Fig. 2: TRPV1 antagonist CPZ inhibits NLRP3 inflammasome activation in microglia.

A–K Primary mouse microglia were seeded in the 96-well or 6-well plate overnight. The cells were pretreated with 10 μM Capsazepine (CPZ) for 1 h and then primed with 100 ng/ml LPS for 3 h and subsequently challenged with 5 mM ATP for 30 min or 10 μM nigericin for 1 h to activate NLRP3 inflammasome. Cleaved IL-1β p17, caspase-1 p10 and ASC specks were used as the indicators of NLRP3 inflammasome activation. A–D IL-1β and TNF-α release in the supernatants were measured using ELISA method. E, F Concentrated culture supernatants and cell lysates were immunoblotted with IL-1β p17 and Caspase-1 p10 antibody, and the results are summarized respectively in (G) and (H). Cells were fixed, permeabilized and blocked, and then stained with anti-ASC antibody. I ASC speck formation was detected using confocal microscopy. J Statistics of the percentage of cells with ASC specks. K Quantitative analysis of LDH released from primary microglia by LDH Cytotoxicity Assay Kit. L–N Primary mouse microglia were primed with LPS (100 ng/ml) for 3 h, and then CPZ was added for 1 h prior to the 30-min challenge of ATP to activate the NLRP3 inflammasome. L Drug-administration scheme. M Cell lysates were immunoblotted with IL-1β p17 and Caspase-1 p10 antibody, and the result is summarized respectively in (N). Scale bar: 10 μm. Data are representative of three independent experiments. *p < 0.05. **p < 0.01. ***p < 0.001.