Nutr Res Pract.  2017 Oct;11(5):430-434. 10.4162/nrp.2017.11.5.430.

Luteolin and fisetin suppress oxidative stress by modulating sirtuins and forkhead box O3a expression under in vitro diabetic conditions

• Kim A
• Lee W
• Yun JM

• KMID: 2390136
• DOI: http://doi.org/10.4162/nrp.2017.11.5.430

Abstract

BACKGROUND
/OBJECTIVE: Chronic hyperglycemia induces oxidative stress via accumulation of reactive oxygen species (ROS) and contributes to diabetic complications. Hyperglycemia induces mitochondrial superoxide anion production through the increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This study aimed to determine whether fisetin and luteolin treatments suppress the oxidative stress by modulating the expression of sirtuins (SIRTs) and forkhead box O3a (FOXO3a) under hyperglycemic conditions in human monocytes.
MATERIALS/METHODS
Human monocytic cells (THP-1) were cultured under osmotic 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 and luteolin for 48 h. To determine the effect of fisetin and luteolin treatments on high glucose-induced oxidative stress, western blotting and intracellular staining were performed.
RESULTS
Hyperglycemic conditions increased the ROS production, as compared to normoglycemic condition. However, fisetin and luteolin treatments inhibited ROS production under hyperglycemia. To obtain further insight into ROS production in hyperglycemic conditions, evaluation of p47phox expression revealed that fisetin and luteolin treatments inhibited p47phox expression under hyperglycemic conditions. Conversely, the expression levels of SIRT1, SIRT3, SIRT6, and FOXO3a were decreased under high glucose conditions compared to normal glucose conditions, but exposure to fisetin and luteolin induced the expression of SIRT1, SIRT3, SIRT6, and FOXO3a. The above findings suggest that fisetin and luteolin inhibited high glucose-induced ROS production in monocytes through the activation of SIRTs and FOXO3a.
CONCLUSIONS
The results of our study supports current researches that state fisetin and luteolin as potential agents for the development of novel strategies for diabetes.

Keyword

Diabetes mellitus; oxidative stress; monocytes

MeSH Terms

Blotting, Western
Diabetes Complications
Diabetes Mellitus
Glucose
Humans
Hyperglycemia
In Vitro Techniques*
Luteolin*
Monocytes
NADP
Oxidative Stress*
Oxidoreductases
Reactive Oxygen Species
Sirtuins*
Superoxides
Glucose
Luteolin
NADP
Oxidoreductases
Reactive Oxygen Species
Sirtuins
Superoxides

Figure

• 

  Fig. 1 Cytotoxicity of fisetin and luteolin under HG conditions.Effect of fisetin and luteolin on cell viability after 48 h was evaluated by the CCK-8 assay. Human monocyte THP-1 cells (4 × 103 cells/well) were cultured under normoglycemic (NG, 5.5 mM/L glucose) or hyperglycemic (HG, 20mM/L glucose) conditions in a 96-well plate. Cells were treated with 3, 5 and 10 µM luteolin and fisetin for 48 h. Results are shown as mean ± SE of three independent experiments.

• 

  Fig. 2 Effects of luteolin and fisetin on O2- production in HG conditions by immunofluorescence. Cells were treated with appropriate concentrations of the phytochemicals, and fixed with 10% formaldehyde. The signal quantification was assessed by ImageJ software; magnification ×400. Man, mannitol condition; NG, normoglycemic condition; HG, hyperglycemic condition; L, luteoline; F, fisetin.

• 

  Fig. 3 Effects of luteolin and fisetin on p47phox expression in HG conditions.Cells were treated with 3, 5 and 10 µM luteolin (A) and fisetin (B) for 48 h. Protein levels were evaluated by western blot for p47phox. Equal loading of protein was confirmed by stripping the immunoblot and reprobing for β-actin protein. The immunoblots shown here are representative of three independent experiments. NG, normoglycemic condition; HG, hyperglycemic condition.

• 

  Fig. 4 Effects of luteolin and fisetin on SIRTs and FOXO3a gene expression under HG conditionsCells were treated with 3, 5 and 10 µM luteolin (A) and fisetin (B) for 48 h. Protein levels were evaluated by western blot for SIRT1, SIRT3, SIRT6 and FOXO3a. Equal loading of protein was confirmed by stripping the immunoblot and reprobing for TATA binding protein (TBP). The immunoblots shown here are representative of three independent experiments. NG, normoglycemic condition; HG, hyperglycemic condition.

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