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

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.

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