Genistein suppresses tumor necrosis factor α-induced inflammation via modulating reactive oxygen species/Akt/nuclear factor κB and adenosine monophosphate-activated protein kinase signal pathways in human synoviocyte MH7A cells

Abstract

Aims: Genistein, an isoflavone derivative found in soy, is known as a promising treatment for rheumatoid arthritis (RA). However, the detailed molecular mechanism of genistein in suppression of proinflammatory cytokine production remains ambiguous. The aim of this work was to evaluate the signal pathway by which genistein modulates inflammatory cytokine expression.

Materials and methods: MH7A cells were stimulated with tumor necrosis factor (TNF)-α and incubated with genistein, and interleukin (IL)-1β, IL-6, and IL-8 production was measured by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor (NF)-κB was measured by a confocal fluorescence microscopy. The intracellular accumulation of reactive oxygen species (ROS) was monitored using the fluorescent probe 5-6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Signal-transduction protein expression was measured by Western blot.

Results: Genistein decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. Genistein prevented TNF-α-induced NF-κB translocation as well as phosphorylation of IκB kinase-α/β and IκBα, and also suppressed TNF-α-induced AMPK inhibition. The production of IL-1β, IL-6, and IL-8 induced by TNF-α was decreased by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting that inhibition of Akt activation might inhibit IL-1β, IL-6, and IL-8 production induced by TNF-α. In addition, we also found that pretreatment with the adenosine monophosphate-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside obviously inhibited TNF-α-induced proinflammatory cytokine production. These observations suggest that the inhibitory effect of genistein on TNF-α-induced proinflammatory cytokine production is dependent on AMPK activation.

Conclusion: These findings indicate that genistein suppressed TNF-α-induced inflammation by inhibiting the ROS/Akt/NF-κB pathway and promoting AMPK activation in MH7A cells.

Keywords: cytokine; genistein; inflammation; rheumatoid arthritis; signal transduction.

Figure 1

Cytotoxicity of genistein on MH7A cells. Notes: Cells were treated with different concentrations of genistein for 24 hours, and their viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. **P<0.01 versus group without genistein treatment. The data are presented as the means ± standard deviation from three independent experiments.

Figure 2

(A–C) Effects of genistein (Gen) on tumor necrosis factor (TNF)-α-induced proinflammatory cytokine production. Notes: MH7A cells were pretreated with Gen or not for 2 hours, and then stimulated with TNF-α. After 24 hours, the levels of interleukin (IL)-1β, IL-6, and IL-8 were measured in the culture medium by enzyme-linked immunosorbent assay. *P<0.05 and **P<0.01 versus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments.

Figure 3

(A and B) Effects of genistein (Gen) on nuclear factor (NF)-κB activation in MH7A cells. Notes: (A) MH7A cells were pretreated with Gen or not for 2 hours, and then incubated with tumor necrosis factor (TNF)-α for the indicated times. Phosphorylated IκB kinase (p-IKK)-α/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. (B) After being pretreated with Gen or not for 2 hours, MH7A cells were then incubated with TNF-α for 15 minutes. The nuclei translocation of NF-κB p65 was assessed by confocal fluorescence microscopy. Cells were immunostained with NF-κB p65 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Results representative of three independent experiments. Bar 10 μm. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

(A–E) Involvement of Akt in nuclear factor (NF)-κB activation by tumor necrosis factor (TNF)-α. Notes: (A) MH7A cells were pretreated with genistein (Gen) or LY294002 for 2 hours, and then incubated with TNF-α for the indicated times. Phosphorylated (p)-Akt levels were determined by Western blot analysis. (B) After being pretreated with LY294002 for 2 hours, MH7A cells were then incubated with TNF-α for 15 minutes. p-IκB kinase (p-IKK)-α/β, p-IκBα, and p-NF-κB p65 levels were determined by Western blot analysis. (C–E) Interleukin (IL)-1β, IL-6,and IL-8 production was determined in MH7A cells treated with TNF-α in the presence or absence of LY294002 for 24 hours by enzyme-linked immunosorbent assay. *P<0.05 and **P<0.01 vsersus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

(A–E) The role of reactive oxygen species (ROS) in tumor necrosis factor (TNF)-α-mediated inflammatory responses. Notes: (A) MH7A cells were stimulated with TNF-α (10 ng/mL) for 15 minutes in the presence of genistein (Gen; 20 μM) or N-acetyl-L-cysteine (NAC; 10 mM). 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate was used to determine the generation of intracellular ROS. Intracellular ROS levels were examined by fluorescence microscopy. (B–D) The levels of interleukin (IL)-1β, IL-6, and IL-8 were determined in cells treated with TNF-α alone or with NAC for 24 hours by enzymelinked immunosorbent assay. (E) MH7A cells pretreated with NAC for 2 hours before incubating with TNF-α for 15 minutes. Phosphorylated IκB kinase (p-IKK)-α/β, p-IκBα, and p-nuclear factor κB p65 levels were determined by Western blot analysis. *P<0.05 and **P<0.01 versus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments. Bar 100 μm. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6

(A–D) The role of adenosine monophosphate-activated protein kinase (AMPK) in the anti-inflammatory effects of genistein (Gen). Notes: (A) MH7A cells were either exposed to tumor necrosis factor (TNF)-α for 15 minutes, cotreated with TNF-α and Gen, treated with TNF-α and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), or left untreated for 15 minutes. Phosphorylation (p-) of AMPK was determined by Western blotting. (B–D) The levels of interleukin (IL)-1β, IL-6, and IL-8 were determined in cells treated with TNF-α alone or with AICAR for 24 hours by enzyme-linked immunosorbent assay. *P<0.05 and **P<0.01 versus group with TNF-α treatment alone. The data are presented as the means ± standard deviation from three independent experiments. Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 7

Schematic model illustrating the potential pathway associated with genistein’s inhibition of tumor necrosis factor (TNF)-α-induced inflammation. Notes: Genistein suppresses TNF-α-induced proinflammatory cytokine production through 1) reducing intracellular reactive oxygen species (ROS) accumulation triggered by TNF-α, which represses Akt/nuclear factor (NF)-κB signaling, and 2) promoting adenosine monophosphate-activated protein kinase (AMPK) activation. Abbreviations: IKK, IκB kinase; PI3K, phosphoinositide-3 kinase; TNFR, tumor necrosis factor receptor.