The triterpenoid CDDO-Me inhibits bleomycin-induced lung inflammation and fibrosis

Abstract

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p≤0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p≤0.001), and excess production of the pro-fibrotic cytokine TGFβ by 50%. CDDO-Me also inhibited α-smooth muscle actin and fibronectin mRNA by 50% (p≤0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and αSMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.

Figure 1. CDDO-Me reduces total BALF protein and cellular infiltration.

Groups of mice (6 per group) were treated with bleomycin or PBS by inhalation on day 0, and with either CDDO-Me (400 ng/day) or control vehicle (Veh) by inhalation on days -1, 1, 3 and 5 and harvested on day 7. Lungs were lavaged and BALF was analyzed for total protein (A), total cell number (B), neutrophils (C) and alveolar macrophages (D). CDDO-Me potently reduces BALF protein, total cells, neutrophils and alveolar macrophages. These data are the mean ± SE of n = 6 mice per group (*p≤0.05, ** p≤0.01, compared to bleomycin+vehicle and #p≤0.01 compared to PBS+veh by one-way ANOVA).

Figure 2. CDDO-Me inhibits production of IL6, KC and TGFβ in total lung homogenate.

Mice were treated either with bleomycin or CDDO-Me as described and harvested on day 7. Cytokines in total lung homogenate were measured by ELISA and normalized to total protein. Bleomycin induces, and CDDO-Me significantly inhibits, KC (A), IL6 (B) and TGFβ (C). These data represent six independent animals and analyzed using one-way ANOVA (mean ± S.E. shown, *** p≤0.001, compared to bleomycin+vehicle and #p≤0.001 compared to PBS+veh).

Figure 3. CDDO-Me decreases fibrotic gene expression.

Mice were treated with bleomycin with or without CDDO-Me (as described in methods and materials). On day 7, the mice were euthanized and RNA prepared from a right lung lobe analyzed by RT-qPCR. Bleomycin significantly upregulates expression of fibronectin (A), αSMA (B) and type 1 collagen (C) at day 7. CDDO-Me significantly inhibited expression αSMA and FN. There was also a trend toward inhibition of collagen expression but this was not significant. These data represent six independent animals and analyzed using one-way ANOVA (mean ± S.E. shown, *p≤0.05, ** p≤0.01, compared to bleomycin+vehicle and #p≤0.01 compared to PBS+veh).

Figure 4. CDDO-Me reduces fibrosis in mice treated with bleomycin.

Mice received bleomycin on day 0, and CDDO-Me or vehicle was given from day -1 to day 9 and lungs were harvested on day 21 (A). The left lungs were processed for histology and stained with Gomori's trichrome to visualize collagen deposition (blue) and overall fibrosis. Representative sections from each group are shown (B). Sections were scored by a blinded reviewer; CDDO-Me significantly reduced fibrosis score (C). Results shown are the mean± SEM for 14–16 mice per group and were analyzed using a two-sided t-test as described in methods and materials. A portion of the right lung was homogenized and hydroxyproline content was determined. Hydroxyproline contents increased in bleomycin-treated lungs and was reduced by CDDO-Me treatment (D) (mean ± SEM for n = 14–16, p = 0.0578 using two-sided t-test as described in the statistical supplement. Scale bar  = 100 μm.

Figure 5. CDDO-Me inhibits expression of collagen I and fibronectin.

Mice received bleomycin on day 0, and 350 ng of CDDO-Me or vehicle was given from day -1 to day 9 and lungs were harvested on day 21, a portion of right lung was used to measure mRNA and analyzed by RT-qPCR for collagen I (Col1A1) specific primers and normalized to 18S mRNA. CDDO-Me inhibited bleomycin–induced up-regulation of collagen I mRNA *p≤0.05 (A). CDDO-Me also reduced bleomycin–induced up-regulation of fibronectin expression, *p≤0.05 (B). These data represent 8–10 independent animals and analyzed using one-way ANOVA (mean ± S.E. shown, *p≤0.05, ** p≤0.01, compared to bleomycin+vehicle and #p≤0.01 compared to PBS+veh).

Figure 6. CDDO-Me reduces αSMA deposition and expression.

Treatment of mice was carried out as described in Figure 4. Left lung sections processed for histological analysis and stained using an antibody against αSMA (brown) and counterstained with hematoxylin (blue). CDDO-Me reduces amount and distribution of αSMA positive cells in the treatment schedule. Scale bar  = 100 μm (A). Immunoblots were performed on total lung homogenate to detect expression of αSMA. CDDO-Me inhibited bleomycin–induced up-regulation of αSMA (B). Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment (n = 2) are shown here. Relative changes in the average expression of αSMA/GAPDH (R.E.) are as indicated in the figure for PBS, Bleo and Bleo+CDDO-Me.

Figure 7. CDDO-Me improves lung functions of bleomycin treated mice.

Mice (n = 13–16) were treated with bleomycin and CDDO-Me as described in Figure 4. (A) Respiratory rates (respirations/minute) were monitored on day 20 prior to harvesting the lungs on day 21.(B) Specific dynamic lung compliance was measured on day 21 immediately prior to euthanasia. (p≤0.05, using two-sided t-test as described in methods and materials).