Abstract

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.

摘要

镉 (Cd) 对骨骼有直接的毒性作用。辛伐他汀等他汀类药物对各种疾病具有保护作用，包括对组织损伤的保护作用。目前的研究揭示了辛伐他汀对镉诱导的前成骨细胞损伤的疗效。前成骨细胞 MC3T3-E1 细胞与不同剂量的 CdCl ₂孵育12 h、24 h 和 48 h，然后分别使用 MTT 法和流式细胞术评估细胞毒性。通过蛋白质印迹和qRT-PCR评估Nox4的表达水平。显微镜下观察MC3T3-E1细胞形态。暴露于 CdCl _2的细胞(5 µM) 用不同剂量的辛伐他汀进一步处理，随后测量细胞活力、细胞凋亡和 Nox4 的表达。此外，为了确认辛伐他汀对 Cd 诱导的前成骨细胞损伤的保护作用，在用辛伐他汀 (10 ^-8 M)、CdCl ₂ (5 µM) 和过表达 Nox4 进行相应的细胞处理后进行了功能性拯救测定。通过蛋白质印迹和qRT-PCR测量细胞凋亡相关标志物的表达。结果表明，CdCl ₂引起MC3T3-E1细胞损伤，因为细胞活力降低，细胞凋亡增加。_{Nox4的表达随着CdCl 2}的增加而上调浓度。辛伐他汀增加了细胞活力，缓解了细胞凋亡和先前因 CdCl ₂增加的 Nox4 表达。CdCl ₂对 MC3T3-E1 细胞和 Nox4 表达的影响可被辛伐他汀减弱，并被 Nox4 过表达促进。目前的研究发现辛伐他汀通过Nox4保护Cd诱导的前成骨细胞损伤，因此可作为治疗镉诱导的骨损伤的潜在药物。