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. 2022 Dec 4;11(12):2405.
doi: 10.3390/antiox11122405.

Cathelicidin Attenuates Hyperoxia-Induced Lung Injury by Inhibiting Ferroptosis in Newborn Rats

Hsiu-Chu Chou  1 , Chung-Ming Chen  2   3
Affiliations

Cathelicidin Attenuates Hyperoxia-Induced Lung Injury by Inhibiting Ferroptosis in Newborn Rats

Hsiu-Chu Chou et al. Antioxidants (Basel). .

Abstract

High oxygen concentrations are often required to treat newborn infants with respiratory distress but have adverse effects, such as increased oxidative stress and ferroptosis and impaired alveolarization. Cathelicidins are a family of antimicrobial peptides that exhibit antioxidant activity, and they can reduce hyperoxia-induced oxidative stress. This study evaluated the effects of cathelicidin treatment on lung ferroptosis and alveolarization in hyperoxia-exposed newborn rats. Sprague Dawley rat pups were either reared in room air (RA) or hyperoxia (85% O2) and then randomly given cathelicidin (8 mg/kg) in 0.05 mL of normal saline (NS), or NS was administered intraperitoneally on postnatal days from 1-6. The four groups obtained were as follows: RA + NS, RA + cathelicidin, O2 + NS, and O2 + cathelicidin. On postnatal day 7, lungs were harvested for histological, biochemical, and Western blot analyses. The rats nurtured in hyperoxia and treated with NS exhibited significantly lower body weight and cathelicidin expression, higher Fe2+, malondialdehyde, iron deposition, mitochondrial damage (TOMM20), and interleukin-1β (IL-1β), and significantly lower glutathione, glutathione peroxidase 4, and radial alveolar count (RAC) compared to the rats kept in RA and treated with NS or cathelicidin. Cathelicidin treatment mitigated hyperoxia-induced lung injury, as demonstrated by higher RAC and lower TOMM20 and IL-1β levels. The attenuation of lung injury was accompanied by decreased ferroptosis. These findings indicated that cathelicidin mitigated hyperoxia-induced lung injury in the rats, most likely by inhibiting ferroptosis.

Keywords: cathelicidin; ferroptosis; hyperoxia; newborn; radial alveolar count.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1
Body weights of the RA- or hyperoxia-reared rats treated with NS or cathelicidin from postnatal days 1 to 7. n = 9–10 rats in each group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. RA + NS and RA + cathelicidin groups. # p < 0.01 vs. O2 + NS group.
Figure 2
Figure 2
(A) Representative photomicrographs of immunohistochemistry of cathelicidin staining and (B) semi-quantitative analysis of cathelicidin immunoreactivity in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. Treatment with cathelicidin significantly increased the hyperoxia-induced decrease in cathelicidin immunoreactivity. n = 9–10 rats in each group. *** p < 0.001.
Figure 3
Figure 3
Ferroptosis biomarkers in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. (A) Fe2+ level, (B) MDA level, (C) GSH level, (D) GPX4 activity, and (E) GPX4 protein expression. Treatment with cathelicidin significantly reversed the hyperoxia-induced changes in the ferroptosis biomarkers. n = 6–8 rats in each group. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 4
Figure 4
(A) Representative photomicrographs of Prussian blue staining and (B) positive cells per high-power field in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. Prussian blue staining was primarily localized in type II alveolar cells and alveolar macrophages (black arrow). Cathelicidin treatment significantly reduced the hyperoxia-induced increase in the iron deposition. n = 8 rats in each group *** p < 0.001.
Figure 5
Figure 5
(A) Representative photomicrographs of immunohistochemistry of TOMM20 staining and (B) representative Western blots and quantitative data determined with densitometry for TOMM20 proteins in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. The positive cells (black arrows) were present in the cytoplasm of the epithelial cell of bronchi, bronchioles, and alveoli, as well as vascular endothelial cells. Cathelicidin treatment significantly reduced TOMM20 expression in the hyperoxia-exposed group. n = 6 rats in each group. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 6
Figure 6
Lung IL-1β levels in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. Treatment with cathelicidin significantly reduced the hyperoxia-induced increase in lung IL-1β levels. n = 6 rats in each group. *** p < 0.001.
Figure 7
Figure 7
(A) Representative H&E-stained lung sections and (B) RAC in 7-day-old rats exposed to RA or hyperoxia and treated with NS or cathelicidin. The rats reared in RA and treated with NS or cathelicidin exhibited normal lung morphology and comparable RAC. Treatment with cathelicidin significantly reversed the hyperoxia-induced decrease in the RAC. n = 9–10 rats in each group. *** p < 0.001.
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