Identification of Potential Anti-Neuroinflammatory Inhibitors from Antarctic Fungal Strain Aspergillus sp. SF-7402 via Regulating the NF-κB Signaling Pathway in Microglia

Abstract

Microglia play a significant role in immune defense and tissue repair in the central nervous system (CNS). Microglial activation and the resulting neuroinflammation play a key role in the pathogenesis of neurodegenerative disorders. Recently, inflammation reduction strategies in neurodegenerative diseases have attracted increasing attention. Herein, we discovered and evaluated the anti-neuroinflammatory potential of compounds from the Antarctic fungi strain Aspergillus sp. SF-7402 in lipopolysaccharide (LPS)-stimulated BV2 cells. Four metabolites were isolated from the fungi through chemical investigations, namely, 5-methoxysterigmatocystin (1), sterigmatocystin (2), aversin (3), and 6,8-O-dimethylversicolorin A (4). Their chemical structures were elucidated by extensive spectroscopic analysis and HR-ESI-MS, as well as by comparison with those reported in literature. Anti-neuroinflammatory effects of the isolated metabolites were evaluated by measuring the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in LPS-activated microglia at non-cytotoxic concentrations. Sterigmatocystins (1 and 2) displayed significant effects on NO production and mild effects on TNF-α and IL-6 expression inhibition. The molecular mechanisms underlying this activity were investigated using Western blot analysis. Sterigmatocystin treatment inhibited NO production via downregulation of inducible nitric oxide synthase (iNOS) expression in LPS-stimulated BV2 cells. Additionally, sterigmatocystins reduced nuclear translocation of NF-κB. These results suggest that sterigmatocystins present in the fungal strain Aspergillus sp. are promising candidates for the treatment of neuroinflammatory diseases.

Keywords: Antarctica; BV2 cells; NF-κB; fungi metabolites; neuroinflammation; sterigmatocystin.

Figure 1

Structure of isolated metabolites (1–4).

Figure 2

The effects of compounds on cell viability (A) and nitrite content (B). BV2 cells were incubated for 24 h with various concentrations of compounds 1–4. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. BV2 cells were pretreated for 3 h with indicated concentrations of compounds and stimulated for 24 h with lipopolysaccharide (LPS) (0.5 μg/mL). Bars represent means ± standard deviation of three independent experiments. *** p < 0.001 compared with LPS-treated group.

Figure 3

Protein expression levels of inducible nitric oxide synthase (iNOS) (A,C) and cyclooxygenase-2 (COX-2) (B,D) in lipopolysaccharide (LPS)-stimulated BV2 cells. Cells were pretreated for 3 h with indicated concentrations of compounds 1 and 2 and stimulated for 24 h with LPS (0.5 μg/mL). Representative blots from three independent experiments are shown. Immunoblots were quantified using ImageJ software. Band intensities were normalized to β-actin. * p < 0.05, ** p < 0.01 compared with LPS-treated group.

Figure 4

Effects of the isolated compounds on TNF-α (A), IL-6 (B), and PGE₂ (C) in LPS-stimulated BV2 cells. Cells were pretreated for 3 h with indicated concentrations of compounds 1–4 and stimulated for 24 h with LPS (0.5 μg/mL). Bars represent means ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with LPS-treated group.

Figure 5

Effects of the isolated compounds on the NF-κB (p65) pathway in BV2 cells (A–D). Cells were pretreated with indicated concentrations of compounds 1 and 2 for 3 h and stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 1 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using ImageJ software. p-IκBα intensities were normalized to β-actin. P65 intensities were normalized to PCNA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with LPS-treated group.