Anti-apoptotic effect of taxodione on serum/glucose deprivation-induced PC12 cells death

Abstract

Taxodione, a diterpenoid from the roots of Salvia chorassanica Bunge, possesses cytotoxic, apoptotic, and antimicrobial activity. This study was designed to investigate the protective effects of taxodione on serum/glucose deprivation-induced ischemic injury in PC12 cells and related mechanisms. In an in vitro model of ischemia, PC12 cells were exposed to serum and glucose deprivation for 6 and 18 h. The protective effects of the methanol extract of S. chorassanica and taxodione were assessed using alamarBlue(®) assay. Intracellular ROS production was measured by fluorimetry using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The levels of PARP, Bcl-2, and Bax proteins were detected after western blot analysis. It was shown that taxodione (0.2-1.5 μM) significantly increased cell viability in a dose-dependent manner after ischemic insult. Taxodione has antioxidant activity and protects PC12 cells against oxidative stress-induced apoptotic cell death. Meanwhile, pretreatment with taxodione significantly induced an increase in Bcl-2 and a decrease in Bax protein level. The results of this study confirmed the protective effect of taxodione in serum/glucose deprivation-induced ischemic injury and the putative role of apoptosis as a underling mechanisms. Thus, it would be fair to consider taxodione as a promising ingredient of S. chorassanica for the expansion on novel class of anti-ischemic agents.

Fig. 1

Cytotoxicity of S. chorassanica methanol extract and taxodione on PC12 cells. S. chorassanica methanol extract (6.2–25.0 µg/ml) and taxodione (0.1–50.0 μM) were prepared in high-glucose (4.5 g/ml) DMEM supplemented with FBS. PC12 cells exposed to S. chorassanica methanol extract taxodione for 6 and 18 h. The incubation in the high-glucose (4.5 g/ml) DMEM supplemented with FBS during the whole treatment period served as control group, while the treatment only with serum-/glucose-free DMEM for 6 and 18 h served as SGD alone group. The cell viability was expressed as the percent (%) of the control value using alamarBlue^® assay. The data presented are mean ± SEM of three independent experiments (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control

Fig. 2

Protective effects of S. chorassanica methanol extract (6.2–25.0 µg/ml) and taxodione (0.1–50.0 μM) on SGD-induced cytotoxicity in PC12 cells. PC12 cells exposed to SGD for 6 and 18 h exhibited toxicity, reaching a maximal cytotoxicity after 18 h. Concomitantly, PC12 cells were pretreated with S. chorassanica methanol extract (6.2–25.0 µg/ml) and taxodione (0.1–50.0 μM) for 2 h, and then were exposed to SGD for an additional 6 and 18 h along with S. chorassanica methanol extract (6.2–25.0 µg/ml) and taxodione (0.1–50.0 μM). The incubation in the high-glucose (4.5 g/ml) DMEM supplemented with FBS during the whole treatment period served as control group, while the treatment only with serum-/glucose-free DMEM for 6 and 18 h served as SGD alone group. The cell viability was expressed as the percent (%) of the control value using alamarBlue^® assay. The data presented are mean ± SEM of three independent experiments (n = 3). ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 compared to SGD

Fig. 3

Fluorimetry with DCFH-DA staining for measuring ROS production for cultured PC12 cells. PC12 cells were pretreated with taxodione (0.1–50.0 μM) for 2 h, and then were exposed to SGD for an additional 2 and 4 h along with taxodione (0.1–50.0 μM). The incubation in the high-glucose medium during the whole treatment period served as control group, and the treatment only with serum-/glucose-free medium for 2 and 4 h served as SGD alone group. The ROS production was assessed according to changes in the fluorescence intensity of DCF, the oxidation product of DCFH-DA. The data presented are mean ± SEM of three independent experiments (n = 3). Results are mean ± SEM (n = 3). ^† P < 0.05, ^†† P < 0.01, and ^††† P < 0.001 compared to SGD

Fig. 4

Effect of taxodione on the expression of cleaved Bcl-2, and Bax protein by Western blot analysis. PC12 cells were pretreated with taxodione (0.1–50.0 μM) for 2 h, and then were exposed to SGD for an additional 18 h along with taxodione (0.1–50.0 μM). The incubation in the high-glucose medium during the whole treatment period served as control group, and the treatment only with serum-/glucose-free medium for 18 h served as SGD alone group. At the end of treatment, cells were harvested for western blotting and exposed to Bcl-2, and Bax antibodies. β-Actin was used as a loading control. The data presented are mean ± SEM of three independent experiments (n = 3). ^†† P < 0.01 compared to SGD