Autophagy mediates the inhibitory effect of CB2R on NLRP3 inflammasome initiation and activation stimulated with LPS/DSS in vitro.

(A) The quantitative expression of mRNA in RAW264.7 cells after transfection with control siRNA or Atg5 siRNAs. n = 6 per group. *P<0.05 vs. the control siRNA, **P<0.01 vs. the control siRNA. (B and C) After transfection with control siRNA or Atg5 siRNA, RAW264.7 cells were primed with LPS (10 ng/ml) for 1 h, and then were stimulated with 3% DSS in the presence or absence of HU 308 (10 μM) for 24 h. Cells were fixed and colocalization of NLRP3 with ASC and NLRP3 with Casp-1 were observed using a confocal laser scanning microscope. Representative images of the colocalization of NLRP3 (green) with ASC or Casp-1 (red). (D and E) Quantitative analysis showing the fold change in Pearson coefficient correlation (PCC) for the colocalization of NLRP3 with ASC and NLRP3 with Casp-1. Atg5 siRNA attenuated the inhibitory effect of HU 308 the colocalization of NLRP3 with ASC and NLRP3 with Casp-1. n = 6 per group. *P<0.05 vs. control, **P<0.01 vs. control, #P<0.05 vs. LPS/DSS. (F and G) After transfection with control siRNA or Atg5 siRNA, RAW246.7 cells were primed with LPS (10 ng/ml) for 1 h, and then were stimulated with 3% DSS in the presence or absence of HU 308 (10 μM) for 24 h. Western blot was conducted to test Casp-1 p20 in supernatants and Casp-1 p45 and GAPDH in lysates, and the expression of Casp-1 p20/Casp-1 p45 ratio was analyzed. IL-1β secretion in the supernatant was tested by ELISA. HU 308 decreased the level of Casp-1 p20/Casp-1 p45 ratio and IL-1β secretion. Atg5 siRNA attenuated the inhibitory effect of HU 308 on Casp-1 p20/Casp-1 p45 ratio and IL-1β secretion. n = 8 per group. **P<0.01 vs. LPS/DSS.