Hyperbaric Oxygen Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Mice

The prevalence of pulmonary fibrosis is increasing with an aging population and its burden is likely to increase following COVID-19, with large financial and medical implications. As approved therapies in pulmonary fibrosis only slow disease progression, there is a significant unmet medical need. Hyperbaric oxygen (HBO) is the inhaling of pure oxygen, under the pressure of greater than one atmosphere absolute, and it has been reported to improve pulmonary function in patients with pulmonary fibrosis. Our recent study suggested that repetitive HBO exposure may affect biological processes in mice lungs such as response to wounding and extracellular matrix. To extend these findings, a bleomycin-induced pulmonary fibrosis mouse model was used to evaluate the effect of repetitive HBO exposure on pulmonary fibrosis. Building on our previous findings, we provide evidence that HBO exposure attenuates bleomycin-induced pulmonary fibrosis in mice. In vitro, HBO exposure could reverse, at least partially, transforming growth factor (TGF)-β–induced fibroblast activation, and this effect may be mediated by downregulating TGF-β–induced expression of hypoxia inducible factor (HIF)-1α. These findings support HBO as a potentially life-changing therapy for patients with pulmonary fibrosis, although further research is needed to fully evaluate this.


Supplementary Figure 1. Bleomycin treatment induces pulmonary fibrosis in mice. (A and B)
Lungs from saline-treated (Saline) or bleomycin-challenged mice (Bleomycin) collected at day 21 post instillation were stained with H/E (A) or Masson's trichrome stain (B, collagen shown in blue). Top panels show the whole left lung lobe (scale bar: 1 mm) with higher-magnification images in bottom panels (scale bar: 100 µm). (C) Graph showing Ashcroft scores in lungs from saline-treated (Saline) or bleomycin-challenged (Bleomycin) mice. (D) Graph showing relative hydroxyproline content in lungs from saline-treated (Saline) or bleomycin-challenged (Bleomycin) mice. Lung tissue massnormalised hydroxyproline levels in saline group were used to set the baseline value at unity. Data are mean ± s.d., with P values analysed with unpaired t-test.

Supplementary Figure 2. Body weight changes in bleomycin-challenged mice (Bleo) or bleomycin-challenged mice treated with repetitive HBO (Bleo + HBO).
Body weights were measured every third day. A Two-way ANOVA test with repeated measure data was used to analyse the difference with no significant (ns) difference identified. Data are mean ± s.d., with numbers of mice within each group indicated.

Supplementary Figure 3. Effects of bleomycin on fibroblast activation and ECM deposition in mice lungs.
Fold change in the mRNA levels of Acta2 (α-SMA), Col1a1 (collagen I) and Fn1 (fibronectin) in lungs from control (Vehicle) or bleomycin-challenged (Bleo) mice. Actb (β-actin)normalised mRNA levels in control mice lungs were used to set the baseline value at unity. Data are mean ± s.d., with numbers of mice within each group and P values indicated. Data were analysed with multiple t-test.

Supplementary Figure 4. Effects of TGF-β on fibroblast activation in HFL1 cells.
Fold change in the mRNA levels of ACTA2 (α-SMA), COL1A1 (collagen I) and FN1(fibronectin) in HFL1 cells with indicated treatments. ACTB (β-actin)-normalised mRNA levels in control cells (Vehicle) were used to set the baseline value at unity. Data are mean ± s.d., with P values indicated. n = 3 samples each group. Data were analysed with multiple t-test.

Supplementary Figure 5. Effects of HBO treatment on TGF-β-induced fibroblast differentiation and HIF-1α expression. (A)
Schematic diagram of the experimental procedure. In brief, TGF-β (5 ng/mL) was added to HFL1 cells for 48 hours to induce fibroblast activation. At the beginning of TGFβ treatment, HFL1 cells were exposed to 2.5 ATA HBO for 90 minutes immediately. The HBO exposure was repeated for another time with a 24-hours interval (about 26 h post TGF-β treatment). Samples were collected at 48 hours after the beginning of TGF-β treatment. (B) Fold change in the mRNA levels of ACTA2 (α-SMA) in HFL1 cells with indicated treatments. ACTB (β-actin)-normalised mRNA levels in control cells (Vehicle) were used to set the baseline value at unity. (C) Schematic diagram of the experimental procedure. In brief, HFL1 cells were exposed to 2.5 ATA HBO for 90 minutes right after TGF-β (5 ng/mL) treatment. At the end of HBO exposure, samples were collected immediately. (D) Protein expression of HIF-1α in HFL1 cells with indicated treatments. β-actin was used as a loading control. (E) Fold change in the protein level of HIF-1α in HFL1 cells with indicated treatments. In the graph, β-actin-normalised protein levels in control cells (Vehicle) were used to set the baseline value at unity. Data in (B and E) are mean ± s.d., with P values indicated. Data were analysed with one-way ANOVA.