Chalcones Display Anti-NLRP3 Inflammasome Activity in Macrophages through Inhibition of Both Priming and Activation Steps-Structure-Activity-Relationship and Mechanism Studies

Abstract

Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1β, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1β, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K⁺ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K⁺ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1β. Next, 11Cha1 blocked ATP-mediated K⁺ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1β and IL-18.

Keywords: ATP; K+ efflux; NF-ĸB; NLRP3 inflammasome; chalcone; structure-activity relationship.

Figure A1

General procedure for the synthesis of compound 11Cha1–11.

Figure 1

Effects of chalcone derivatives and flavonoids on the inhibition of lipopolysaccharide (LPS)-primed ATP-induced pro-caspase-1 processing in differentiated THP-1 cells. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h. Then, the cells were primed with 1 μg/mL LPS for 4 h and were pre-treated with or without the indicated compound for 30 min and then treated with 5 mM ATP for another 2 h. After treatment, the concentrations of p20 caspase-1 subunit in the medium were determined. Data are expressed as mean±SEM of three to four independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with compound-free control.

Figure 2

Effects of 11Cha1 on the release reaction and protein expressions of caspase-1 and cytokines. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h and then primed with 1 μg/mL LPS for 4 h. The cells were pre-treated with or without the indicated agent (200 µM glyburide as the positive control) for 30 min before a two-hour exposure to 5 mM ATP. After treatment, the concentrations (A) and protein expressions (B) of p20 caspase-1 subunit and cytokines in the medium were determined. Data are expressed as mean±SEM of three independent experiments. One-way ANOVA by Newman Keuls post hoc test was used. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with ATP-treated control. The analyses of P (ANOVA) indicated P < 0.001 in all tests.

Figure 3

Effect of 11Cha1 on LPS-induced IĸBα phosphorylation, p65 NF-ĸB nuclear translocation and several protein expressions. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h. Then, the cells were pre-treated in the absence or presence of 30 µM 11Cha1 for 30 min and then treated with 0.3 µg/mL LPS for 30 min (A,B) or 2 h (C). The cells were harvested for the detection of p-IkBα and IkBα (A), nuclear translocation of p65 NF-ĸB (B), or several protein expressions (C) using Western blot analysis. Data are expressed as mean ± SEM of at least three independent determinations.

Figure 4

Effect of 11Cha1 on ATP-induced ASC dimerization and oligomerization. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h and then primed with 1 μg/mL LPS for 4 h. The cells were pre-treated with or without the indicated agent for 30 min (200 µM glyburide as the positive control) and then treated with 5 mM ATP for another 2 h. After treatment, the cells were harvested for the detection of the protein expressions of ASC dimerization and oligomerization. Data are expressed as mean ± SEM of at least three independent determinations. One-way ANOVA by Newman Keuls post hoc test was used. * P < 0.05 and ** P < 0.01 compared with ATP-treated control. The analyses of P (ANOVA) indicated P < 0.01 in all tests.

Figure 5

Effect of 11Cha1 on ATP-induced interaction between NLRP3, pro-caspase-1, and ASC. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h and then primed with 1 μg/mL LPS for 4 h. The cells were pre-treated with or without 11Cha1 (30 µM) for 30 min and then treated with 5 mM ATP for another 2 h. After treatment, immunoprecipitation and immunoblotting experiments were performed. Data are expressed as mean ± SEM of four independent determinations. One-way ANOVA by Newman Keuls post hoc test was used. The analyses of P (ANOVA) indicated P < 0.001 in all tests.

Figure 6

Effect of 11Cha1 on ATP-induced K⁺ outflow from cells. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h and then primed with 1 μg/mL LPS for 4 h. The cells were pre-treated with or without 11Cha1 (30 µM) for 30 min and then treated with 5 mM ATP for another 30 min. After treatment, intracellular potassium content was determined using Potassium Assay Kit (MyBioSource, San Diego, CA, USA) according to manufacturer’s protocols. Data are expressed as mean ± SEM of five independent determinations. One-way ANOVA by Newman Keuls post hoc test is used. The analyses of P (AVOVA) indicates P < 0.001.

Figure 7

Effect of 11Cha1 on ATP-induced pyroptosis reaction. THP-1 cells were seeded and differentiated by 50 nM PMA for 48 h and then primed with 1 μg/mL LPS for 4 h. The cells were pre-treated with or without the indicated agent for 30 min and then treated with 5 mM ATP for another 2 h. After the treatment, the LDH levels in medium was determined using LDH assay kit. Data are expressed as mean ± SEM of five independent determinations.