based screen identified cannabinoid receptor 1 (CB1R) as a critical regulator of cell pyroptosis. a Dual omics-based screen workflow. b Analysis of overlapped pathways by the transcriptomic and proteomic analyses. The overlapped pathways were highlighted in the right upper quadrant with the pathway ID and full names annotated. c Small-molecule compounds against the overlapped pathways were designed (upper panel) and added to the HL-1 myocytes for 24 h at a final dose of 1 μM. Western blot analysis was performed to assess the effects of pathway inhibition on the Olz-activated pyroptosis (lower panel). d, e LC-MS/MS detection of serum major endocannabinoids 2-arachiodonoylglycerol (2-AG) and anandamide (AEA) (n = 8–10/group) in Veh (PBS) or Olz (5 mg/kg)-treated mice. LC-MS/MS detection of cellular 2-AG levels in H9c2 cells (f) and HL-1 cells (g) receiving Veh (PBS), low dose (LD, 1 μM), medium dose (MD, 4 μM), and high dose (HD, 16 μM) of Olz from three independent assays. h Western blot analysis of CB1R in primary cardiomyocytes. i qRT-PCR analysis of Cb1r mRNA in Veh (PBS) or Olz (5 mg/kg)-treated mouse hearts (n = 5/group). j Molecular docking of ligand binding with human CB1R (hCB1R). The receptor is shown in gray cartoon representation. Ligands are shown in stick representation with indicated colors. The inset shows magnified view of ligand binding with CB1R. The binding energies were tabulated. k Surface plasmon resonance (SPR) measurements illustrating binding of Olz to the hCB1R. l Western blot detection of CB1R subcellular localization in response to Veh (PBS), LD (1 μM), MD (4 μM), and HD of Olz (16 μM) treatments of mouse HL-1 myocytes. m Subcellular localization of CB1R in response to clozapine (Clz, 20 μM) or Olz (4 μM) treatments of mouse HL-1 cells for 24 h. White arrowheads indicate membrane location. Scale bar = 20 μM. n Wild type (WT) Cb1r plasmid, Cb1r with C416A (CB1R-C416A), S426A (CB1R-S426A), or S430A (CB1R-S430A) mutants were individually transfected into Cb1r-knockout HL-1 myocytes and were then treated with Olz (4 μM) or Clz (20 μM) for 24 h. Images were taken under confocal immunofluorescence microscope. White arrowheads indicate CB1R membrane localization. Scale bar = 20 μM. o, p Cardiac HL-1 cells with stable overexpression of Cb1r (Lv-CB1R) and its control cells (Lv-vector), or CRISPR-mediated Cb1r knockout (Cb1r^−/−) cells and the WT (Cb1r^+/+) cells were cultured with indicated treatments for 24 h. Final doses of drugs were Olz (4 μM), Clz (20 μM), ACEA (2.7 μM), and each CB1R antagonist (1 μM). Western blot analysis was performed to detect pyroptosis proteins. q, r Rat H9c2 cells were pretreated with PBS or AM 281 (1 μM) for 1 h. The cells were then subject to Olz (4 μM) or Clz (20 μM) treatments in medium containing PI dye (30 μM). Time duration = h: min: s: ms. The percent of pyroptosis cells was quantified from three independent assays. Scale bar = 50 μm. All quantification represents the mean and SEM of each group. Students’ t-test was used for analysis in (d, e, I, q, r) and one-way ANOVA with Bonferroni post hoc test was used for analysis in (f, g). *p < 0.05; **p < 0.01; ***p < 0.001 as indicated