Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarin

Abstract

The present study investigated the protective actions of the antioxidant scutellarin against the cytotoxicity produced by exposure to H 2O 2 in PC12 cells. This was done by assaying for MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide) reduction and lactate dehydrogenase (LDH) release. Reactive oxygen species (ROS) and Ca 2+ in cells were evaluated by fluorescent microplate reader using DCFH and Fura 2-AM, respectively, as probes. Lipid peroxidation was quantified using thiobarbituric acid-reactive substances (TBARS). Mitochondrial membrane potential (MMP) was assessed by the retention of rhodamine123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. The DNA content and percentage of apoptosis were monitored with flow cytometry. Vitamin E, a potent antioxidant, was employed as a comparative agent. Preincubation of PC12 cells with scutellarin prevented cytotoxicity induced by H 2O 2. Intracellular accumulation of ROS, Ca 2+ and products of lipid peroxidation, resulting from H 2O 2 were significantly reduced by scutellarin. Incubation of cells with H 2O 2 caused a marked decrease in MMP, which was significantly inhibited by scutellarin. PC12 cells treated with H 2O 2 underwent apoptotic death as determined by flow cytometric assay. The percentage of this H 2O 2-induced apoptosis in the cells was decreased in the presence of different concentrations of scutellarin. Scutellarin exhibited significantly higher potency compared to the antioxidant vitamin E. The present findings showed that scutellarin attenuated H 2O 2-induced cytotoxicity, intracellular accumulation of ROS and Ca 2+, lipid peroxidation, and loss of MMP and DNA, which may represent the cellular mechanisms for its neuroprotective action.