Abstract
Elevated homocysteine (Hcy) levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on cortical neurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG). Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO). Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive) and hardly in astrocytes (GFAP-positive). 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA). Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.
Highlights
Stroke is one of the world’s leading causes of death and disability
Data are expressed as xs (n = 5). a p < 0.05 vs. the sham operation control (SHAM) group, b p < 0.05 vs. middle cerebral artery occlusion-reperfusion (MCAO) group, * p < 0.05 vs. the concentration of Hcy before intervention
Our results suggested that autophagy plays a crucial role in Hcy-induced injury of cells, and the oxidative damage may be involved in the mechanism
Summary
Stroke is one of the world’s leading causes of death and disability. Ischemic stroke, which accounts for approximately 85% of all strokes, occurs when there is an acute blockage of arterial blood flow to the brain tissue [1]. It has been reported that the underlying molecular mechanisms for the neurotoxic effects of Hcy may involve the excessive activation of glutamate receptors, a rise in free calcium concentration, the disruption of DNA and the generation of reactive oxygen species (ROS) [6,7,8]. Despite this valuable study evidence, the understanding of Hcy toxicity remains incomplete. We hypothesize that the changes in autophagy level may be involved in Hcy-induced brain damage after ischemia stroke, and ROS may be a possible link between Hcy and autophagy. We provide evidence that Hcy-triggered autophagy contributes to brain neuronal cell injury and show it was accompanied by oxidative damage
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