The protective effects of simvastatin in Cadmium-Induced preosteoblast injury through Nox4.

Cadmium (Cd) has a direct toxic effect on bones. Statins such as simvastatin have protective effects on various diseases, including on tissue injury. The current study revealed the efficacy of simvastatin on Cd-induced preosteoblast injury. Preosteoblast MC3T3-E1 cells were incubated with various doses of CdCl₂ for 12 h, 24 h and 48 h, and then the cell cytotoxicity was assessed using MTT assay and flow cytometry, respectively. The expression level of Nox4 was assessed by Western blot and qRT-PCR. The morphological appearance of MC3T3-E1 cells was observed under a microscope. Cells exposed to CdCl₂ (5 µM) were further treated by simvastatin at various doses, subsequently cell viability, apoptosis and the expression of Nox4 were measured. Furthermore, to confirm the protective effects of simvastatin on Cd-induced pre-osteoblast injury, functional rescue assays were performed after corresponding cell treatment by simvastatin (10^-8 M), CdCl₂ (5 µM), and overexpression of Nox4. Expressions of cell apoptosis-related markers were measured by Western blot and qRT-PCR. The results revealed that CdCl₂ caused MC3T3-E1 cell injury because the cell viability was decreased and the apoptosis was increased. Nox4 expression was up-regulated with the increase of CdCl₂ concentrations. Simvastatin increased the cell viability, relieved the cell apoptosis and Nox4 expression previously increased by CdCl₂. The effects of CdCl₂ on MC3T3-E1 cells and Nox4 expression could be attenuated by simvastatin, and promoted by Nox4 overexpression. The current study found that simvastatin protects Cd-induced preosteoblast injury via Nox4, thus, it can be used as a potential drug for treating cadmium-induced bone injury.