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ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death.

Oxidative medicine and cellular longevity (2020-10-31)
Qing Tian, Bo Qin, Yufan Gu, Lijun Zhou, Songfeng Chen, Song Zhang, Shuhao Zhang, Qicai Han, Yong Liu, Xuejian Wu
ABSTRACT

Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2',7'-ジクロロフルオレセイン二酢酸, ≥97%
Sigma-Aldrich
クエン酸アンモニウム, 三塩基酸, ≥97% (titration)
Sigma-Aldrich
JC-1, solid
Sigma-Aldrich
DL-システイン, technical grade
Sigma-Aldrich
アミロイドタンパク質非Aβ成分, ≥80% (HPLC)