Baicalein protects against retinal ischemia by antioxidation, antiapoptosis, downregulation of HIF-1α, VEGF, and MMP-9 and upregulation of HO-1

J Ocul Pharmacol Ther. Jul-Aug 2013;29(6):539-49. doi: 10.1089/jop.2012.0179. Epub 2013 Mar 28.

Abstract

Purpose: Retinal ischemia-associated ocular disorders are vision threatening. This study examined whether the flavonoid baicalein is able to protect against retinal ischemia/reperfusion.

Methods: Using rats, the intraocular pressure was raised to 120 mmHg for 60 min to induce retinal ischemia. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating dissociated retinal cells with 100 μM ascorbate and 5 μM FeSO4 (iron) for 1 h. The rats or the dissociated cells had been pretreated with baicalein (in vivo: 0.05 or 0.5 nmol; in vitro: 100 μM), vehicle (1% ethanol), or trolox (in vivo: 5 nmol; in vitro: 100 μM or 1 mM). The effects of these treatments on the retina or the retinal cells were evaluated by electrophysiology, immunohistochemistry, terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) staining, Western blotting, or in vitro dichlorofluorescein assay. In addition, real-time-polymerase chain reaction was used to assess the retinal expression of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), vascular endothelium growth factor (VEGF), and heme oxygenase-1 (HO-1).

Results: The retinal changes after ischemia included a decrease in the electroretinogram b-wave amplitude, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, an increase in vimentin immunoreactivity, which is a marker for Müller cells, an increase in apoptotic cells in the retinal ganglion cell layer linked to a decrease in the Bcl-2 protein, and changes in the mRNA levels of HIF-1α, VEGF, MMP-9, and HO-1. Of clinical importance, the ischemic detrimental effects were concentration dependently and/or significantly (0.05 nmol and/or 0.5 nmol) altered when baicalein was applied 15 min before retinal ischemia. Most of all, 0.5 nmol baicalein significantly reduced the upregulation of MMP-9; in contrast, 5 nmol trolox only had a weak attenuating effect. In dissociated retinal cells subjected to ascorbate/iron, there was an increase in the levels of reactive oxygen species, which had been significantly attenuated by 100 μM baicalein and trolox (100 μM or 1 mM; a stronger antioxidative effect at 1 mM).

Conclusions: Baicalein would seem to protect against retinal ischemia via antioxidation, antiapoptosis, upregulation of HO-1, and downregulation of HIF-1α, VEGF, and MMP-9. The antioxidative effect of baicalein would appear to play a minor role in downregulation of MMP-9.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / administration & dosage
  • Antioxidants / metabolism
  • Antioxidants / pharmacology
  • Antioxidants / therapeutic use*
  • Apoptosis / drug effects*
  • Cell Line
  • Down-Regulation
  • Flavanones / administration & dosage
  • Flavanones / pharmacology
  • Flavanones / therapeutic use*
  • Heme Oxygenase-1 / biosynthesis*
  • Hypoxia-Inducible Factor 1, alpha Subunit / biosynthesis*
  • Intravitreal Injections
  • Ischemia / metabolism
  • Ischemia / pathology
  • Ischemia / prevention & control*
  • Matrix Metalloproteinase 9 / biosynthesis*
  • Rats
  • Rats, Wistar
  • Retina / drug effects
  • Retina / metabolism
  • Retina / pathology
  • Retinal Diseases / metabolism
  • Retinal Diseases / pathology
  • Retinal Diseases / prevention & control*
  • Retinal Vessels / drug effects
  • Up-Regulation
  • Vascular Endothelial Growth Factor A / biosynthesis*

Substances

  • Antioxidants
  • Flavanones
  • Hif1a protein, rat
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, rat
  • baicalein
  • Heme Oxygenase-1
  • Matrix Metalloproteinase 9
  • Mmp9 protein, rat