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.