NADPH oxidase isoforms are involved in glucocorticoid-induced osteoblast apoptosis

[Speaker] Wenhao Cui:1
[Co-author] Shucai Bai:2, Huafeng Zhang:2
1:Kyoto Prefectural University of Medicine, Japan, 2:Tianjin Medical University General Hospital, China

Oxidative stress induced by long-term glucocorticoids (GCs) use weakens the repair capacity of bone tissue. Accumulating evidence has indicated that reactive oxygen species (ROS) are closely related to osteoblast cell death. However, the specific mechanism remains unclear.
Methods: Murine pre-osteoblast MC3T3-E1 cells were used to establish a model of osteoblast culture in vitro. To establish the high-dose dexamethasone environment, the cells were incubated with 100 nM, 250 nM, 500 nM, or 1000 nM dexamethasone for 24 hr.
MTT assay showed that dexamethasone could induce cell death in a dose-dependent manner in MC3T3-E1 cells, accompanied with ROS generation of same pattern. Addition of the ROS inhibitor NAC (N-acetyl-L-cysteine) or NOX inhibitor DPI (diphenylene iodonium) significantly deceased the apoptosis of osteoblast, indicating that dexamethasone induced cell death is NOX-derived-ROS dependent. mRNA expression level of Nox1, Nox2, and Nox4 and protein expression of NOX2 and NOX4 were analyzed. Nox1 and Nox4 mRNA expression was elevated in a dose-dependent manner after treated by dexamethasone, where knockdown of NOX1 and NOX4 by siRNAs significantly inhibit the apoptosis of osteoblast. To clarify the signaling pathway in ROS-induced osteoblast apoptosis, mitogen-activated protein kinase (MAPK) signaling molecules were analyzed. Phosphorylated ASK1 and p38 levels were significantly elevated after dexamethasone stimulation, which were markedly attenuated by NAC or DPI. However, there was no change in the mRNA and total protein levels of ASK1 and p38 with/without dexamethasone stimulation.
NOX1- and NOX4-derived ROS plays a pivotal role in the dexamethasone-induced osteoblast apoptosis via regulating phosphorylation of ASK1 and p38. This study provide a new therapeutic strategy for glucocorticoids induced bone diseases.

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