Curcumin Represses NLRP3 Inflammasome Activation via TLR4/MyD88/NF-κB and P2X7R Signaling in PMA-Induced Macrophages

Abstract

Aims: In the NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) inflammasome is closely related to the progression of atherosclerosis. This study aimed to assess the effects of curcumin on NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages and explore its underlying mechanism. Methods: Human monocytic THP-1 cells were pretreated with curcumin for 1 h and subsequently induced with PMA for 48 h. Total protein was collected for Western blot analysis. Cytokine interleukin (IL)-1β release and nuclear factor kappa B (NF-κB) p65 translocation were detected by ELISA assay and cellular NF-κB translocation kit, respectively. Results: Curcumin significantly reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion in PMA-induced macrophages. Moreover, Bay (a NF-κB inhibitor) treatment considerably suppressed the expression of NLRP3 inflammasome in PMA-induced THP-1 cells. Curcumin also markedly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκB-α, and activation of NF-κB in PMA-induced macrophages. In addition, purinergic 2X7 receptor (P2X7R) siRNA was administered, and it significantly decreased NLRP3 inflammasome expression in PMA-induced macrophages. Furthermore, curcumin reversed PMA-stimulated P2X7R activation, which further reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion. Silencing of P2X7R using siRNA also suppressed the activation of NF-κB pathway in PMA-induced macrophages, but P2X7R-silenced cells did not significantly decrease the expression of TLR4 and MyD88. Conclusion: Curcumin inhibited NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB and P2X7R pathways in PMA-induced macrophages.

Keywords: Curcumin; NLRP3 inflammasome; P2X7R; TLR4/MyD88/NF-κB; macrophages.

FIGURE 1

Effects of curcumin on cell viability and apoptosis. THP-1 monocytes were incubated with various curcumin concentrations (0–100 μM) for 1 h and exposed to 100 nM of phorbol 12-myristate 13-acetate (PMA) for 48 h. (A) Cell proliferation was assessed using the CCK8 assay. Cells incubated in a medium without curcumin and PMA were defined as control and considered to have a 100% proliferation rate. (B) Chemical structure of curcumin. (C) Representative Western blot analysis of Bax and Bcl-2 in curcumin (6.25–25 μM)-treated THP-1 cells. (D) Densitometric analysis was used to quantify the ratio of Bax/Bcl-2. The results represent the mean ± SEM for three experiments. ^∗P < 0.05 vs. PMA group, ^#P < 0.05 vs. Control group.

FIGURE 2

Curcumin attenuates the activation of the NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome. THP-1 macrophages were stimulated by incubation with curcumin (Cur) at the indicated concentration (6.25–25 μM) for 1 h, followed by PMA for 48 h. The condition referred to as control refers to THP-1 treated with vehicle or dimethyl sulfoxide (DMSO) for 48 h. (A) Representative Western blot analysis of NLRP3 and the cleavage of caspase-1 and interleukin (IL)-1β protein expression after PMA-induced inflammasome activation. (B) Densitometric analysis was used to quantify the level of NLRP3 and cleavage of caspase-1 and IL-1β. (C) Concentrations of IL-1β in cell culture supernatants were detected by ELISA. The results represent the mean ± SEM for three experiments. ^∗P < 0.05 vs. PMA group, ^#P < 0.05 vs. Control group.

FIGURE 3

Nuclear factor kappa B (NF-κB) pathway activation participates in the activation of the NLRP3 inflammasome in PMA-induced macrophages. Cells were incubated with 6.25 μM of curcumin (Cur) for 1 h or 5 μM of NF-κB specific inhibitor Bay 11-7082 (Bay) for 30 min and exposed to 100 nM of PMA for 48 h before collection. (A) Representative Western blot analysis of NLRP3 and the cleavage of caspase-1 and IL-1β protein expression after PMA-induced inflammasome activation. (B) Densitometric analysis was used to quantify the level of NLRP3 and cleavage of caspase-1 and IL-1β. The results represent the mean ± SEM for three experiments. ^∗P < 0.05 vs. PMA group, ^#P < 0.05 vs. Control group.

FIGURE 4

Curcumin inhibits the activation of the TLR4/MyD88/NF-κB-signaling pathways. (A) Representative Western blot analysis of TLR4, MyD88, p-IκB-α, IκB-α, p-P65, and P65 was normalized based on the internal control GAPDH. (B–E) Densitometry measurements of protein analysis. The results represent the mean ± SEM for three experiments. ^∗P < 0.05 vs. PMA group, ^#P < 0.05 vs. Control group. (F) Differentiated THP-1 cells were treated with indicated agent’s immunostained with DAPI (Blue) and anti-NF-κB p65 (Red) and observed using an inverted fluorescence microscope, 200×.

FIGURE 5

Curcumin decreases the expression of P2X7R in PMA-induced macrophages. (A,B) THP-1 cells silenced for purinergic 2X7 receptor (P2X7R) were processed to obtain a whole-cell extract as described under the Section “Materials and Methods”. The expression of P2X7R in cells were transfected with P2X7R or negative control (NC) siRNA. (C,D) The expression of NLRP3 and cleavage of caspase-1 and IL-1β in cells were transfected with P2X7R or negative control (NC) siRNA. (E,F) THP-1 cells were pretreated with curcumin in the presence of PMA for 48 h. The expression of P2X7R was determined by Western blot analysis. ^∗P < 0.05 vs. PMA group, ^#P < 0.05 vs. Control group.

FIGURE 6

Purinergic 2X7 receptor inhibition reduces the phosphorylation of p65 in PMA-induced macrophages. THP-1 cells silenced for P2X7R were used to obtain a whole-cell extract as described under Materials and Methods. (A) Representative Western blot analysis of TLR4, MyD88, p-P65, and P65 was normalized based on the internal control GAPDH. (B,C) Densitometry measurements of protein analysis. The results represent the mean ± SEM for three experiments. ^∗P < 0.05 vs. PMA group.

FIGURE 7

Schematic model for the reduction of NLRP3 inflammasome expression in PMA-stimulated macrophages treated with curcumin. The curcumin-downregulated P2X7R expression and TLR4/MyD88/NF-κB signaling, which together inhibited NLRP3 expression, caspase-1 activation, and the secretion of IL-1β.