Ethyl Pyruvate Attenuates Microglial NLRP3 Inflammasome Activation via Inhibition of HMGB1/NF-κB/miR-223 Signaling

Abstract

Ethyl pyruvate is a molecule with anti-inflammatory and pro-metabolic effects. Ethyl pyruvate has been shown to ameliorate the clinical and pathological findings of neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases in rodents. Its anti-inflammatory and neuroprotective effects are widely investigated in animal and cellular models. Our study aimed to investigate the mechanism of the impact of Ethyl pyruvate on NLRP3 inflammasome activation in the N9 microglial cell line. Our results indicated that ethyl pyruvate significantly suppressed LPS and ATP-induced NLRP3 inflammasome activation, decreased active caspase-1 level, secretion of IL-1β and IL-18 cytokines, and reduced the level of pyroptotic cell death resulting from inflammasome activation. Furthermore, ethyl pyruvate reduced the formation of total and mitochondrial ROS and suppressed inflammasome-induced HMGB1 upregulation and nuclear NF-κB translocation and reversed the inflammasome activation-induced miRNA expression profile for miR-223 in N9 cells. Our study suggests that ethyl pyruvate effectively suppresses the NLRP3 inflammasome activation in microglial cells regulation by miR-223 and NF-κB/HMGB1 axis.

Keywords: HMGB1; NF-κB; NLRP3 inflammasome; ethyl pyruvate; microRNA; microglia.

Figure 1

EP reduced mRNA and protein levels of IL-1β and IL-18. N9 microglial cells were pretreated with EP (10 mM) for one hour, then treated with LPS (1 μg/mL) for four hours and ATP (5 mM) for 1 h. (A) The toxicity of EP was determined. (B–D) Protein levels of pro-IL-1β and secreted IL-1β were reduced by EP compared to LPS- and ATP-induced cells. (E,F) The suppressor effect of EP on secreted IL-1β and IL-18 was measured with ELISA. (G,H) mRNA levels of IL-1β and IL-18 reduced by EP pre-treatment compared to LPS and ATP induction group. Data are presented as mean ± S.E.M, n = 5. * p < 0.05, ** p < 0.01 compared to control and # p < 0.05, ## p < 0.01 compared to LPS- and ATP-induced cells.

Figure 2

EP reduced NLRP3, caspase-1, and ASC speck formation. N9 microglial cells were pretreated with EP (10 mM) for one hour, then treated with LPS (1 μg/mL) for four hours, and ATP (5 mM) for 1 h. (A–C) Pro-caspase-1 shows no difference among groups. EP reduced cleaved caspase-1 in EP-pretreated cells compared to LPS- and ATP-induced cells. (D–F) EP suppressed NLRP3 on both protein and mRNA levels compared with LPS- and ATP-induced cells. (G) ASC speck formation was determined by confocal microscopy. (H) EP significantly prevented ASC speck formation compared to LPS- and ATP-induced cells. Data are presented as mean ± S.E.M, n = 5. ** p < 0.01 compared to control and # p < 0.05, ## p < 0.01 compared to LPS- and ATP-induced cells.

Figure 3

EP reduced pyroptotic cell death. N9 microglial cells were pretreated with EP (10 mM) for one hour, then treated with LPS (1 μg/mL) for four hours, and ATP (5 mM) for 1 h. (A,B) EP reduced pyroptotic cell death and decreased PI-positive cells. All the data are presented as mean ± S.E.M, n = 5. ** p < 0.01 compared to control and ## p < 0.01 compared to LPS- and ATP-induced cells.

Figure 4

EP inhibited intracellular and mitochondrial ROS production and restored mitochondrial membrane potential. N9 microglial cells were pretreated with EP (10 mM) for one hour, then treated with LPS (1 μg/mL) for four hours, and ATP (5 mM) for 1 h. (A) EP pre-treatment reduced intracellular ROS production. (B,C) EP also decreased mitochondrial ROS production in EP-pretreated cells compared with LPS- and ATP-induced cells. (D,E) EP pre-treatment restored mitochondrial membrane potential. Data are presented as mean ± S.E.M, n = 5. * p < 0.05, ** p < 0.01 compared to control; # p < 0.05 compared to LPS- and ATP-induced cells and ## p < 0.01 compared to LPS- and ATP-induced cells.

Figure 5

EP suppressed HMGB1 levels and inhibited activation of NF-κB. N9 microglial cells were pretreated with EP (10 mM) for one hour, then treated with LPS (1 μg) for 30 min. (A,B) EP reduced LPS- and ATP-induced HMGB1 levels. (C,D) EP pre-treatment reduced phosphorylation of p65 subunit of NF-κB. (C,E) EP pre-treatment significantly reduced the p50 subunit of NF-κB. (F) EP rescued LPS and ATP induced an increase of nuclear/cytoplasmic ratio of NF-κB p65 subunit. (G,H) EP pre-treatment increased expression of IκBα, which was downregulated after LPS and ATP induction. Data are presented as mean ± S.E.M, n = 5. ** p < 0.01 compared to control and # p < 0.05 and ## p < 0.01 compared to LPS-induced cells.

Figure 6

miR-223 inhibition impairs the protective role of EP in inflammasome activation. N9 cells were treated with antagomiR-223 before EP (10 mM) for one hour, then treated with LPS (1 μg/mL) for four hours, and ATP (5 mM) for 1 h. (A) miR-223 expression without any modification decreased in LPS- and ATP-treated group and increased in the EP pre-treatment group. (B) miR-223 levels were suppressed following miR-223 antagomiR transfection. (C) After mir-223 inhibition, the difference in NLRP3 between the LPS and ATP and EP pre-treatment groups decreased. (D) After mir-223 inhibition, the difference in IL-1β between the LPS and ATP group and EP pre-treatment group was decreased. (E) After mir-223 inhibition, the difference in IL-18 between the LPS and ATP group and the EP pre-treatment group was decreased. Data are presented as mean ± S.E.M, n = 5. * p < 0.05 and ** p < 0.01 compared to control and # p < 0.05 and ## p < 0.01 compared to LPS-induced cells.