Nutr Res Pract.  2023 Feb;17(1):164-173. 10.4162/nrp.2023.17.1.164.

Sodium butyrate inhibits high glucose-induced inflammation by controlling the acetylation of NF-κB p65 in human monocytes

• Moon HR
• Yun JM

• KMID: 2539449
• DOI: http://doi.org/10.4162/nrp.2023.17.1.164

Abstract

BACKGROUND/OBJECTIVES
Hyperglycemia is a major cause of diabetes and diabetesrelated diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells.
MATERIALS/METHODS
THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0–25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay.
RESULTS
HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α.
CONCLUSIONS
These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

Keyword

Sodium butyrate; cytokines; inflammation; histone deacetylase; high glucose

Figure

• 

  Fig. 1 Cytotoxicity of NaB to cultured HG-treated THP-1 cells. (A) Chemical structure of NaB. (B) The effect of NaB on the cell viability after 48 h was evaluated by the CCK-8 assay. Human monocytic THP-1 cells (1 × 105 cells/mL) were cultured in the presence of an osmolar control (14.5 mmol/L mannitol), under NG (5.5 mmol/L glucose) or HG (20 mmol/L glucose) conditions, or in the absence or presence of NaB (0, 1, 3, 10, 30 μM) for 48 h. The cell culture media was collected for the measurement of cytokine release. The data represent the mean ± SD of 3 independent experiments.CCK-8, Cell Counting Kit-8; NaB, sodium butyrate; Man, mannitol; NG, normoglycemia; HG, high glucose.

• 

  Fig. 2 NaB-mediated inhibition of cytokine release in HG-treated THP-1 cells. (A) Cells (1 × 105 cells/mL) were treated with NaB for 48 h, and the mRNA levels were evaluated by a quantitative real-time polymerase chain reaction. (B) Cell lysates were prepared, and TNF-α levels were evaluated by western blot analysis, as described in the Materials and Methods section. Equal protein loading was confirmed by stripping the immunoblot and reprobing it for β-actin protein. The immunoblots shown here are representative of 3 independent experiments. (C) TNF-α in the cell media was measured using an ELISA assay kit. The cytokine levels in the media were measured using an ELISA assay kit according to the manufacturer’s instructions. The values were calculated based on the assay’s standard curve. The data represent the mean ± SD of 3 independent experiments.NaB, sodium butyrate; Man, mannitol; NG, normoglycemia; HG, high glucose; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; ELISA, enzyme-linked immunosorbent assay.##P < 0.01 compared with NG; *P < 0.05, **P < 0.01 compared with HG.

• 

  Fig. 3 Effect of NaB on the HAT and HDAC activity as well as p300 and Acetyl CBP/p300 levels in HG-treated THP-1 cells. The cells were harvested after 48 h of the NaB treatment, and nuclear lysates were prepared. The samples were analyzed to determine the HAT (A) and HDAC activity (B). After nuclear protein extraction, the p300 and Acetyl CBP/p300 levels (C) were evaluated by western blot analysis. Densities of (D) Acetyl CBP/p300, (E) p300 normalized to β-actin using ImageJ software. The data represent the mean ± SD of 3 independent experiments.NaB, sodium butyrate; Man, mannitol; NG, normoglycemia; HG, high glucose; HAT, histone acetyltransferase; HDAC, histone deacetylase; CBP/p300, CREB-binding protein/p300; OD, optical density; Acetyl, acetylated.#P < 0.05, ##P < 0.01 compared with NG; *P < 0.05 compared with HG.

• 

  Fig. 4 NaB-induced suppression of NF-κB activation in HG-treated THP-1 cells. (A) The protein levels were evaluated by western blot for NF-κB p65 and Acetyl p65. An equal protein loading was confirmed by stripping the immunoblot and reprobing it for the histone 2A protein. Densities of (B) NF-κB p65, (C) Acetyl p65 normalized to β-actin using ImageJ software. The data represent the mean ± SD for 3 independent experiments.NaB, sodium butyrate; Man, mannitol; NG, normoglycemia; HG, high glucose; NF-κB, nuclear factor-κB; Acetyl, acetylated.##P < 0.01 compared to NG; **P < 0.01 compared with HG.

• 

  Fig. 5 NaB-induced interaction of p300 with Acetyl p65 and TNF-α in HG-treated THP-1 cells. (A) The cells were treated with NaB for 48 h, and nuclear lysates were prepared. p300 was immunoprecipitated, and the interaction with Acetyl p65 and TNF-α was assessed by western blotting. The immunoblots shown here are representative of 3 independent experiments. (B) Quantitative representation of Acetyl p65 and TNF-α in p300. The data represent mean ± SD for 3 independent experiments.NaB, sodium butyrate; Man, mannitol; NG, normoglycemia; HG, high glucose; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; Acetyl, acetylated.##P < 0.01 compared with NG; **P < 0.01 compared with HG.

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