Fisetin inhibits hyperglycemia-induced proinflammatory cytokine production by epigenetic mechanisms

Abstract

Diabetes is characterized by a proinflammatory state, and several inflammatory processes have been associated with both type 1 and type 2 diabetes and the resulting complications. High glucose levels induce the release of proinflammatory cytokines. Fisetin, a flavonoid dietary ingredient found in the smoke tree (Cotinus coggygria), and is also widely distributed in fruits and vegetables. Fisetin is known to exert anti-inflammatory effects via inhibition of the NF-κB signaling pathway. In this study, we analyzed the effects of fisetin on proinflammatory cytokine secretion and epigenetic regulation, in human monocytes cultured under hyperglycemic conditions. Human monocytic (THP-1) cells were cultured under control (14.5 mmol/L mannitol), normoglycemic (NG, 5.5 mmol/L glucose), or hyperglycemic (HG, 20 mmol/L glucose) conditions, in the absence or presence of fisetin. Fisetin was added (3-10 μM) for 48 h. While the HG condition significantly induced histone acetylation, NF-κB activation, and proinflammatory cytokine (IL-6 and TNF-α) release from THP-1 cells, fisetin suppressed NF-κB activity and cytokine release. Fisetin treatment also significantly reduced CBP/p300 gene expression, as well as the levels of acetylation and HAT activity of the CBP/p300 protein, which is a known NF-κB coactivator. These results suggest that fisetin inhibits HG-induced cytokine production in monocytes, through epigenetic changes involving NF-κB. We therefore propose that fisetin supplementation be considered for diabetes prevention.

Figure 1

The cytotoxicity of fisetin to cultured high glucose-induced THP-1 cells. (a) Chemical structure of fisetin. (b) Effect of fisetin on cell viability after 48 h was evaluated by the CCK-8 assay, as described in the methods. Human monocytic (THP-1) cells (1 × 10⁵ cells/mL) were cultured in presence of osmolar control (14.5 mmol/L mannitol) or normal glycemic (NG, 5.5 mmol/L glucose) or hyperglycemic (HG, 20 mmol/L) conditions in absence or presence of fisetin (0, 3, 6, 10 μM) for 48 h as described in the methods and the media was collected. Results are shown as mean ± SD of five different experiments.

Figure 2

Fisetin-mediated inhibition of cytokine release in HG-treated THP-1 cells. (a) Cells (1 × 10⁵ cells/mL) were treated with fisetin for 48 h and then mRNA levels were evaluated by quantitative real-time PCR. (b) Cell media were collected for TNF-α measurement by ELISA assay kit. Cytokine levels in the media were measured with ELISA assay kit according to the manufacturer's instructions. Values were calculated based on a standard curve constructed for the assay. Results are shown as mean ± SD of five different experiments. ^†† P < 0.01 compared to NG; *P < 0.05; **P < 0.01 compared to HG. (c) Cell lysates were prepared and TNF-α level was evaluated by western blot analysis as described in the methods. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin protein. The immunoblots shown here are representative of 3 independent experiments.

Figure 3

Effect of fisetin on HAT and HDAC activity as well as p300 and acetylated CBP/p300 levels in HG-treated THP-1 cells. Cells were harvested after 48 h of fisetin treatment and nuclear lysates were prepared. Samples were analyzed for determination of HAT (a) and HDAC activity (b). Results are shown as mean ± SD for 3 different experiments. ^†† P < 0.01 compared to NG; *P < 0.05; **P < 0.01 compared to HG. (c) After nuclear protein extraction, p300 and acetylated CBP/p300 levels were evaluated by western blot. The immunoblots shown here are representative of 3 independent experiments.

Figure 4

Fisetin-induced suppression of NF-κB activation in HG-treated THP-1 cells. Protein levels were evaluated by western blot for NF-κB p65 and acetylated p65. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for histone 2A protein. The immunoblots shown here are representative of 3 independent experiments.

Figure 5

Effect of fisetin on the interaction of p300 with acetylated p65 and TNF-α. Cells were treated with fisetin for 48 h and then nuclear lysates were prepared. p300 was immunoprecipitated, and interaction with acetylated p65 and TNF-α was assessed by western blotting. The immunoblots shown here are representative of 3 independent experiments.

Figure 6

Effect of fisetin on chromatin event at the promoter of inflammatory gene. ChIP assays were performed using MAGnify ChIP according to the manufacturer's instructions. Immunoprecipitations were performed overnight at 4°C with 5 μg of p300 antibody. DNA was subjected to PCR. ChIP assays showed the recruitment of p300 to the TNF-α promoters. Results of 1 typical experiment of 3 are shown. Values from ChIP with anti-p300 antibody represent the fold difference relative to those from IgG control antibody. ^†† P < 0.01 compared to NG; *P < 0.05; **P < 0.01 compared to HG. Results are shown as mean ± SD for 3 different experiments.