Advanced oxidation protein products inhibit proliferation and differentiation of rat osteoblast-like cells via NF-κB pathway

Abstract

Background/Aims: Advanced oxidation protein products (AOPPs), a novel marker for oxidative protein damage, regulated cell behavior in many cell types. Although previous reports have revealed that oxidative stress resulted in an increased AOPPs content in cultured osteoblast-like cells, the effects of AOPPs on osteoblast-like cell function remain unclear. This study aimed to investigate the effect of AOPPs on the proliferation and differentiation of rat osteoblast-like (ROB) cells in vitro. Methods: ROB cells isolated from calvarias of newborn rats were incubated with AOPPs-modified rat serum albumin (AOPPs-RSA) prepared in vitro by incubation of RSA with hypochlorous acid. Cell proliferation was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Alkaline phosphatase activity was measured. Expression of osteocalcin mRNA and protein were examined using reverse transcriptase-polymerase chain reaction and Western blot, respectively. Reactive oxygen species (ROS) generation was detected by flow cytometry and fluorescent microscope. Phosphorylation of nuclear factor (NF)- κB p65 subunit was detected by Western blot. Results: Exposure of ROB cells to AOPPs-RSA significantly inhibited cell proliferation, decreased alkaline phosphatase activity, down-regulated the expression of osteocalcin mRNA and protein in both a concentration- and time-dependent manner. AOPPs challenge induced ROS generation and NF-κB p65 phosphorylation, which were inhibited by superoxide dismutase, catalase and NADPH oxidase inhibitors diphenyleneiodonium and apocynin. Furthermore, NF-κB inhibitor SN50 could reverse the AOPPs-induced inhibition of ROB cell proliferation and differentiation. Conclusion: These results suggest that AOPPs can inhibit proliferation and differentiation of ROB cells through ROS-dependent NF-κB pathway. © 2009 S. Karger AG, Basel.