Anti-inflammatory effects of N-cyclooctyl-5-methylthiazol-2-amine hydrobromide on lipopolysaccharide-induced inflammatory response through attenuation of NLRP3 activation in microglial cells

Abstract

Microglial activation is closely associated with neuroinflammatory pathologies. The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasomes are highly organized intracellular sensors of neuronal alarm signaling. NLRP3 inflammasomes activate nuclear factor kappa-B (NF-κB) and reactive oxygen species (ROS), which induce inflammatory responses. Moreover, NLRP3 dysfunction is a common feature of chronic inflammatory diseases. The present study investigated the effect of a novel thiazol derivative, N-cyclooctyl-5-methylthiazol-2-amine hydrobromide (KHG26700), on inflammatory responses in lipopolysaccharide (LPS)-treated BV-2 microglial cells. KHG26700 significantly attenuated the expression of several pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, in these cells, as well as the LPS-induced increases in NLRP3, NF-κB, and phospho-IkBα levels. KHG26700 also suppressed the LPS-induced increases in protein levels of autophagy protein 5 (ATG5), microtubule- associated protein 1 light chain 3 (LC3), and beclin-1, as well as downregulating the LPS-enhanced levels of ROS, lipid peroxidation, and nitric oxide. These results suggest that the anti-inflammatory effects of KHG26700 may be due, at least in part, to the regulation of the NLRP3-mediated signaling pathway during microglial activation. [BMB Reports 2021; 54(11): 557-562].

Fig. 1

Effects of KHG26700 on the production of IL-1b (A), IL-6 (B), and TNF-α (C) in LPS-treated BV-2 cells. BV-2 cells were treated with KHG26700 for 30 min followed by treatment with LPS (1 μg/ml) for 24 h. Data are presented as means ± S.D. (n = 3). *P < 0.01 for comparisons between cells treated with LPS and LPS plus KHG26700.

Fig. 2

Effects of KHG26700 on NF-κB (A), NLRP3 (B), and Iκb-α (C) in LPS-treated BV-2 cells, as determined by immunofluorescence assays and western blot analysis. BV-2 cells were treated with KHG26700 for 30 min followed by treatment with LPS (1 μg/ml) for 24 h. Images presented are from a single experiment and are representative of three independent experiments. β-Actin was used as a loading control. Scale bars were 10 μm in (A) and 50 μm in (B).

Fig. 3

Western blot analysis of the effects of KHG26700 on protein levels of ATG5, beclin-1, and LC3 in LPS-treated BV-2 cells. (A) Western blot analysis was performed using antibodies against ATG5, beclin-1, and LC3, with β-actin used as a loading control. (B-D) Relative protein levels were quantified by densitometry and normalized relative to the expression of β-actin. Data are presented as means ± S.D. (n = 3). *P < 0.01 for comparisons between cells treated with LPS and LPS plus KHG26700.

Fig. 4

Effects of KHG26700 on ROS (A), NO (B), and MDA (C) production in LPS-treated BV-2 cells. BV-2 cells were treated with KHG26700 for 30 min followed by treatment with LPS (1 μg/ml) for 24 h. In (A), images presented are from a single experiment and are representative of three independent experiments. Data are presented as means ± S.D. (n = 3). *P < 0.01 for comparisons between cells treated with LPS and LPS plus KHG26700.