Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

Abstract

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.

Fig. 1.

Effect of curcumin on alveolar maturation by examination of embryonic day 19 (e19) fetal rat lung alveolar interstitial fibroblast (AIF) proliferation and differentiation. Treatment of cultured e19 AIFs with curcumin for 24 h significantly increased thymidine incorporation (A), which was accompanied by increased parathyroid hormone-related protein (PTHrP) receptor (PTHrP Rec; B), peroxisome proliferator-activated receptor (PPAR)-γ (C), and adipocyte differentiation-related protein (ADRP; D) protein levels and triolein uptake (E). Values are means ± SE; n = 6 (A and E) and 3 (B–D). All biomarker responses were curcumin-dose-dependent; increases were significant at 5 μM curcumin.

Fig. 2.

Hyperoxia-induced pulmonary damage markers and cytoprotective effect of curcumin against hyperoxia in cultured e19 AIFs. When exposed to 95% O₂ for 24 h, PPARγ (A) and ADRP (B) protein levels decreased significantly, α-smooth muscle actin (α-SMA, C) and fibronectin (D) protein levels increased significantly, and triolein uptake decreased significantly (E). These hyperoxia-induced effects were completely blocked by 1 h of pretreatment with 5 μM curcumin. Values are means ± SE; n = 3 (A–D) and 6 (E).

Fig. 3.

Effect of curcumin on hyperoxia-induced apoptosis in e19 AIFs. Exposure of cultured e19 AIFs to 95% O₂ for 24 h resulted in decreased Bcl-2-to-Bax ratio (A), an effect that was blocked by 1 h of pretreatment with 5 μM curcumin. Under normoxic conditions, 24 h of treatment with 20 μM curcumin increased fibroblast apoptosis (decreased Bcl-2-to-Bax ratio), while 5 and 10 μM curcumin had no effect (B). Values are means ± SE.

Fig. 4.

Hyperoxia-induced increase in oxidative stress and activation of the TGF-β pathway and their blockage by curcumin pretreatment. In cultured e19 AIFs exposed to 95% O₂ for 24 h (to determine oxidative stress) and 30 min (to determine TGF-β activation), NADP-to-NADPH ratio and phosphorylated-to-total Smad3 ratio (P-Smad3/T-Smad3) were significantly increased. Similarly, 95% O₂ exposure of e19 AIFs for 24 h caused a significant increase in activin-like receptor kinase (ALK)-5 expression. All these effects were completely blocked by 5 μM curcumin pretreatment. Values are means ± SE; n = 3.

Fig. 5.

Hyperoxia-induced increase in Smad3 and its nuclear translocation and their blockage by curcumin pretreatment. In cultured e19 AIFs exposed to 95% O₂ for 6 h, Smad3 translocated to the nucleus (white arrows), an effect that was at least partially blocked by 1 h of pretreatment with 10 μM curcumin. Original magnification ×20.

Fig. 6.

In vivo protective effects of curcumin against hyperoxia. Exposure to 95% O₂ for 7 days caused a significant decrease in the alveolar count of experimental rats, an effect that was effectively blocked by concurrent treatment with curcumin (5 mg/kg). Values are means ± SE; n = 3.

Fig. 7.

Molecular mechanism by which hyperoxia exposure inhibits alveolarization and how curcumin blocks this effect. Exposure of newborn rat pups to 95% O₂ for 7 days significantly reduced PPARγ protein level (A) and increased α-SMA (B) and fibronectin (C) protein levels. All these effects were blocked by concurrent treatment with 5 mg/kg curcumin. Values are means ± SE; n = 3.

Fig. 8.

Molecular mechanism by which hyperoxia exposure inhibits alveolarization and how curcumin blocks this effect. Increase in α-SMA and decreases in PPARγ and platelet endothelial cell adhesion molecule (PECAM, CD31) protein levels induced by 7 days of hyperoxia (95% O₂) in vivo were corroborated by immunostaining. All these effects were blocked by concurrent treatment with 5 mg/kg curcumin. Representative images are shown, and locations of the specific proteins are indicated by white arrows; nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images are representative of results from 3 experiments.

Fig. 9.

In vivo hyperoxia-induced activation of TGF-β pathway and blockage by curcumin treatment. A and B: phosphorylated-to-total Smad3 ratio and expression of ALK-5 were significantly increased in newborn rats exposed to 95% O₂ for 7 days; these effects were completely blocked by concurrent treatment with 5 mg/kg curcumin. Under normoxic conditions, treatment with curcumin did not affect basal phosphorylated Smad3 levels. C: Smad7 was significantly induced by hyperoxia exposure and blocked by concurrent treatment with 5 mg/kg curcumin. Values are means ± SE; n = 3.

Fig. 10.

Effect of curcumin on in vivo hyperoxia-induced increase in lung tissue nitrosative stress. After 7 days of exposure to 95% O₂, Western blotting on lung tissue from newborn rat pups showed increased 3-nitrotyrosine protein level, an effect that was significantly attenuated by concurrent treatment with 5 mg/kg curcumin. Values are means ± SE; n = 3.

Fig. 11.

Effect of curcumin on in vivo hyperoxia-induced increase in lung cellular apoptosis. Exposure of newborn rat pups to 95% O₂ for 7 days significantly increased lung cellular apoptosis, as reflected by a decrease in Bcl-2 and an increase in Bax protein levels by Western blotting (A) and immunostaining (B). Both effects were blocked by concurrent treatment with 5 mg/kg curcumin. Values are means ± SE; n = 3. Original magnification ×20 for representative images in B.