Korean J Physiol Pharmacol.  2024 Nov;28(6):559-568. 10.4196/kjpp.2024.28.6.559.

Hemin attenuates bleomycin-induced lung fibrosis in mice by regulating the TGF-ββ1/MAPK and AMPK/SIRT1/PGC-1αα/HO-1/ NF-κκB pathways

• Hao W
• Yu Tt
• Li W
• Wang Gg
• Hu Hx
• Zhou Pp

• KMID: 2560735
• DOI: http://doi.org/10.4196/kjpp.2024.28.6.559

Abstract

The objective of this study was to investigate the protective effect and potential mechanism of action of hemin on bleomycin-induced pulmonary fibrosis in mice. Male C57BL/6 mice were randomly divided into control, bleomycin and bleomycin + hemin groups. Mice in the bleomycin and bleomycin + hemin groups were injected intratracheally with bleomycin to establish the pulmonary fibrosis model. The bleomycin + hemin group mice were injected intraperitoneally with hemin starting 7 days before modeling until the end of Day 21 after modeling. Pathological changes in lung tissue were assessed by HE and Masson staining. Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined in lung tissue. Immunohistochemistry was performed to assess the expression of α-SMA and collagen I. The serum levels of IL-6 and TNF-α were measured via ELISA. Western blotting was used to determine the expression of TGF-β1, SIRT1, PGC-1α and HO-1 and the phosphorylation levels of p38, ERK1/2, JNK, AMPK and NF-κB p65 in lung tissue. Hemin significantly reduced lung indices, increased terminal body weight. It also significantly increased SOD and CAT activities; decreased MDA, IL-6 and TNF-α levels; reduced the levels of α-SMA and collagen I-positive cells; upregulated SIRT1, PGC-1α and HO-1 expression; promoted AMPK phosphorylation; and downregulated TGF-β1 expression and p38, ERK1/2, JNK and NF-κB p65 phosphorylation. Hemin might attenuate oxidative damage and inflammatory responses and reduces extracellular matrix deposition by regulating the expression and phosphorylation of proteins associated with the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/ NF-κB pathways, thereby alleviating bleomycin-induced pulmonary fibrosis.

Keyword

Bleomycin; Hemin; Inflammation; Oxidative stress; Pulmonary fibrosis

Figure

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  Fig. 1 Effect of hemin on the survival rate, terminal body weight and lung indices of mice with pulmonary fibrosis. (A) Survival rate. The survival rates were recorded, estimated by the Kaplan–Meier method and were compared by a log-rank test. There were fifteen mice in each group. (B) Terminal body weight. (C) Lung indices. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. There were six mice in each group. **p < 0.01 compared with the control group. ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 2 Effect of hemin on morphological and pathological changes in the lung tissue of mice with pulmonary fibrosis. (A) Gross morphology of lung tissue. (B) HE staining (400×). The black arrow in the figure indicates the location of pulmonary inflammation. (C) Masson staining (200×). The black arrow in the figure indicates collagen fibers. (D) Quantification of collagen expression levels by area percentage of positively stained collagen. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. There were six mice in each group. **p < 0.01 compared with the control group. ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 3 Immunohistochemical staining of α-SMA and collagen I in the lung tissue of mice with pulmonary fibrosis (400×). (A) Immunohistochemical staining of α-SMA. (B) Immunohistochemical staining of collagen I. The black arrow in the figure indicates positive cells. (C) and (D) present the areas of positively stained α-SMA and collagen I to represent their expression levels, respectively. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. There were six mice in each group. **p < 0.01 compared with the control group. ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 4 Effect of hemin on the levels of MDA, SOD and CAT in the lung tissue of mice with pulmonary fibrosis. (A) MDA levels in the lung tissue of mice in each group. (B) The activity of SOD in the lung tissue of the mice in each group. (C) The activity of CAT in the lung tissue of the mice in each group. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. There were six mice in each group. MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase. **p < 0.01 compared with the control group. #p < 0.05 and ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 5 Effect of hemin on the serum levels of interleukin (IL)-6 and tumor necrosis factor- α (TNF-α) in mice with pulmonary fibrosis. (A) Serum IL-6 levels of the mice in each group. (B) Serum TNF-α levels of the mice in each group. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. There were six mice in each group. **p < 0.01 compared with the control group. ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 6 Effect of hemin on the TGF- β1/MAPK signaling pathway in the lung tissue of mice with pulmonary fibrosis (PF). (A) Western blotting detection of the expression of proteins related to the TGF-β1/MAPK pathway in the lung tissue of mice with PF. (B) TGF-β1/GAPDH. (C) p-p38/p38. (D) p-ERK1/2/ERK1/2. (E) p-JNK/JNK. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. Three mice were in each group. TGF-β1, transforming growth factor-β1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun-n-terminal kinase. **p < 0.01 compared with the control group. #p < 0.05 and ##p < 0.01 compared with the bleomycin group.

• 

  Fig. 7 Effect of hemin on the AMPK/SIRT1/PGC-1 α/HO-1/NF-κB signaling pathway in the lung tissue of mice with pulmonary fibrosis (PF). (A) Western blotting detection of the expression of proteins related to the AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathway in the lung tissue of mice with PF. (B) p-AMPK/AMPK ratio. (C) SIRT1/GAPDH ratio. (D) PGC-1α/GAPDH ratio. (E) HO-1/GAPDH ratio. (F) p-NF-κB p65/NF-κB p65 ratio. The significance of difference was determined by one-way ANOVA followed by Tukey’s test. The data are presented as the means ± standard deviations. Three mice were in each group. AMPK, adenosine monophosphate-activated protein kinase; SIRT1, silent information regulator 1; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator-1α; HO-1, heme oxygenase-1; NF-κB, nuclear transcription factor-κB. **p < 0.01 compared with the control group. #p < 0.05 and ##p < 0.01 compared with the bleomycin group.

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