Therapeutic effects of C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO-Me; bardoxolone methyl) on radiation-induced lung inflammation and fibrosis in mice

Abstract

The C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO-Me), one of the synthetic triterpenoids, has been found to have potent anti-inflammatory and anticancer properties in vitro and in vivo. However, its usefulness in mitigating radiation-induced lung injury (RILI), including radiation-induced lung inflammation and fibrosis, has not been tested. The aim of this study was to explore the therapeutic effect of CDDO-Me on RILI in mice and the underlying mechanisms. Herein, we found that administration of CDDO-Me improved the histopathological score, reduced the number of inflammatory cells and concentrations of total protein in bronchoalveolar lavage fluid, suppressed secretion and expression of proinflammatory cytokines, including transforming growth factor-β and interleukin-6, elevated expression of the anti-inflammatory cytokine interleukin-10, and downregulated the mRNA level of profibrotic genes, including for fibronectin, α-smooth muscle actin, and collagen I. CDDO-Me attenuated radiation-induced lung inflammation. CDDO-Me also decreased the Masson's trichrome stain score, hydroxyproline content, and mRNA level of profibrotic genes, and blocked radiation-induced collagen accumulation and fibrosis. Collectively, these findings suggest that CDDO-Me ameliorates radiation-induced lung inflammation and fibrosis, and this synthetic triterpenoid is a promising novel therapeutic agent for RILI. Further mechanistic, efficacy, and safety studies are warranted to elucidate the role of CDDO-Me in the management of RILI.

Keywords: CDDO-Me; cytokine; fibrosis; inflammation; mouse; radiation-induced lung injury; radiotherapy; transforming growth factor-β.

Figure 1

Chemical structures of CDDO and CDDO-Me. Abbreviations: CDDO, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

Figure 2

Effects of CDDO-Me treatment on inflammatory cell infiltration and total protein concentration in BALF of mice treated with radiation. Notes: Groups of mice (n=6) were irradiated with a single dose of 12.5 Gy over their right hemithoraces or had sham irradiation on day 0, and either CDDO-Me (600 ng/day) or control vehicle by gavage on days –1, 1, 3, and 5. BALF was harvested at 3 weeks post-irradiation. BALF was collected and analyzed for May-Grünwald-Giemsa staining (magnification ×200) (A), total cell number (B), proportion of inflammatory cells (C), and total protein content (D). The data are shown as the mean ± standard error of the mean of six mice per group. **P<0.01 and ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviations: BALF, bronchoalveolar lavage fluid; CDDO, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

Figure 3

Effects of CDDO-Me treatment on histopathological changes in lung tissues of radiation-treated mice. Notes: After 3 weeks of radiation, mouse lung tissues were collected, and histologic sections were prepared and stained with H&E. Images of the stained lung sections were taken using an Olympus light microscope. One representative lung section from six mice per group is shown (magnification ×100) (A). The histopathological slides were evaluated using a semiquantitative scoring method (B). The data are shown as the mean ± standard error of the mean of six mice per group. **P<0.01 and ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviations: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; H&E, hematoxylin and eosin.

Figure 4

Effects of CDDO-Me treatment on proinflammatory and anti-inflammatory cytokine expression in radiation-treated mice. Notes: Proinflammatory and anti-inflammatory cytokines in total lung homogenates were measured by enzyme-linked immunosorbent assay and normalized to total protein. The data are shown as the mean ± standard error of the mean of six independent animals. ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviations: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; IL-6, interleukin-6; IL-10, interleukin-10; TGF-β, transforming growth factor-β.

Figure 5

Effects of CDDO-Me treatment on selected profibrotic gene expression in radiation-treated mice. Notes: After 3 weeks of radiation, the mice were euthanized, and RNA was prepared from a right lung lobe and analyzed by real time-polymerase chain reaction. Expression levels of fibronectin (A), α-SMA (B), and collagen I (C) at 3 weeks post-irradiation were determined using real-time polymerase chain reaction. The data are shown as the mean ± standard error of the mean of six animals. ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviations: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; α-SMA, α-smooth muscle actin.

Figure 6

Effects of CDDO-Me treatment on radiation-induced collagen deposition and lung fibrosis in mice. Notes: Groups of mice (n=10) were irradiated with a single dose of 22.5 Gy over their right hemithoraces or had sham irradiation on day 0, with either CDDO-Me (600 ng/day) or control vehicle by gavage on days –1, 1, 3, 5, 7, and 9. The lung tissues were harvested at 12 weeks post-irradiation. The right lungs were processed and stained with Masson’s trichrome stain to visualize collagen deposition (blue) and overall fibrosis. Representative sections from each group are shown in (A). The fibrosis in lung sections was scored by a blinded reviewer (B). The data are shown as the mean ± standard error of the mean of ten mice per group. *P<0.05 and ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviation: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

Figure 7

Effects of CDDO-Me treatment on hydroxyproline content in radiation-treated mice. Notes: A portion of the right lung was homogenized and the hydroxyproline content was determined. The results are shown as the mean ± standard error of the mean of ten mice per group. ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviation: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid.

Figure 8

Effects of CDDO-Me treatment on expression of selected profibrotic genes in radiation-treated mice. Notes: Expression levels of three profibrotic genes including fibronectin (A), α-SMA (B) and collagen I (C) were analyzed by real time-polymerase chain reaction in mouse lungs at 12 weeks post-irradiation. The results are shown as the mean ± standard error of the mean of ten mice per group. *P<0.05 and ***P<0.001 by one-way analysis of variance followed by Tukey’s post hoc test. Abbreviations: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; α-SMA, α-smooth muscle actin.

Figure 9

Possible mechanisms for the protective effects of CDDO-Me against RILI in mice. Notes: Irradiation causes acute and delayed damage to resident lung cells, resulting in necrosis, apoptosis, and autophagy of bronchiolar epithelial cells. With secretion of proinflammatory cytokines and chemokines, including TGF-β, TNF-α, and ILs from activated macrophages, lymphocytes, and neutrophils, lung epithelial cells undergo EMT to form myofibroblasts that can produce collagens I and III, fibronectins, and other matrix molecules. Alveolar type II epithelial cells also express high levels of EMT-associated protein markers. Thorax irradiation triggers recruitment of various immune cells into the lung, such as monocytes, neutrophils, and lymphocytes, which produce large amounts of cytokines and chemokines. CDDO-Me can block or inhibit these molecular events and cascades, and thus shows potent radioprotective effects in mice. Abbreviations: CDDO-Me, C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; EMT, epithelial to mesenchymal transition; ILs, interleukins; RILI, radiation-induced lung injury; RNS, radical nitrogen species; ROS, radical oxygen species; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.