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Objective@#To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action.@*Methods@#MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e-D/D0)N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3’-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data.@*Results@#Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003).@*Conclusion@#Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.
RESUMO
<p><b>OBJECTIVE</b>To investigate the effects and related mechanism of simvastatin on oxidized low density lipoprotein(ox-LDL) induced oxidative stress in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>HUVECs were cultured in 6 different culture media: control, ox-LDL, ox-LDL+vehicle, ox-LDL+0.1 µmol/L simvastatin, ox-LDL+0.5 µmol/L simvastatin, ox-LDL+1.0 µmol/L simvastatin. HUVECs were incubated with ox-LDL (120 µg/ml) for 24 h in the presence or absence of different concentrations of simvastatin (0.1,0.5, 1.0 µmol/L) . The fluorescence intensity for reactive oxygen species (ROS) in HUVECs was measured by a laser confocal scanning microscopy and a microplate reader.NADPH oxidase activity was measured by lucigenin chemiluminescence. p22(phox), gp91(phox), p47(phox) and p67(phox) mRNA expression of HUVECs post various treatments was detected by RT-PCR. p22(phox) immunoprecipitates were immunoblotted for p47(phox) and total p22(phox) levels to identify p47(phox)/p22(phox) interaction.</p><p><b>RESULTS</b>Simvastatin attenuated ox-LDL induced ROS generation and NADPH oxidase activity in a concentration dependent manner (all P < 0.05). In addition, simvastatin significantly downregulated mRNA expression of p22(phox), gp91(phox), p47(phox) and p67(phox) (all P < 0.05), as well as the interaction of p47(phox)/p22(phox) (P < 0.01).</p><p><b>CONCLUSIONS</b>Simvastatin is an important regulator on NADPH subunits mRNA expressions and p47(phox)/p22(phox) interaction. Simvastatin attenuates ox-LDL-induced oxidative stress in HUVECs via reducing NADPH oxidase activity.</p>
