Luteolin Attenuates Foam Cell Formation and Apoptosis in Ox-LDL-Stimulated Macrophages by Enhancing Autophagy

Cell Physiol Biochem. 2016;39(5):2065-2076. doi: 10.1159/000447902. Epub 2016 Oct 31.

Abstract

Background: Our previous studies demonstrated that luteolin, which is rich in flavones, has various biological properties and can exert anti-oxidant, anti-inflammatory and anti-apoptotic activities. However, its effect on ox-LDL-induced macrophage lipid accumulation and apoptosis has not been revealed.

Aims: This study aimed to explore the role of luteolin in ox-LDL-induced macrophage-derived foam cell formation and apoptosis and to delineate the underlying mechanism.

Methods: Murine RAW264.7 cells were stimulated with oxidized low-density lipoprotein (ox-LDL) (50 µg/ml) for 24 h and then pretreated with 25 µM luteolin for another 24 h. The effects of luteolin on lipid accumulation in RAW264.7 cells induced by ox-LDL were assayed using Oil red O staining and high performance liquid chromatography (HPLC). Apoptosis was confirmed by acridine orange/ethidium bromide (AO/EB) staining, flow cytometric analysis and the TUNEL assay. Immunofluorescence, Western blot and monodansylcadaverine (MDC) staining analyses were then used to further investigate the molecular mechanisms by which luteolin protects macrophages from ox-LDL-induced foam cell formation and apoptosis. 3-Methyladenine (3-MA), an autophagy inhibitor, was used as a positive control.

Results: Treatment with 25 µM luteolin not only significantly attenuated ox-LDL-induced macrophage lipid accumulation but also decreased the apoptotic rate of RAW264.7 cells, the number of TUNEL-positive macrophages and the expression of Bax, Bak, cleaved caspase-9 and cleaved caspase-3. In addition, luteolin pretreatment significantly increased autophagosome formation and Beclin-1 activity, thus increasing the ratio of LC3-II/LC3-I. Moreover, these effects were abolished by 3-MA.

Conclusions: Taken together, these results highlight that luteolin treatment attenuates foam cell formation and macrophage apoptosis by promoting autophagy and provide new insights into the molecular mechanism of luteolin and its therapeutic potential in the treatment of atherosclerosis.

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / pharmacology
  • Animals
  • Anti-Inflammatory Agents / pharmacology*
  • Antioxidants / pharmacology*
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • Autophagy / drug effects*
  • Autophagy / genetics
  • Azo Compounds
  • Beclin-1 / genetics
  • Beclin-1 / metabolism
  • Caspase 3 / genetics
  • Caspase 3 / metabolism
  • Caspase 9 / genetics
  • Caspase 9 / metabolism
  • Cell Differentiation / drug effects
  • Cell Line
  • Foam Cells / drug effects
  • Foam Cells / metabolism
  • Foam Cells / pathology
  • Gene Expression Regulation
  • Lipoproteins, LDL / antagonists & inhibitors*
  • Lipoproteins, LDL / pharmacology
  • Luteolin / pharmacology*
  • Macrophage Activation
  • Macrophages / cytology
  • Macrophages / drug effects*
  • Macrophages / metabolism
  • Mice
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Signal Transduction
  • bcl-2 Homologous Antagonist-Killer Protein / genetics
  • bcl-2 Homologous Antagonist-Killer Protein / metabolism
  • bcl-2-Associated X Protein / genetics
  • bcl-2-Associated X Protein / metabolism

Substances

  • Anti-Inflammatory Agents
  • Antioxidants
  • Azo Compounds
  • Bak1 protein, mouse
  • Bax protein, mouse
  • Beclin-1
  • Becn1 protein, mouse
  • Lipoproteins, LDL
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • bcl-2 Homologous Antagonist-Killer Protein
  • bcl-2-Associated X Protein
  • oxidized low density lipoprotein
  • 3-methyladenine
  • Casp3 protein, mouse
  • Casp9 protein, mouse
  • Caspase 3
  • Caspase 9
  • oil red O
  • Adenine
  • Luteolin