Fig 2: Effect of cannabinoids on the expression of NLRP3 inflammasome proteins in THP-1 macrophages. (A): CBD and THC decreased the expression of NLRP3 after LPS + ATP stimulation in THP-1 macrophages: Representative Western blots of NLRP3 and GAPDH bands and the quantification of the same using one-way ANOVA with Tukey’s multiple-comparison test. Four independent experiments (n = 3–4) of THP-1 cell lysates obtained after pre-treatment with CBD or THC for 30 min and LPS stimulation for 3 h followed by ATP for 1 h were analyzed, and data are expressed as means ± SD; * p < 0.05, ** p < 0.01. (Abbreviation: LA: LPS + ATP). (B): CBD and THC decreased the ratio of the expressions of caspase-1 p20/p45 after LPS + ATP stimulation in THP-1 macrophages: Representative Western blots of caspase-1 p45 (pro-caspase1), p20, and GAPDH bands and the quantification of caspase-1 p20/p45 ratio using one-way ANOVA with Tukey’s multiple-comparison test. Four independent experiments (n = 4) of THP-1 cell lysates obtained after pre-treatment with CBD or THC for 30 min and LPS stimulation for 3 h followed by ATP for 1 h were analyzed, and data are expressed as means ± SD; * p < 0.05, ** p < 0.01. (Abbreviation: LA: LPS + ATP). (C): CBD and THC decreased the expression of pro-IL-1β after LPS + ATP stimulation in THP-1 macrophages: Representative Western blots of pro-IL-1β and GAPDH bands and the quantification of the same using one-way ANOVA with Tukey’s multiple-comparison test. Four independent experiments (n = 4) of THP-1 cell lysates obtained after pre-treatment with CBD or THC for 30 min and LPS stimulation for 3 h followed by ATP for 1 h were analyzed, and data are expressed as means ± SD; ** p < 0.01, *** p < 0.001. (Abbreviation: LA: LPS + ATP). (D): CBD and THC decreased the expression of mature IL-1β after LPS + ATP stimulation in cell supernatants of THP-1 macrophages: Representative Western blots of mature-IL-1β and β-actin bands and the quantification of the same using one-way ANOVA with Tukey’s multiple-comparison test. Four independent experiments (n = 4) of THP-1 cell supernatants obtained after pre-treatment with CBD or THC for 30 min and LPS stimulation for 3 h followed by ATP for 1 h were analyzed, and data are expressed as means ± SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (Abbreviation: LA: LPS + ATP).

Fig 3: SUCNR1 and inflammasome components are increased and positively correlated in surgical resections from UC patients. (a) Graphs show mRNA expression of SUCNR1, NLPR3, CASP-1, IL1B, IL18 and ASC in intestinal resections from UC patients (n = 25) and non-IBD patients (n = 30). (b) Graphs show protein expression of SUCNR1, NLRP3 and Caspase-1/pro-Caspase-1 (n = 15). Bars in graphs represent mean ± SEM. * p < 0.05 vs. non-IBD patients. (c) Graphs show the correlations between data relative to mRNA expression of SUCNR1 vs. mRNA expression of the inflammasome components NLRP3, CASP1, IL1B and IL18 (expressed as ΔCt). In each correlation, the value of the Spearman’s correlation coefficient (R) and p value are shown.

Fig 4: SUCNR1 mediates inflammasome priming in intestinal epithelial cells. (a) HT29 cells treated with succinate 1 mM and/or an inflammatory cocktail. Graphs show mRNA (n = 7) and protein (n = 3) expression of SUCNR1. Image of a representative Western Blot of one independent experiment. Bars in graphs represent mean ± SEM; * p < 0.05 and ** p < 0.01 vs. vehicle cells. (b–e) HT29 cells transiently transfected with a specific siRNA against SUCNR1 or ctrl; (b) Graphs show mRNA and protein expression of SUCNR1 (n = 5). (c) Graphs show mRNA expression of NLRP3, CASP1, IL1B, ASC and IL18 (n = 5). (d) Graph shows protein expression of pro-Caspase-1 (n = 4). Image of a representative Western Blot of one independent experiment. (e) Graph shows secreted protein levels of IL-1β detected in the supernatant of HT29 cells (n = 3). (f) Graph shows succinate levels in supernatant of HT29 cells (n = 6). In all cases, bars in graphs represent mean ± SEM. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. siCtrl vehicle cells. # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. the respective siCtrl cells. $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. siCtrl cocktail treated cells.

Fig 5: High expression of PRMT5 and low expression of CASP1 associate with poor clinical outcomes in MM patients.A, B Representative images of IHC staining of PRMT5 and CASP1 in MM bone marrow tissues, which were divided into a high-expression group (upper panel) and a low-expression group (lower panel). Scale bar = 50 μm. C Overall survival of high PRMT5 (n = 78) and low PRMT5 (n = 30) MM patients performed by Kaplan-Meier survival analysis. D Overall survival of high CASP1 (n = 39) and low CASP1 (n = 69) MM patients performed by Kaplan-Meier survival analysis. E Kaplan-Meier survival analysis of MM patients based on both PRMT5 and CASP1 expression. MM patients were divided into four groups: high PRMT5 and low CASP1 expression (n = 51), high PRMT5 and high CASP1 expression (n = 27), low PRMT5 and high CASP1 expression (n = 12), and low PRMT5 and low CASP1 expression (n = 18). P-values were calculated using the group of high PRMT5 and low CASP1 expression as the reference group. F A model for PRMT5-dependent cell proliferation and apoptosis by silencing CASP1 in MM.