Fig 2: Nano-ZnO-induced triglyceride (TG) deposition was closely related to the MTF-1-SHP pathway with or without Dynasore incubation. (A) The mRNA expression of MTF-1-SHP pathway genes. (B) Protein levels of SHP. (C) Quantification of SHP protein. Asterisks (*) indicate the significant differences between the two groups (p < 0.05). For each value, three biological replicates are undertaken.

Fig 3: Nano-Zn-induced up-regulation of PPAR? protein levels and TG content is related to nano-Zn absorption. (A) Protein levels of MTF1 after 40 µM nano-Zn incubation for 24 h. (B) Free Zn2+ was quantified by calculating the FL1 (green) mean fluorescence intensity of intestinal epithelial cells incubated for 24 h in 40 µM nano-Zn with 10 µg/mL CHL pretreatment for 2 h. (C) Representative confocal microscopy stained with Zn2+ fluorescent probe (Newport Green DCF), and the presence of DCF-stained Zn2+ was demonstrated by flow cytometric analysis of green (FL1) fluorescence intensity. The primary intestinal epithelial cells from P. fulvidraco were incubated for 24 h in ZnSO4 or 40 µM nano-Zn containing medium with or without 10 µg/mL CHL pretreatment for 2 h. (D) Protein levels of PPAR? of intestinal epithelial cells incubated for 24 h in control or 40 µM nano-Zn containing medium with or without 10 µg/mL CHL pretreatment for 2 h. (E) TG content after 24 h treatments. (F) Free fatty acid content after 24 h treatments. Values indicate means ± SEMs, n = 3–6. Asterisks (*) indicate significant differences between two groups (p < 0.05, n = 3). CHL, chlorpromazine.

Fig 4: TBHP treatment aggravated FPN downregulation in human NPCs by MTF1 suppression. (a, b) The human NPCs were treated with DMSO or TBHP (50 µM) for 12 h. The quantitative values are expressed as mean ± SD (n = 3); *p < 0.05. (a) MTF1 protein levels in cytosolic and nuclear fractions of human NPCs were determined using western blotting, which was, respectively, normalized to GADPH and Histone 3. (b) The human NPCs were fixed and stained with DAPI (nuclear) and MTF1 antibody. Representative images were shown to visualize the subcellular locations of MTF1. (c) The subcellular localization of MTF1 was performed in human NP tissues from the control (NC) groups and IVDD groups by immunohistochemistry analysis. (d, e) Lenti-Vector or Lenti-MTF1 infection was performed in human NPCs. The quantitative values are expressed as mean ± SD (n = 3); *p < 0.05. (d) MTF1 protein levels in cytosolic and nuclear fractions of human NPCs were determined using western blotting, which was, respectively, normalized to GADPH and Histone 3. (e) Representative western blotting assay and quantitation of the level of MTF1, FPN, and FTL proteins, which was normalized to ß-actin. (f–i) Lenti-Vector or Lenti-MTF1 infection and scrambled siRNA (siCON) or FPN siRNA (siFPN) transfection were performed before DMSO or TBHP treatment (50 µM) for 12 h. Data are expressed as mean ± SD (n = 3); *p < 0.05. (f) Intracellular total free iron levels in human NPCs were assayed using iron assay kit. (g, h) Lipid ROS levels were assayed using C11-BODIPY 581/591 using flow cytometry. (i) Cell viability was examined by the absorbance of CCK-8.

Fig 5: Ferroptosis occurred during IVDD, while administration of hinokitiol reduced the degree of disc degeneration in vivo. (a, b) Representative HE and SO staining images of the midsagittal sections of IVD specimens. (c) Histologic scores of the midsagittal sections of IVD specimens. Data are expressed as mean ± SD (n = 3); *p < 0.05, the saline group versus the control group; *p < 0.05, the DFO group/Fer-1 group/hinokitiol group versus the saline group. The interobserver error of each group is, respectively, 0.05 (control group), 0.02 (saline group), 0.05 (hinokitiol group), 0.03 (DFO group), and 0.07 (Fer-1 group). (d) Immunohistochemical staining for FTH, FPN, and MTF1 expressions in the rats' NP samples. Data are expressed as mean ± SD (n = 3); *p < 0.05, the saline group versus the control group; *p < 0.05, the hinokitiol group versus the saline group.