L‐Citrulline Decreases LPS‐Induced Inflammation and Oxidative Stress in Newborn Rat Lungs

Abstract

Introduction Extremely low birth weight infants have immature lungs at birth as well as low levels of anti-inflammatory and antioxidative defences, placing them at high risk of developing bronchopulmonary dysplasia (BPD). Adiponectin (APN) is produced by adipocytes and has potent anti-inflammatory and antioxidative properties, and anti-proliferative effects; APN levels are quite low in premature infants. We showed that exogenous APN markedly attenuated lipopolysaccharide (LPS)-induced lung injury (Ivanovska et al. 2020) in newborn rats. Recombinant APN is not available for human use; however, L-citrulline (L-CIT) increases APN gene expression in adult rats (Joffin et al., 2015) and upregulates expression of peroxisome proliferator-activated receptor-gamma coactivator alpha (PGC-1α). PGC-1α is a master coactivator of genes involved in mitochondrial biogenesis, inflammation and oxidative stress. L-CIT levels are also low in premature infants. Thus, we sought to determine the effect of exogenous L-CIT on LPS-induced changes in protein expression of PGC-1α, APN, APN receptors (AdipoR1/2), cytokines, and superoxide dismutase (SOD) in lung and pulmonary artery smooth muscle cells (PASMC) homogenates, and on LPS-induced changes of intracellular ROS levels in PASMC. Methods in-vivo: Sprague Dawley rat pups were treated with L-CIT (2.5 g/kg) or saline intraperitoneally from postnatal day (PND) 1-5. On PND 5, intra-pharyngeal LPS (5mg/kg) or saline was given to L-CIT (n=4-6) or saline (n=4-6) treated animals; pups were euthanized 24 h after LPS treatment and lungs were removed and processed for western blot. In-vitro: PASMCisolated from PND 4 rats were treated with L-CIT (4 mM/ml), LPS (5μg/ml) and a combination of the two, for 3 h; cells were harvested and processed for protein expression (n=4 replicate experiments). PASMC were assayed for the level of LPS-induced intracellular ROS with fluorescence dye in the presence and absence of blocker for PGC-1α (SR 18292) Results In vivo, L-CIT, in the presence of LPS, increased lung protein expression of APN (1.36-fold), AdipoR1/2 (2-fold), PGC-1α (1.8-fold), phosphorylated AMP-kinase (2.1-fold), SOD1 (2.3-fold), SOD2 (1.7-fold), SOD3 (1.43-fold), and decreased tumor necrosis factor α (1.2-fold), and interleukin-8 (1.28-fold) compared to saline and LPS treated animals (p<0.05 L-CIT+LPS vs saline+LPS for all proteins). In vitro, in the presence of LPS, L-CIT increased PASMC protein expression of PGC-1α (1.55-fold), SOD1 (2-fold), SOD2 (1.57-fold), and SOD3 (2.2-fold) compared to cells only exposed to LPS (p<0.05; L-CIT+LPS vs medium+LPS). L-CIT decreased LPS-induced ROS production in PASMC, an effect that was inhibited in the presence of PGC-1α blocker. Conclusion L-CIT upregulates APN signaling in presence of LPS and decreases LPS-induced lung inflammation and oxidative stress in newborn rats through activation of PGC1-α. The translational significance of these findings warrants further clinical studies of the L-CIT potential preventative/curative benefit in BPD.