DL-3-n-butylphthalide protects endothelial cells against oxidative/nitrosative stress, mitochondrial damage and subsequent cell death after oxygen glucose deprivation in vitro.

DL-3-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Recently, we found that NBP can reduce the incidence of stroke and have protective action on cerebral microvessels, suggesting a direct action of NBP on endothelial cells. However, it is difficult to evaluate the direct action of NBP on endothelial cells in vivo because of the interactions of endothelial cells with other types of neuronal cells. Therefore, we investigated whether NBP protects against oxygen glucose deprivation (OGD)-induced cell injury in an immortalized human umbilical vein endothelial cells (HUVEC) in vitro. Cells were exposed to OGD, leading to endothelial damage. Endothelial injury was assessed by measuring MTT and the changes in chromatin morphology. Mitochondrial superoxide, mitochondrial membrane potential and mitochondrial morphology were assessed using MitoSOX Red. Rhodamine 123 and MitoTracker, respectively. Nitrosative stress was assessed by measuring the production of peroxynitrite. The activity of superoxide dismutase (SOD) is evaluated using SOD assay kit-WST. The expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was assessed at the protein level by immunofluorescence and Western blotting. NBP at doses between 0.01 and 100 micromol/L dose-dependently protected against OGD-induced cell death. In addition, NBP attenuated OGD-induced mitochondria superoxide, cellular formation of peroxynitrite, and decrease in SOD activity, mitochondria fragmentation and loss of mitochondrial membrane potential. In parallel, NBP enhanced OGD-induced HIF-1alpha expression. This study demonstrates that NBP can protect HUVEC against OGD-induced oxidative/nitrosative stress, mitochondrial damage and subsequent cell death. This protective effect is, at least in part, associated with its enhancement on OGD-induced HIF-1alpha expression.