Simvastatin attenuates TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells

Abstract

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Simvastatin is a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor known as a stimulator for bone formation via promoting osteoblast differentiation and mineralization. This study was designed to examine the role of simvastatin on TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in DMEM essential medium supplemented with 10% fetal bovine serum and antibiotics, and 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. A trypan blue exclusion assay was used to determine the level of cytotoxicity. Hoechst staining for cells treated with TNF-α was visualized under a microscope equipped with an epifluorescence illumination. Apoptosis of MC3T3-E1 cells was further examined by flow cytometric analysis after Annexin V/7-AAD double staining. MC3T3-E1 cells exposed to low concentrations (10−10–10−7 M) of simvastatin increased in both growth and viability. In contrast, high concentrations of simvastatin (10−6–10−5 M) have no effect on growth and viability of MC3T3-E1 cells. TNF-α-induced decrease in cell viability and growth were antagonized by simvastatin in a concentration-dependent manner. MC3T3-E1 cells treated with TNF-α at 10 ng/ml showed typical pyknotic fragmented nuclei and apoptotic bodies assessed by Hoechst staining. Moreover, Annexin V/7-AAD double staining indicated that MC3T3-E1 cells treated with 10 ng/ml of TNF-α resulted in a significant increase of the proportion of apoptotic cells up to 39.5% of total cell population, and the proportion of apoptotic cells were dose-dependently decreased after simvastatin treatment (10−10 – 10−7 M); the inhibitory rates were approximately 31.34, 48.5, 52.11, and 65.73%, respectively. Our data demonstrate that simvastatin increases growth and viability of osteoblastic cells and decreases TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells.