Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation
Abstract
Aim: To investigate the protective effects of simvastatin (Sim) combined with nifedipine (Nif) on endothelial cells and elucidate the action mechanism.
Methods: Human umbilical vein endothelial cells (HUVEC) were used. mRNA and protein levels were measured by using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Intracellular calcium and reactive oxygen species (ROS) were detected using confocal microscopy. The Griess assay was used to evaluate nitric oxide (NO) release.
Results: Treatment of HUVEC with H2O2 100 μmol/L for 30 min inhibited the mRNA and protein expression of endothelial nitric oxide synthase (eNOS). With increased concentrations of Nif, eNOS mRNA and protein levels increased (P<0.05). Combined treatment with Sim 1.0 μmol/L and Nif 1.0 μmol/L significantly increased the mRNA and protein expression of eNOS and NO release compared with Sim or Nif alone (P<0.05). The combination significantly lowered the intracellular ROS level (P<0.05), which was correlated with the increase in eNOS and NO, but there was no visible change in intracellular calcium (P>0.05). Compared with individual drug treatment, Akt phosphorylation and the ratio of p-eNOS/eNOS were up-regulated in the combination group, and this effect was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002.
Conclusion: The Sim-Nif combination effectively protects HUVEC against H2O2 injury by inhibiting intracellular ROS generation, increasing the ratio of p-eNOS/eNOS and up-regulating Akt phosphorylation.
Keywords:
Methods: Human umbilical vein endothelial cells (HUVEC) were used. mRNA and protein levels were measured by using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Intracellular calcium and reactive oxygen species (ROS) were detected using confocal microscopy. The Griess assay was used to evaluate nitric oxide (NO) release.
Results: Treatment of HUVEC with H2O2 100 μmol/L for 30 min inhibited the mRNA and protein expression of endothelial nitric oxide synthase (eNOS). With increased concentrations of Nif, eNOS mRNA and protein levels increased (P<0.05). Combined treatment with Sim 1.0 μmol/L and Nif 1.0 μmol/L significantly increased the mRNA and protein expression of eNOS and NO release compared with Sim or Nif alone (P<0.05). The combination significantly lowered the intracellular ROS level (P<0.05), which was correlated with the increase in eNOS and NO, but there was no visible change in intracellular calcium (P>0.05). Compared with individual drug treatment, Akt phosphorylation and the ratio of p-eNOS/eNOS were up-regulated in the combination group, and this effect was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002.
Conclusion: The Sim-Nif combination effectively protects HUVEC against H2O2 injury by inhibiting intracellular ROS generation, increasing the ratio of p-eNOS/eNOS and up-regulating Akt phosphorylation.