Beneficial effects of δ-tocotrienol against oxidative stress in osteoblastic cells: studies on the mechanisms of action

Purpose Natural antioxidants are considered as promising compounds in the prevention/treatment of osteoporosis. We studied the ability of purified delta-tocotrienol (delta-TT) isolated from a commercial palm oil (Elaeis guineensis) fraction to protect osteoblast MC3T3-E1 and osteocyte MLO-Y4 cells againsttert-butyl hydroperoxide (t-BHP)-induced oxidative damage and the mechanisms involved in its protective action in MC3T3-E1. Methods MC3T3-E1 and MLO-Y4 cells were treated with delta-TT (1.25-20 mu g/ml for 2 h) followed byt-BHP at 250 mu M or 125 mu M for 3 h, respectively. MTT test was used to measure cell viability. Apoptotic cells were stained with Hoechst-33258 dye. Intracellular ROS levels were measured by dichlorofluorescein CM-DCFA. The OPT fluorimetric assay was used to detect the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) contents. Results delta-TT significantly prevented the effects oft-BHP on cell viability and apoptosis reaching a maximum protective activity at 10 and 5 mu g/ml in MC3T3-E1 and MLO-Y4 cells, respectively. This protective effect was due to a reduction of intracellular ROS levels and an increase in the defense systems shown by the increase in the GSH/GSSG. GSH loss induced by an inhibitor of GSH synthesis significantly reduced the delta-TT-positive effect on ROS levels. delta-TT prevention of oxidative damage was completely removed by combined treatment with the specific inhibitors of PI3K/AKT (LY294002) and Nrf2 (ML385). Conclusions The delta-TT protective effect against oxidative damage in MC3T3-E1 cells is due to a reduction of intracellular ROS levels and an increase of the GSH/GSSG ratio, and involves an interaction between the PI3K/Akt-Nrf2 signaling pathways.