Cadmium Promotes Ferroptosis in Osteoblasts by Activating the Ferritinophagy
LOU Kai1#, SHEN Fangmin1#, ZHANG Tongtong2, ZHANG Zhiming1, CHEN Yihang1, WU Xinran1, ZHANG Yun1*
This study aimed to investigate whether Cd (cadmium) induced ferroptosis of rat primary osteoblasts and to explain the underlying molecular mechanisms. Rat primary osteoblasts isolated in vitro were treated with Cd (0.25-10 μmol/L). Cell viability and cytotoxicity were evaluated by MTT and colorimetric assays to determine the optimal concentrations of Cd. Based on the IC50 value, osteoblasts were randomly assigned to six groups: Control (normal) group, L-Cd (low-dose Cd, 0.5 μmol/L) group, M-Cd (medium-dose Cd, 1.0 μmol/L) group, H-Cd (high-dose Cd, 2.0 μmol/L) group, Fer-1+H-Cd (the ferroptosis inhibitor ferrostatin-1+H-Cd) group, and BafA1+H- Cd (the autophagy inhibitor bafilomycin A1+H-Cd) group. To elucidate the regulatory effects of Fer-1 or BafA1 on Cd-induced ferritinophagy and ferroptosis in osteoblasts, cells in the H-Cd group were pretreated with the two in- hibitors, respectively. Intracellular levels of free Fe2+ and Lip ROS (lipid reactive oxygen species) were determined by FeRhoNox-1 and C11-BODIPY 581/591 staining, respectively. Intracellular contents of MDA (malondialdehyde) and GSH (glutathione) were detected by colorimetric assays. The mRNA expression levels of SLC7A11 (solute carrier family 7 member 11), GPX4 (glutathione peroxidase 4), and ACSL4 (acyl-CoA synthetase long-chain family member 4) were measured via RT-qPCR (reverse transcription-quantitative polymerase chain reaction). Protein expression of SLC7A11, GPX4, ACSL4, LC-3 (microtubule-associated protein 1 light chain 3), NCOA4 (nuclear receptor coactivator 4), LAMP2 (lysosome-associated membrane protein 2) and FTH1 (ferritin heavy chain 1) were examined by Western blot. The punctate accumulation of LC-3B and the colocalization of FTH1 with lysosomes were observed by immunofluorescence staining. Results showed that compared with the control group, the osteoblasts viability, GSH content, mRNA levels of SLC7A11 and GPX4, as well as protein expression levels of SLC7A11, GPX4, LAMP2 and FTH1 were significantly decreased in the M-Cd and H-Cd-treated groups (P<0.01); whereas, the intracellular levels of free Fe2+, Lip ROS and MDA, mRNA level of ACSL4, protein expression levels of ACSL4 and NCOA4, as well as the LC-3II/LC-3I value were significantly increased (P<0.01); meanwhile,the number of autophagic vacuoles, LC-3B protein expression, and the accumulation of FTH1 in lysosomes were remarkably enhanced in the Cd-treated cells. Compared with the H-Cd group, the intracellular levels of free Fe2+ and Lip ROS, as well as the LC-3II/LC-3I value, were significantly reduced in the Fer-1 or BafA1-treated groups; protein expression of NCOA4, FTH1, and GPX4, along with osteoblasts viability, was markedly restored (P<0.01). Collectively, these results suggest that Cd induces ferroptosis in osteoblasts, which is closely associated with the activation of the ferritinophagy.
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