Nebulized PPARγ agonists: a novel approach to augment neonatal lung maturation and injury repair in rats - PubMed (original) (raw)

Nebulized PPARγ agonists: a novel approach to augment neonatal lung maturation and injury repair in rats

Edith Morales et al. Pediatr Res. 2014 May.

Abstract

Background: By stimulating lipofibroblast maturation, parenterally administered peroxisome proliferator-activated receptor γ (PPARγ) agonists promote lung homeostasis and injury repair in the neonatal lung. In this study, we determined whether PPARγ agonists could be delivered effectively via nebulization to neonates, and whether this approach would also protect against hyperoxia-induced lung injury.

Methods: One-day old Sprague-Dawley rat pups were administered PPARγ agonists rosiglitazone (RGZ, 3 mg/kg), pioglitazone (PGZ, 3 mg/kg), or the diluent, via nebulization every 24 h; animals were exposed to 21% or 95% O2 for up to 72 h. Twenty-four and 72 h following initial nebulization, the pups were sacrificed for lung tissue and blood collection to determine markers of lung maturation, injury repair, and RGZ and PGZ plasma levels.

Results: Nebulized RGZ and PGZ enhanced lung maturation in both males and females, as evidenced by the increased expression of markers of alveolar epithelial and mesenchymal maturation. This approach also protected against hyperoxia-induced lung injury, since hyperoxia-induced changes in bronchoalveolar lavage cell and protein contents and lung injury markers were all blocked by nebulized PGZ.

Conclusion: Nebulized PPARγ agonist administration promotes lung maturation and prevents neonatal hyperoxia-induced lung injury in both males and females.

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Conflict of interest statement

Disclosure : No financial interests in this study or potential/perceived conflicts of interest

Figures

Figure 1

Figure 1. Effects of nebulized RGZ or PGZ on alveolar epithelial and mesenchymal differentiation markers in the female and male rat pup lung

Twenty-four hour following either RGZ or PGZ nebulization resulted in increased expression of SP-B and SP-C in both females and males (A). Similarly, either PGZ or RGZ stimulated the alveolar mesenchymal differentiation markers PPARγ, ADRP and leptin in both females and males (B); n=4; *, p<0.05, control vs. treated animals. Either nebulized RGZ or PGZ also increased [3H]choline incorporation (C) and triolein uptake (D); n=6; *, p<0.05, control vs. treated animals. White bars, controls; black bars, PGZ group; and gray bars, RGZ group.

Figure 2

Figure 2. Comparative effects of nebulized RGZ or PGZ on the expression of alveolar epithelial and mesenchymal differentiation markers in the female and male neonatal rat pup

There were no significant differences in the lung protein levels of epithelial, SPB and SPC (A), and mesenchymal, PPARγ, ADRP and leptin, (B), differentiation markers in the female (white bars) and male (black bars) newborn rat pup, 24h following nebulized RGZ or PGZ (n=4; p>0.05, control vs. treated animals).

Figure 3

Figure 3. Effect of nebulized PGZ on total bronchoalveolar lavage cell count (A), macrophage count (B), and total protein (C) content after exposure of neonatal rat lung to hyperoxia (95% O2) for 72h

When exposed to hyperoxia, the bronchoalveolar lavage total cell counts increased 6-fold, macrophage count and total protein content increased 3-fold each. In contrast, with nebulized PGZ, all of these BAL changes were blocked. White bars, 21% O2; black bars, 95% O2; and gray bars, 95% O2 + PGZ. n=4; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2.

Figure 4

Figure 4. Effect of nebulized PGZ on neonatal rat lung triolein uptake and lung injury repair protein levels

When neonatal rat pups were exposed to hyperoxia for 72h, triolein uptake was diminished compared to controls. In contrast, triolein uptake was clearly stimulated by treatment with nebulized PGZ after 72h exposure to hyperoxia (A). Similarly, PPARγ (B) and ADRP (C) protein levels decreased and fibronectin (D) protein levels increased in the hyperoxia-exposed group (p<0.05 vs. control); all of these changes were blocked in the PGZ nebulized group. White bars, 21% O2; black bars, 95% O2; and gray bars, 95% O2 + PGZ (n=4 for all; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2).

Figure 5

Figure 5. Effect of nebulized PGZ on hyperoxia-induced alterations in neonatal rat lung vascular markers VEGF and PECAM-1

As determined by immunostaining, 72h hyperoxia-induced decreases in vascular markers VEGF and PECAM-1 were blocked with PGZ nebulization.

Figure 6

Figure 6. Effect of nebulized PGZ on hyperoxia-induced changes in neonatal rat lung inflammation-related cytokines and apoptosis (determined by BcL2/Bax protein ratio)

Seventy-two hour hyperoxia exposure-induced alterations in lung cytokines, IL-6, (A), IL-1β (B), CCL-2 (C), and MIF (D), were blocked in the PGZ nebulized group, as was the decrease in BcL2/Bax protein ratio (E). White bars, 21% O2; black bars, 95% O2; and gray bars, 95% O2 + PGZ (n=4 for all; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2).

Figure 7

Figure 7. Effect of nebulized PGZ on hyperoxia-induced changes in lung morphometry

Seventy-two hour hyperoxia exposure-induced lung morphometric changes, as determined by mean linear intercept and radial alveolar count, were blocked in the PGZ nebulized group. White bars, 21% O2; black bars, 95% O2; and gray bars, 95% O2 + PGZ (n=4 for all; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2; magnification = 10×).

Figure 8

Figure 8. Comparison of the efficacy for protection against hyperoxia-induced lung injury using nebulized PGZ (1 mg/kg body weight) with that afforded by intraperitoneal PGZ (3 mg/kg body weight)

Nebulized PGZ at a lower dose (1 mg/kg/ body weight) was equally effective in blocking hyperoxia induced decreases in PPARγ, ADRP, and BcL2, and increase in fibronectin protein levels compared to that achieved by the higher intraperitoneally administered dose (3 mg/kg body weight). White bars, 21% O2; black bars, 95% O2; gray bars, 95% O2 + nebulized PGZ; hatched bars, 95% O2 + intraperitoneal PGZ. N=3 for all; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2.

Figure 9

Figure 9. Effect of nebulized PGZ on hyperoxia-induced changes in key intermediates of TGF-β activation

When newborn rat lung was exposed to 72h hyperoxia, P-Smad 3 (A), Smad 7 (B) and ALK 5 (C) expression increased. In contrast, treatment with nebulized PGZ daily during the 72 h hyperoxia exposure prevented the stimulation of all of these TGF-β activation markers. White bars, 21% O2; black bars, 95% O2; and gray bars, 95% O2 + PGZ. N=4; *, p<0.05 vs. control; **, p<0.05 vs. 95% O2.

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