Aiouea padiformis extract exhibits anti-inflammatory effects by inhibiting the ATPase activity of NLRP3

Abstract

Inflammation is implicated as a cause in many diseases. Most of the anti-inflammatory agents in use are synthetic and there is an unmet need for natural substance-derived anti-inflammatory agents with minimal side effects. Aiouea padiformis belongs to the Lauraceae family and is primarily found in tropical regions. While some members of the Aiouea genus are known to possess anti-inflammatory properties, the anti-inflammatory properties of Aiouea padiformis extract (AP) have not been investigated. In this study, we aimed to examine the anti-inflammatory function of AP through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and elucidate the underlying mechanisms. Treatment with AP inhibited the secretion of interleukin-1 beta (IL-1β) mediated by NLRP3 inflammasome in J774A.1 and THP-1 cells without affecting the viability. In addition, AP treatment did not influence NF-κB signaling, potassium efflux, or intracellular reactive oxygen species (ROS) production-all of which are associated with NLRP3 inflammasome activation. However, intriguingly, AP treatment significantly reduced the ATPase activity of NLRP3, leading to the inhibition of ASC oligomerization and speck formation. Consistent with cellular experiments, the anti-inflammatory property of AP in vivo was also evaluated using an LPS-induced inflammation model in zebrafish, demonstrating that AP hinders NLRP3 inflammasome activation.

Keywords: Aiouea padiformis; Anti-inflammation; Lauraceae; NLRP3 inflammasome; Plant extracts.

Figure 1

AP specifically reduces NLRP3 inflammasome activation. (a,b) Cell viability assays of J774A.1 cells (a) and differentiated THP-1 cells (b) treated with AP (10 μg/mL, 50 μg/mL, 100 μg/mL) for 2 h. Cell viability was analyzed by EZ-CYTOX, measured at a wavelength of 450 nm. Ns not significant. (c–e) Western blots of IL-1β in the supernatants (Sup), pro-IL-1β and α-tubulin in the soluble lysates (Lys) of treated cells. LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) (c) or ATP (5 mM) (d) with or without MCC950, differentiated, and LPS-primed THP-1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM) with or without MCC950 (e). (f,g) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 3 h with transfection of dsDNA (2 μg/mL) (f) or flagellin (1.25 μg/mL) (g).

Figure 2

AP does not affect the NF-κB signaling pathway. (a) 293 T cells were transfected with the pGL4.32 luciferase reporter vector and treated with TNF-α (20 ng/mL) for 5 h with or without AP. Luciferase activity was analyzed using the Promega Bright-Glo™ Luciferase Assay System. (b) LPS-primed J774A.1 cells were treated with AP for 2 h and activated for 30 min with nigericin (10 μM). IL-1β in the supernatants (Sup), phospho-NF-κB and NF-κB in the soluble lysates (Lys) were analyzed using a Western blot.

Figure 3

AP inhibits NLRP3 inflammasome, regardless of K+ efflux, intracellular ROS, and mitochondrial membrane potential. (a) LPS-primed J774A.1 cells were treated with AP for 2 h and activated with Imiquimod (200 μM) for 1 h. (b,c) LPS-primed J774A.1 cells treated with AP (50 μg/mL) for 2 h and activated with ATP (5 mM) for 5 min. Representative immunofluorescence images of ROS (DCFDA; green color) (b) and mitochondrial membrane potential (JC-1 monomer; green color, J-aggregates; red color) (c) were taken by a confocal laser-scanning microscope (Carl Zeiss, LSM710, scale bar, 20 μm).

Figure 4

AP blocks NLRP3 inflammasome assembly by hindering ATPase activity of NLRP3. (a) HEK 293FT cells were transfected with NLRP3-Myc and treated with or without AP for 2 h. The interaction between NLRP3 and NEK7 was analyzed by immunoprecipitation and immunoblot. (b) The ATPase activity of NLRP3 with or without AP was measured by luminescence using the ADP-Glo™ Max assay. (c) LPS-primed THP-1 cells were treated with AP for 2 h and activated with nigericin (10 μM) for 30 min. The cell pellet was cross-linked by DSS (2.5 mM) for 30 min. ASC oligomerization was analyzed by immunoblot. (d,e) LPS-primed J774A.1 cells treated with AP for 2 h and activated with nigericin (10 μM) for 30 min. Representative immunofluorescence images of ASC speck formation (indicated by arrows) were taken by a confocal laser-scanning microscope (Carl Zeiss, LSM710, scale bar, 20 μm) (d). Graph representing the number of cells containing ASC specks in all treatments (e).

Figure 5

AP alleviates inflammatory response induced by LPS treatment in zebrafish. (a) Representative images of posterior areas of Sudan Black B-stained zebrafish embryos after LPS induction and AP treatment at 3 dpf. The upper panel shows the control group with DMSO (0.025%) treatment, the middle panel shows LPS inflammation group, and the bottom panel shows the LPS inflammation group after treatment with 10 µg/mL AP. (b) Quantification of neutrophils in the posterior region of zebrafish after LPS induction and AP treatment. (c) Representative images of the caudal hematopoietic tissues after LPS induction and AP treatment at 3 dpf. Whole-mount in situ hybridization was conducted using an mpx probe. (d) Representative images of anterior Sudan Black B-stained zebrafish embryos following direct LPS injection and AP treatment at 3 dpf. (e) Quantification of the number of neutrophils in the anterior zebrafish embryos after LPS induction and AP treatment. All graphs represent the mean ± S.E.M. of individual values. P-values were calculated using an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.