YDZG inhibits NLRP3 inflammasome assembly by inhibiting NLRP3 expression.
The ability of YDZG to inhibit NLRP3 inflammasome activation by promoting protein degradation of inflammasome components was investigated. In resting J774A.1 cells, YDZG treatment was associated with reduced NLRP3 protein expression, however there was no influence on ASC and caspase-1 protein expression (Figure 2A). When J774A.1 cells were stimulated with LPS for different time, YDZG greatly decreased NLRP3 protein expression, with no effects on ASC and caspase-1 protein expression (Figure 2B). These results suggested that YDZG could greatly decrease NLRP3 expression before the functional NLRP3 inflammasome is activated. An essential step for NLRP3 inflammasome assembly is via simultaneous exposure to a second NLRP3-specific trigger, such as extracellular ATP, alum, or the pore-forming toxin nigericin. We evaluated the effects of YDZG on the components of NLRP3 inflammasome after the formation of NLRP3 inflammasome complexes. When J774A.1 cells were stimulated with YDZG before LPS and ATP treatment, YDZG led to a sharp decrease in NLRP3 expression (Figure 2C). YDZG consistently led to a large decrease in NLRP3 protein expression in a dose-dependent manner in THP-1 cells (Figures 2D- E). Importantly, using NLRP3-flag plasmid, we found that YDZG treatment led to much less NLRP3 protein expression in HEK293T cells (Figure 2F). Furthermore, immunofluorescence staining analysis revealed that YDZG reversed LPS-induced high expression of NLRP3 in HEK293T cells (Figure 2G). Taken together, our data showed that YDZG inhibited NLRP3 expression to block NLRP3 inflammasome assembly.