Aquilariae Lignum Methylene Chloride Fraction Attenuates IL-1β-Driven Neuroinflammation in BV2 Microglial Cells

Abstract

Microglial hyperactivation and neuroinflammation are known to induce neuronal death, which is one of the main causes of neurodegenerative disorders. We previously found that Aquilariae Lignum extract attenuated both neuronal excitotoxicity and neuroinflammation in vivo and in vitro. For further analysis, we extracted the methylene chloride fraction of Aquilariae Lignum to determine the bioactive compounds. In this study, we investigated the anti-neuroinflammatory effects and underlying mechanisms of the Aquilariae Lignum fraction (ALF) using lipopolysaccharide (LPS)-stimulated BV2 microglial cells. BV2 cells were pretreated with ALF (0.5, 1, and 2.5 μg/mL) before treatment with LPS (1 μg/mL). Pretreatment with ALF significantly attenuated the LPS-induced overproductions of nitric oxide (NO), cyclooxygenase-2 (COX-2), prostaglandin E₂ (PGE₂), and interleukin (IL)-1β. These anti-inflammatory effects were supported by ALF-mediated modulation of the nuclear factor-kappa B (NF-κB) pathway. Furthermore, ALF exerted strong anti-inflammasome effects, as shown by IL-1β-specific inhibitory activity, but not activity against tumor necrosis factor (TNF)-α, along with inhibition of caspase-1 activity and NACHT, LRR, and PYD domain-containing protein 3 (NLRP3)-related molecules. These results indicate the potent anti-neuroinflammatory activity of ALF and that its underlying mechanism may involve the regulation of NLRP3 inflammasome-derived neuroinflammation in microglial cells.

Keywords: Aquilariae Lignum; NLRP3 inflammasome; microglia; neuroinflammation.

Figure 1

Fingerprinting analysis of the Aquilariae Lignum fraction (ALF). Five peaks were detected at each retention time (A), and their areas were semiquantified (B). ALF was subjected to UHPLC-MS, and a chromatogram was obtained at a wavelength of 280 nm.

Figure 2

Cell viability and nitric oxide levels. BV2 cells were pretreated with ALF for 2 h before exposure to lipopolysaccharide (LPS) (1 μg/mL) for 24 h. The cytotoxicity of ALF (A) and the levels of nitric oxide (B) in BV2 cells were determined. The data are expressed as the mean ± SD (n = 6). ## p < 0.01 compared with the vehicle-treated cells; ** p < 0.01 compared with the LPS-treated BV2 cells.

Figure 3

Prostaglandin E₂ (PGE₂) production and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in BV2 cells. Cell supernatant PGE₂ levels (A), protein expression of iNOS and COX-2 (B), and gene expression of iNOS and COX-2 relative to that of GAPDH (C) were determined using ELISA (n = 6), Western blot (n = 3), or real-time PCR (n = 3) methods. Semiquantification of protein expression was performed with normalization to β-actin (D). The data are expressed as the mean ± SD. # p < 0.05 and ## p < 0.01 compared with the vehicle-treated cells; * p < 0.05 and ** p < 0.01 compared with the LPS-treated BV2 cells.

Figure 4

Nuclear factor-kappa B (NF-κB) signals in BV2 cells. Signals for nuclear translocation of NF-κB (A) and cytosolic and nucleic p65 expression (B) were determined using IF staining or Western blot methods. The intensity of the histological signals (C) and semiquantification of protein expression relative to β-actin or lamin B1 (D) were analyzed. The data are expressed as the mean ± SD (n = 3). ## p < 0.01 compared with the vehicle-treated cells; * p < 0.05 and ** p < 0.01 compared with the LPS-treated BV2 cells.

Figure 5

Tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in BV2 cells. Proinflammatory cytokines TNF-α (A) and IL-1β (B) in the cell supernatant and gene expression relative to GAPDH (C) in the cell lysate were determined using ELISA (n = 6) and real-time PCR (n = 3), respectively. The data are expressed as the mean ± SD. ## p < 0.01 compared with the vehicle-treated cells; * p < 0.05 and ** p < 0.01 compared with the LPS-treated BV2 cells.

Figure 6

NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome pathway in BV2 cells. Protein expression of NLRP3, ASC, and pro and mature-IL-1β was determined using Western blot (A). Caspase-1 activity in cell lysates was determined using ELISA (B). Semiquantification of the protein expression was performed with normalization to β-actin (C). The data are expressed as the mean ± SD (n = 3). ## p < 0.01 compared with the vehicle-treated cells; * p < 0.05 and ** p < 0.01 compared with the LPS-treated BV2 cells.