Nutr Res Pract.  2021 Oct;15(5):591-603. 10.4162/nrp.2021.15.5.591.

Hesperetin suppresses LPS/high glucose-induced inflammatory responses via TLR/MyD88/NF-κB signaling pathways in THP-1 cells

Affiliations
  • 1Department of Food and Nutrition, Chonnam National University, Gwangju 61186, Korea
  • 2Department of Education, Graduate School of Education, Chonnam National University, Gwangju 61186, Korea

Abstract

BACKGROUND/OBJECTIVES
Unregulated inflammatory responses caused by hyperglycemia may induce diabetes complications. Hesperetin, a bioflavonoid, is a glycoside in citrus fruits and is known to have antioxidant and anticarcinogenic properties. However, the effect of inflammation on the diabetic environment has not been reported to date. In this study, we investigated the effect of hesperetin on proinflammatory cytokine secretion and its underlying mechanistic regulation in THP-1 macrophages with co-treatment LPS and hyperglycemic conditions.
MATERIALS/METHODS
THP-1 cells differentiated by PMA (1 μM) were cultured for 48 h in the presence or absence of hesperetin under normoglycemic (5.5 mM/L glucose) or hyperglycemic (25 mM/L glucose) conditions and then treated with LPS (100 ng/mL) for 6 h before harvesting. Inflammation-related proteins and mRNA levels were evaluated by enzyme-linked immunosorbent assay, western blot, and quantitative polymerase chain reaction analyses.
RESULTS
Hesperetin (0–100 μM, 48 h) treatment did not affect cell viability. The tumor necrosis factor-α and interleukin-6 levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions, and these increases were decreased by hesperetin treatment. The TLR2/4 and MyD88 activity levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions; however, hesperetin treatment inhibited the TLR2/4 and MyD88 activity increases. In addition, nuclear factor-κB (NF-κB) and Acetyl-NF-κB levels increased in response to treatment with LPS under hyperglycemic conditions compared to normoglycemic conditions, but those levels were decreased when treated with hesperetin. SIRT3 and SIRT6 expressions were increased by hesperetin treatment.
CONCLUSIONS
Our results suggest that hesperetin may be a potential agent for suppressing inflammation in diabetes.

Keyword

Diabetes mellitus; hesperetin; inflammation; macrophages; NF-kappa B

Figure

  • Fig. 1 Induction of COX-2 expression by LPS (A) or glucose (B) and the cytotoxic range of hesperetin (C) in LPS-treated THP-1 macrophages. COX-2 expression by glucose (0, 5.5, 20, and 25 mM/L) in LPS (100 ng/mL) was determined by western blotting. Effect of hesperetin on cell viability of THP-1 macrophages under LPS and hyperglycemic condition after 48 h was evaluated by performing 5-diphenyltetrazoliumbromide assays. The data are representative of at least three independent experiments.COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose.

  • Fig. 2 Effect of hesperetin on cytokine release in LPS-treated THP-1 macrophages under hyperglycemic conditions. Cytokine levels in cell culture media were measured using enzyme-linked immunosorbent assay kits according to the manufacturer's instructions (A, B). Values were calculated based on the assay's standard curve. IL-6 levels were evaluated by western blot analysis (C). The data are representative of at least three independent experiments.IL-6, interleukin-6; NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; H0, LPS + hyperglycemic + hesperetin 0 μM.##P < 0.01 compared with NG; **P < 0.01 compared with H0.

  • Fig. 3 Effect of hesperetin on TLR2, TLR4, and MyD88 gene expressions in LPS-treated THP-1 macrophage under hyperglycemic conditions. Cells were treated with hesperetin (0, 10, 25, and 50 μM) for 48 h, and mRNA expressions of TLR2, TLR4, and MyD88 were evaluated by quantitative polymerase chain reaction (A, B, C). The data are representative of at least three independent experiments. Data were analyzed using the 2−ΔΔCT method.NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose; LPS, lipopolysaccharide; H0, LPS + hyperglycemic + hesperetin 0 μM.#P < 0.05, ##P < 0.01 compared with NG; **P < 0.01 compared with H0.

  • Fig. 4 Effect of hesperetin on NF-κB protein and mRNA expressions in LPS-treated THP-1 macrophages under hyperglycemic conditions. NF-κB levels were evaluated by western blot analysis of cell lysates (A). The percentage of NF-κB (whole)/β-actin (B) and the percentage of NF-κB (nuclear)/TBP (C) were revealed by image J. Cells (1 × 106 cells/mL) were treated with hesperetin for 48 h, and NF-κB mRNA levels were evaluated by qPCR (D). Immunofluorescence images show downregulation of NF-ĸB (E) and Ac-NF-ĸB (F) in LPS-treated THP-1 macrophages under hyperglycemic conditions following hesperetin treatment. Differentiated THP-1 cells were cultured in the presence of LPS (100 ng/mL) under normoglycemic or hyperglycemic conditions in the absence or presence of hesperetin (50 μM) for 48 h (400 × Magnification). Signal quantification was performed using ImageJ software. The data are representative of at least three independent experiments. Data were analyzed using the 2−ΔΔCT method.NF-κB, nuclear factor-κB; TBP, TATA-binding protein; NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose; LPS, lipopolysaccharide; H0, LPS + hyperglycemic + hesperetin 0 μM; DAPI, 4′,6-diamidino-2-phenylindole.##P < 0.01 compared with NG; **P < 0.01 compared with H0.

  • Fig. 5 Modulation of SIRT3 and SIRT6 protein expressions by hesperetin in LPS-treated THP-1 macrophages under hyperglycemic conditions. SIRT3 and SIRT6 protein levels were evaluated by western blot analysis and normalized to TBP (A). SIRT3 and SIRT6 mRNA levels were evaluated by quantitative polymerase chain reaction (B, C). The data are representative of at least three independent experiments. Data were analyzed using the 2−ΔΔCT method.NG, normoglycemic 5.5 mM/L glucose; HG, hyperglycemic 25 mM/L glucose; LPS, lipopolysaccharide; H0, LPS + hyperglycemic + hesperetin 0 μM; TBP, TATA-binding protein.##P < 0.01 compared with NG; *P < 0.01 compared with H0.


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