Inhibition of the 4-hydroxynonenal-regulated JNK/c-Jun pathway improves bleomycin-induced lung fibrosis

Figures

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  Fig. 1. 4-HNE was upregulated in bleomycin-induced lung fibrosis animal model. (A) Flowchart of experimental procedure. C57BL/6 mice were intratracheally administered 2 U/kg bleomycin (Bleo) or saline (CTR) on day 0. Mice were sacrificed on day 14, and lung tissues were stained with (B) Picro Sirius red or Masson's trichrome or subjected to and immunohistochemical staining to evaluate 4-HNE expression. Scale bars: 200 μm for Picro Sirius Red and Masson's Trichrome; 50 μm for IHC. (C) Quantification of Picro Sirius Red and Masson's trichrome staining. (D) Quantification of hydroxyproline. (E) Western blot analysis of 4-HNE, (F) malondialdehyde (MDA), and (G) GSH/GSSH ratio in mouse lung tissue. n = 6 per group. ∗∗∗p < 0.001.

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  Fig. 2. Ferrostatin-1 (Fer-1) and deferoxamine (DFO) ameliorated bleomycin-induced pulmonary fibrosis by inhibiting lipid peroxidation and 4-HNE expression. (A) Flowchart of experimental procedure. Mice with intratracheal instillation of bleomycin (Bleo, 2 U/kg) or saline (CTR) were treated with or without Fer-1 (2.5 μmol/kg) or DFO (100 mg/kg) from days 1–7 after bleomycin administration. (B) Barometric plethysmography was performed on day 0 before drug administration and on day 14. After sacrifice, mice lung tissues were subjected to (C) Picro Sirius red and Masson's trichrome staining and immunohistochemical staining of 4-HNE expression. Scale bars: 200 μm for Picro Sirius Red and Masson's Trichrome; 50 μm for IHC. (D) Quantification of Picro Sirius red and Masson's trichrome staining. (E) Hydroxyproline assay, (F) western blot analysis, (G) malondialdehyde (MDA) assay, and (H) glutathione (GSH)/glutathione disulfide (GSSH) ratio assay in mice lung tissues. n = 4–5 per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 3. Ferrostatin-1 (Fer-1) and deferoxamine (DFO) reduced bleomycin-induced apoptosis and ferroptosis in MLE-12 cells. (A) Representative immunofluorescence images of mouse lung tissues showing co-staining with CK-18 (red) and 4-HNE (green). Cell nuclei were counterstained with DAPI (blue). (B) Representative immunofluorescence images of mouse lung tissues showing co-staining with 4-HNE (red) and apoptosis cells (green). Cell nuclei were counterstained with DAPI (blue). (C) Cell viability of MLE-12 cells treated with bleomycin (Bleo, 10 mU), Fer-1 (1 μM), and DFO (100 μM). (D) TUNEL assay to detect apoptosis. (E) Lipid peroxidation in MLE-12 cells was assessed by flow cytometry using C11-BODIPY after treatment with Bleo (10 mU), Fer-1 (1 μM), and DFO (100 μM). (F) GSH/GSSH ratio assay in MLE-12 cells treated with Bleo (10 mU), Fer-1 (1 μM), and DFO (100 μM). (G) Western blot analysis of 4-HNE expression in MLE-12 cells treated with Bleo (10 mU), Fer-1 (1 μM), and DFO (100 μM). ∗∗∗p < 0.001.

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  Fig. 4. In vitro exposure to 4-HNE induced apoptosis and ferroptosis in MLE-12 cells. (A) Cell viability of MLE-12 cells treated with indicated doses of 4-HNE. (B) TUNEL assay to detect apoptosis. (C) Lipid peroxidation in MLE-12 cells was assessed by flow cytometry using C11-BODIPY after treatment with 4-HNE (40 μM). (D) GSH/GSSH ratio assay in MLE-12 cells treated with 4-HNE (40 μM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 5. 4-HNE regulated TGF-β expression by activating JNK/c-Jun pathway both in vivo and in vitro. (A) Relative mRNA expression of TGF-β, (B) Serum levels of TGF-β, and (C) western blot analysis of 4-HNE, phospho-JNK, and phospho-c-Jun in mice lung tissues after treatment with Bleo (2 U/kg), Fer-1 (2.5 μmol/kg), and DFO (100 mg/kg). n = 4 per group. (D) Relative mRNA expression of TGF-β, (E) TGF-β levels in the culture medium, and (F) western blot analysis of 4-HNE, phospho-JNK, and phospho-c-Jun in MLE-12 cells treated with Bleo (10 mU), Fer-1 (1 μM), and DFO (100 μM). (G) Relative mRNA expression of TGF-β, (H) TGF-β levels in the culture medium, and (I) western blot analysis of phospho-JNK and phospho-c-Jun in MLE-12 cells treated with 4-HNE (40 μM). (J) ChIP-qPCR analysis using a c-Jun antibody to assess protein–DNA binding at the TGF-β promoter in MLE-12 cells. Data were normalized to input and expressed relative to the CTR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 6. ACSL4 inhibition attenuates bleomycin-induced pulmonary fibrosis by reducing 4-HNE and suppressing TGF-β via JNK/c-Jun activation. In vivo: Mice received intratracheal bleomycin (Bleo, 2 U/kg) or saline (CTR) and were treated with rosiglitazone (0.5 mg/kg, intraperitoneally) every other day for 14 days. (A) Picro Sirius red, Masson's trichrome, and immunohistochemical staining. Scale bars: 200 μm for Picro Sirius Red and Masson's Trichrome; 50 μm for IHC. (B) Hydroxyproline content. (C) Lung TGF-β mRNA expression. (D) Serum TGF-β levels. (E) Western blot analysis of lung tissue. In vitro: MLE-12 cells were treated with Bleo (10 mU), Ro (5 μM), and/or 4-HNE (40 μM), followed by analysis of: (F) Cell viability, (G) TUNEL assay, (H) Lipid peroxidation, (I) TGF-β mRNA expression, (J) TGF-β levels in the culture medium, (K) western blot, and (L) ChIP-qPCR for c-Jun binding at the TGF-β promoter. Data were normalized to input and expressed relative to the CTR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 7. Ferroptosis inhibition attenuates macrophage infiltration and extracellular matrix remodeling in bleomycin-induced lung fibrosis. C57BL/6 mice were intratracheally administered saline (CTR) or bleomycin (Bleo) and treated with Fer-1, DFO, or rosiglitazone (Ro). (A) Relative mRNA expression of MMP2, MMP9, and TIMP1 in lung tissue. (B) Immunofluorescence staining of F4/80 to assess macrophage infiltration in lung tissue. Scale bars: 50 μm ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Fig. 8. 4-HNE expression level was increased in the fibrotic area of IPF patients. Picro Sirius red, Masson's trichrome, and IHC staining of 4-HNE expression in lung tissue of healthy controls and three patients with IPF. Scale bars: 200 μm for Picro Sirius Red and Masson's Trichrome; 50 μm for IHC.

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  Fig. 9. Hypothetical model of 4-HNE in pulmonary fibrosis development. 4-HNE induced epithelial damage and regulated TGF-β expression via JNK/c-Jun pathway activation, which can be inhibited by Fer-1 and DFO treatment.

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  Supplementary Figure S1. Collagen and 4-HNE are upregulated during the progression of bleomycin-induced lung fibrosis in mice. C57BL/6 mice were intratracheally administered bleomycin (Bleo, 2 U/kg) or saline (CTR) on day 0 and sacrificed on days 2, 4, 7, and 14. Lung tissues were analyzed as follows: (A) Representative images of Picro Sirius Red and immunohistochemical staining of 4-HNE at days 0, 2, 4, 7, and 14. Scale bars: 200 μm (Picro Sirius Red), 50 μm (IHC). (B) Quantification of hydroxyproline content. (C) Western blot analysis of 4-HNE. n = 3 per group. ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Supplementary Figure S2. Ferrostatin-1 (Fer-1) and deferoxamine (DFO) reduced bleomycin-induced apoptosis and ferroptosis in A549 cells. (A) Cell viability of A549 cells treated with bleomycin (Bleo, 10 mU), Fer-1 (1 μM), and DFO (100 μM). (B) TUNEL assay to detect apoptosis. (C) Lipid peroxidation in A549 cells was assessed by flow cytometry using C11-BODIPY after treatment with Bleo (10 mU), Fer-1 (1 μM), and DFO (100 μM). ∗∗∗P < 0.001.

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  Supplementary Figure S3. In vitroexposure to 4-HNE induced apoptosis and ferroptosis in A549 cells. (A) Cell viability of A549 cells treated with indicated doses of 4-HNE. (B) TUNEL assay to detect apoptosis. (C) Lipid peroxidation in A549 cells was assessed by flow cytometry using C11-BODIPY after treatment with 4-HNE (40 μM). ∗P < 0.05, ∗∗∗P < 0.001.