Baicalein Protects Against Retinal Ischemia by Antioxidation, Antiapoptosis, Downregulation of HIF-1α, VEGF, and MMP-9 and Upregulation of HO-1

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 mu M ascorbate and 5 mu M 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 mu M), vehicle (1% ethanol), or trolox (in vivo: 5 nmol; in vitro: 100 mu M 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-1 alpha (HIF-1 alpha), 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 Muller 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-1 alpha, 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 mu M baicalein and trolox (100 mu M 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-1 alpha, VEGF, and MMP-9. The antioxidative effect of baicalein would appear to play a minor role in downregulation of MMP-9.