Abstract
Koumine (KM), one of the primary constituents of Gelsemium elegans, has been used for the treatment of inflammatory diseases such as rheumatoid arthritis, but whether KM impacts the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome remains unknown. This study aimed to explore the inhibitory effect of KM on NLRP3 inflammasome activation and the underlying mechanisms both in vitro using macrophages stimulated with LPS plus ATP, nigericin or monosodium urate (MSU) crystals and in vivo using an MSU-induced peritonitis model. We found that KM dose-dependently inhibited IL-1β secretion in macrophages after NLRP3 inflammasome activators stimulation. Furthermore, KM treatment efficiently attenuated the infiltration of neutrophils and suppressed IL-1β production in mice with MSU-induced peritonitis. These results indicated that KM inhibited NLRP3 inflammasome activation, and consistent with this finding, KM effectively inhibited caspase-1 activation, mature IL-1β secretion, NLRP3 formation and pro-IL-1β expression in LPS-primed macrophages treated with ATP, nigericin or MSU. The mechanistic study showed that, KM exerted a potent inhibitory effect on the NLRP3 priming step, which decreased the phosphorylation of IκBα and p65, the nuclear localization of p65, and the secretion of TNF-α and IL-6. Moreover, the assembly of NLRP3 was also interrupted by KM. KM blocked apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and its oligomerization and hampered the NLRP3-ASC interaction. This suppression was attributed to the ability of KM to inhibit the production of reactive oxygen species (ROS). In support of this finding, the inhibitory effect of KM on ROS production was completely counteracted by H2O2, an ROS promoter. Our results provide the first indication that KM exerts an inhibitory effect on NLRP3 inflammasome activation associated with blocking the ROS/NF-κB/NLRP3 signal axis. KM might have potential clinical application in the treatment of NLRP3 inflammasome-related diseases.
Highlights
Inflammation plays a key role in triggering host defense responses by clearing pathogens
The results showed that LPS plus any of the three stimuli promoted a robust increase in IL-1β secretion in the culture supernatants, and NOD-like receptor protein 3 (NLRP3) inflammasome inhibitor MCC950 potently inhibited IL-1β production (Figures 2A–C)
The results indicated that KM (50, 100, and 200 μM) did not affect the vitality and proliferation of Bone marrow-derived macrophages (BMDMs) (Figure 2G) or PMAdifferentiated THP-1 macrophages (Figure 2H), which suggests that the inhibitory effect on IL-1β production was not due to cytotoxicity
Summary
Inflammation plays a key role in triggering host defense responses by clearing pathogens. The activation of NOD-like receptor protein 3 (NLRP3) inflammasome is the major mechanism in IL-1β maturation and secretion (Pedraza-Alva et al, 2015). Inflammasomes, which are multiprotein complexes, are key mediators of inflammatory responses and serve as signaling platforms to orchestrate host defense in response to pathogenassociated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) released by infectious agents or noninfectious damage (Martinon et al, 2002). The classical activation of the NLRP3 inflammasome requires two signals (Guo et al, 2015): for signal 1 ( called priming), toll-like receptors (TLR) agonists such as LPS activate the NF-κB pathway and induce the expression of NLRP3 and pro-IL-1β; and for signal 2, PAMPs or DAMPs, such as extracellular adenosine triphosphates (ATP), nigericin and monosodium urate (MSU) crystals, disrupt cellular physiology, which initiates the assembly of the inflammasome protein complex. Disturbing the molecular compounds of the NLRP3 inflammasome and inhibiting NLRP3 inflammasome activity constitute a promising approach for the treatment of inflammatory disorders
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