L-citrulline Mitigates Systemic Inflammation and Associated Pulmonary Vascular Remodeling in a Neonatal Rat Model of Postnatal Undernutrition

Abstract

Background & Aim: Preterm infants with postnatal growth restriction and undernutrition are at increased risk of developing bronchopulmonary dysplasia (BPD), pulmonary hypertension (PH), metabolic syndrome, and sepsis. Preclinical and clinical studies report increased systemic inflammation, and lung and pancreatic histopathology in growth-restricted neonates. Safe therapies to mitigate these adverse outcomes associated with postnatal growth restriction and undernutrition in preterm infants are needed. L-citrulline is a non-essential amino acid, involved in the urea and nitric oxide cycles, that exhibits anti-inflammatory properties. This study aims to investigate whether L-citrulline may decrease systemic inflammation and improve histopathology in the lung, heart, and pancreas in a neonatal model of postnatal undernutrition. Methods: Neonatal Sprague Dawley rat pups were divided into 2 groups, normal nutrition (litter size n=12) and undernutrition (litter size n=18) by altering the litter size of the nursing dam. Body weight was measured daily and both groups received daily intraperitoneal injections of saline or L-citrulline (2.5g/kg) for 21 days. On postnatal day 21, heart, lung, and pancreatic tissue were collected for histology and mRNA analyses. Lung and pancreatic tissue were processed for qPCR to detect mRNA expression of pro-inflammatory mediators (MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α). Right ventricular hypertrophy was calculated using Fulton’s index (weight of the right ventricle divided by the combined weight of the left ventricle and the intraventricular septum). Lung sections were stained with Movat Pentachrome and examined for the presence of immune cells and medial wall thickening of pulmonary arteries. In a separate group of animals, bronchoalveolar lavage fluid (BALF) and blood obtained by cardiac puncture were cytocentrifuged and stained with hematoxylin & eosin for immune cell counts. Results: Undernourished rats had a lower body weight than normally nourished rats at postnatal day 21 (n=11-17; p<0.0001). Undernourished animals had an increased number of immune cells in BALF and lung tissue and an elevated neutrophil count in the blood which were all decreased with concurrent L-citrulline treatment (n=3-4; p<0.001). L-citrulline treatment also prevented the increased mRNA expression of pro-inflammatory mediators in the lungs [MCP-1/CCL2, TNF-α, and IL-1β] (n=8; p<0.05) and pancreas [MCP-1/CCL2, MIP-1α /CCL3, IL-6, and TNF-α] (n=3; p<0.05) of undernourished animals. These animals had a greater pulmonary artery medial wall area and Fulton’s index when compared to normally nourished animals treated with saline or L-citrulline (n=3-4; p<0.001). Concurrent L-citrulline treatment in undernourished animals prevented pulmonary artery vascular remodeling and heart tissue histopathology such that the medial wall area and Fulton’s index did not differ from that of animals in control groups (n=4; p<0.01). Conclusion: L-citrulline effectively mitigates the adverse effects of postnatal undernutrition, reducing lung, pancreatic, and systemic inflammation and histopathology in the lung and heart associated with PH. As a safe, well-tolerated, and inexpensive treatment, L-citrulline presents promising therapeutic potential that could prevent short and long-term morbidities associated with growth restriction and undernutrition in preterm infants. This work was funded by the Women's Auxiliary of The Hospital for Sick Children. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.