The H₂S Donor GYY4137 Stimulates Reactive Oxygen Species Generation in BV2 Cells While Suppressing the Secretion of TNF and Nitric Oxide

Abstract

GYY4137 is a hydrogen sulfide (H₂S) donor that has been shown to act in an anti-inflammatory manner in vitro and in vivo. Microglial cells are among the major players in immunoinflammatory, degenerative, and neoplastic disorders of the central nervous system, including multiple sclerosis, Parkinson's disease, Alzheimer's disease, and glioblastoma multiforme. So far, the effects of GYY4137 on microglial cells have not been thoroughly investigated. In this study, BV2 microglial cells were stimulated with interferon-gamma and lipopolysaccharide and treated with GYY4137. The agent did not influence the viability of BV2 cells in concentrations up to 200 μM. It inhibited tumor necrosis factor but not interleukin-6 production. Expression of CD40 and CD86 were reduced under the influence of the donor. The phagocytic ability of BV2 cells and nitric oxide production were also affected by the agent. Surprisingly, GYY4137 upregulated generation of reactive oxygen species (ROS) by BV2 cells. The effect was mimicked by another H₂S donor, Na₂S, and it was not reproduced in macrophages. Our results demonstrate that GYY4137 downregulates inflammatory properties of BV2 cells but increases their ability to generate ROS. Further investigation of this unexpected phenomenon is warranted.

Keywords: GYY4137; hydrogen sulfide; inflammation; microglia; reactive oxygen species.

Figure 1

Effects of GYY4137 (GYY) on viability and nitric oxide (NO) and cytokine production in BV2 cells. BV2 cells were stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) and cultivated in the presence of dimethyl sulfoxide (DMSO) (control—Ctrl) as the vehicle or GYY (200 μM in B–F) for 24 h. Subsequently, the cell viability was determined by crystal violet (CV) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests (A), apoptosis was detected by annexin V staining and cytofluorimetry (B), nitrites were measured by Griess reaction (C), cytokine concentrations were determined by ELISA (D,E), and mRNA expression relative to β-actin by real-time RT-PCR (F). Data are presented as mean + standard deviation (SD) from at least three independent experiments. * p < 0.05 refers to Ctrl.

Figure 2

Effects of GYY4137 (GYY) on phenotype and phagocytosis in BV2 cells. BV2 cells were stimulated with IFN-γ and LPS and cultivated in the presence of DMSO (Ctrl) as the vehicle or GYY (200 μM) for 24 h. Subsequently, expression of CD40 (A,B), CD86 (C,D), and phagocytosis (E,F) were determined by cytofluorimetry. Data are presented as mean + SD from at least three independent experiments (A,C,E). Representative plots are also provided (B,D,F). * p < 0.05 refers to Ctrl.

Figure 3

Effects of GYY4137 (GYY) on reactive oxygen species (ROS) production in BV2 cells. BV2 cells were cultivated in the presence of DMSO (Ctrl) as the vehicle or GYY (200 μM). (A) BV2 cells were treated with IFN-γ and LPS and DMSO or GYY for 24 h, stained with DHR, and stimulated with phorbol 12-myristate 13-acetate (PMA) for 90 min (ALL), or they were treated with DMSO or GYY for 24 h, stained with DHR, and stimulated with PMA or IFN-γ and LPS (IFN-γ + LPS) for 90 min. Subsequently, cytofluorimetry was performed. (B) Same as A, except that incubation lasted for 1 h instead of 24 h. (C) Representative DHR plots from (B). (D) BV2 cells were treated with DMSO or GYY for the indicated time periods, stained with DHR, stimulated with PMA, and cytofluorimetry was performed. (E) BV2 cells were treated with IFN-γ and LPS and DMSO or GYY and analyzed by the real-time cell analyzer. Representative plot is provided. (F) DMSO or GYY were mixed with DHR in a cell-free system and fluorescence was detected on a fluorimeter. (G) BV2 cells were treated with Na2S (200 μM) or were untreated (0) for 60 min, stained with DHR, and stimulated with PMA for 90 min. Subsequently, cytofluorimetry was performed. (H) BV2 cells were treated with DMSO or GYY for 60 min, stained with DCFDA, and stimulated with PMA for 90 min. Subsequently, cytofluorimetry was performed. (H) Same as B (PMA), with additional treatment with “spent” GYY4137 (sGYY). (I) same as B (PMA), except that BV2 cells were stained with DCFDA (J). Same as B, except that mouse macrophages were used. (K) Same as B, except that rat macrophages were used. Data are presented as mean + SD from at least three independent experiments. * p < 0.05 refers to Ctrl or 0; ^# p < 0.05 refers to difference between GYY and sGYY.

Figure 3

Effects of GYY4137 (GYY) on reactive oxygen species (ROS) production in BV2 cells. BV2 cells were cultivated in the presence of DMSO (Ctrl) as the vehicle or GYY (200 μM). (A) BV2 cells were treated with IFN-γ and LPS and DMSO or GYY for 24 h, stained with DHR, and stimulated with phorbol 12-myristate 13-acetate (PMA) for 90 min (ALL), or they were treated with DMSO or GYY for 24 h, stained with DHR, and stimulated with PMA or IFN-γ and LPS (IFN-γ + LPS) for 90 min. Subsequently, cytofluorimetry was performed. (B) Same as A, except that incubation lasted for 1 h instead of 24 h. (C) Representative DHR plots from (B). (D) BV2 cells were treated with DMSO or GYY for the indicated time periods, stained with DHR, stimulated with PMA, and cytofluorimetry was performed. (E) BV2 cells were treated with IFN-γ and LPS and DMSO or GYY and analyzed by the real-time cell analyzer. Representative plot is provided. (F) DMSO or GYY were mixed with DHR in a cell-free system and fluorescence was detected on a fluorimeter. (G) BV2 cells were treated with Na2S (200 μM) or were untreated (0) for 60 min, stained with DHR, and stimulated with PMA for 90 min. Subsequently, cytofluorimetry was performed. (H) BV2 cells were treated with DMSO or GYY for 60 min, stained with DCFDA, and stimulated with PMA for 90 min. Subsequently, cytofluorimetry was performed. (H) Same as B (PMA), with additional treatment with “spent” GYY4137 (sGYY). (I) same as B (PMA), except that BV2 cells were stained with DCFDA (J). Same as B, except that mouse macrophages were used. (K) Same as B, except that rat macrophages were used. Data are presented as mean + SD from at least three independent experiments. * p < 0.05 refers to Ctrl or 0; ^# p < 0.05 refers to difference between GYY and sGYY.