Neuroprotective Effects of Pinelliae Rhizoma Water-Extract by Suppression of Reactive Oxygen Species and Mitochondrial Membrane Potential Loss in a Hypoxic Model of Cultured Rat Cortical Cells.

Abstract

stress by free radicals is a major cause of neuronal cell death. Excitotoxicity in hypoxia/ischemia causes an increase in reactive oxygen species (ROS) and a loss of mitochondrial membrane potential (MMP), resulting in dysfunction of the mitochondria and cell death. Pinelliae Rhizoma (PR) is a traditional medicine for incipient stroke. We investigated the effects of PR Water-Extract on the modulation of ROS and MMP in a hypoxic model using cultured rat cortical cells. PR Water-Extract was added to the culture medium at various concentrations (0.25～5, 5.0 µg/ml) on day in vitro 12(DIV12), given a hypoxic shock (2% O2/5% CO2, 37 o C, 3 hr), and cell viability was assessed on DIV15 by Lactate Dehydrogenase Assay (LDH assays). PR Water-Extract showed a statistically significant effect on neuroprotection (10～15% increase in viability; p<0.01) at 1.0 and 2.5 µg/ml in normoxia and hypoxia. Measurement of ROS production by H2DCF-DA stainings showed that PR Water-Extract efficiently reduced the number and intensity of ROS-producing neurons, especially at 1 hr post shock and DIV15. When MMP was measured by JC-1 stainings, PR Water-Extract efficiently maintained high-energy charged mitochondria. These results indicate that PR Water-Extract protects neurons in hypoxia by preventing ROS production and preserving the cellular energy level.