Toxicity and Mechanism of Polygoni Multiflori Radix Alcohol Extract on L02 Cells / 中国实验方剂学杂志

ABSTRACT

Objective::To study the toxic effect of Polygoni Multiflori Radix alcohol extract (PME) on L02 cells and the mechanism of ROS inducing apoptosis via mitochondria pathway, so as to provide a basis for the rational and safe administration of Polygoni Multiflori Radix in clinic. Method::The 4, 5-dimethly-2-thiazolyl-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect the cell viability of PME at different concentrations (5, 10, 20 g·L-1). Nuclear morphology was observed by Hoechst 33342 staining. The apoptosis rate of cells was detected by Annexin V-FITC/PI. The release rate of lactate dehydrogenase (LDH), the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the cells were detected by kit instruction. The changes of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were detected by flow cytometry. The relative protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), cysteinyl aspartate proteinase-9(proCaspase-9) and cysteinyl aspartate proteinase-3 (proCaspase-3) in the PME-administered group were detected by Western blot. Result::After treatment with PME at the concentration of 5, 10, 20 g·L-1, the survival rate of L02 cells were decreased in a concentration and time-depended manner. After treatment with PME for L02 cells, nucleus shrinkage, fragmentation and chromatin condensation were observed under fluorescence after Hoechst 33342 staining. Annexin V-FITC/PI double staining showed a upward cell apoptosis rate in PME 20 g·L-1 group. Compared with the normal control group, the release rate of LDH was significantly increased (P<0.01), the intracellular ROS level was significantly increased (P<0.01), and the SOD activity was significantly decreased (P<0.01), while the MMP rate was significantly decreased in PME 5, 10, 20 g·L-1 groups (P<0.05). With the increase in the concentration of PME, proCaspase-3, proCaspase-9, Bcl-2 protein showed a significantly downward trend in PME 10, 20 g·L-1 groups (P<0.01), while the expression of Bax protein was significantly up-regulated in PME 20 g·L-1 group (P<0.05). Conclusion::The study illustrated that PME have toxic effects on L02 cells, which may destroy the structure of hepatocytes to a certain extent, promote ROS levels, induce oxidative stress, activate the mitochondrial pathway, and then activate apoptosis-related proteins to cause cells damage. It is suggested that ROS-mediated mitochondrial pathway was involved in PME-induced apoptosis.