枸橼酸铁铵通过提高ROS水平激活MAPK通路并诱导成骨细胞凋亡

 目的：探讨铁超载提高人成骨细胞(hFOB1.19)活性氧（ROS）水平在激活丝裂原活化蛋白激酶（MAPK）通路和诱导细胞凋亡中的作用。方法：采用细胞贴壁法培养成骨细胞hFOB1.19，将不同浓度枸橼酸铁铵(100、300、500 μmol/L)加入细胞培养基，用MTT法检测成骨细胞增殖活性；DCFH-DA荧光探针检测成骨细胞ROS水平；Annexin V-FITC/PI法检测细胞凋亡；Western blotting检测MAPK通路相关蛋白。结果：枸橼酸铁铵处理成骨细胞后，细胞增殖活性明显降低，早期凋亡和总死亡率显著增加；不同浓度的枸橼酸铁铵处理成骨细胞后，其ROS水平分别增高至(35.73±2.52)%、(62.89±4.24)%和(76.06±3.55)%，MAPK通路相关蛋白的表达也明显增加并呈剂量效应关系，抗氧化剂N-乙酰半胱氨酸可以在降低ROS水平及MAPK通路相关蛋白表达的同时抑制枸橼酸铁铵所致的细胞凋亡。结论：枸橼酸铁铵能使成骨细胞增殖受到抑制，并且通过增加细胞内ROS水平，激活MAPK通路，诱导成骨细胞凋亡。

Overload of ferric ammonium citrate triggers MAPK pathways by producing high level of reactive oxygen species and induces apoptosis of human hFOB1.19 osteoblast cells

AIM：To investigate the role of reative oxygen species (ROS) generated by iron overload in activating the mitogen-activated protein kinase (MAPK) pathways and apoptosis. METHODS：Cultured human osteoblast cell line hFOB1.19 was treated with ferric ammonium citrate (FAC) at concentrations of 0~500 μmoL/L. The proliferation of hFOB1.19 cells was analyzed by MTT assay. Apoptosis was detected by flow cytometry with Annexin V/PI staining. The expression levels of p-ERK, p-JNK and p-p38 were determined by Western blotting 24 h after treatment with FAC. RESULTS：After treated with FAC, the cell proliferation was inhibited. The early apoptosis and total cell death were significantly increased. The levels of ROS were increased to (35.73±2.52)%, (62.89±4.24)% and (76.06±3.55)% with the increasing doses of FAC treatmen,respectively. The expression levels of p-ERK, p-JNK and p-p38 were also remarkably elevated in FAC groups. CONCLUSION：Iron overload increases intracellular ROS level, thus triggering the MAPK pathways and inducing apoptosis of human hFOB1.19 osteoblast cells.