Relaxin-2 Alleviates Hyperoxia-Induced Acute Lung Injury in Neonatal Rats by Inhibiting TLR4/NF-κB

Abstract

Hyperoxia-induced acute lung injury (ALI) in neonates is driven by oxidative stress and inflammation. This study aims to evaluate the efficacy and underlying mechanisms of Relaxin-2 (RLX-2) in a neonatal rat model of hyperoxia-induced ALI. Neonatal Wistar rats were exposed to 90% O₂ for one week and treated with RLX-2 (0.5 mg/kg/day) via an osmotic pump. Methods included histopathology (hematoxylin–eosin staining), bronchoalveolar lavage fluid (BALF) analysis, Enzyme-Linked Immunosorbent Assay (ELISA) for interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α), oxidative stress markers [malondialdehyde (MDA) and superoxide dismutase (SOD)], and Western blotting for Toll-like Receptor 4 (TLR4), Nuclear Factor-kappa B (NF-κB), and Sirtuin 1 (SIRT1). Human alveolar epithelial cells (HPAEpiCs) underwent assays using Cell Counting Kit-8 (CCK-8), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), and quantitative real-time polymerase chain reaction (qRT-PCR). RLX-2 mitigated alveolar damage and inflammatory infiltration in neonatal rats with hyperoxia-induced acute lung injury. It decreased cytokine levels, reduced MDA levels, and enhanced SOD activity, thereby alleviating oxidative stress. RLX-2 suppressed TLR4/NF-κB signaling by reducing p65 and Myeloid Differentiation Primary Response 88 (MyD88) expression and upregulated SIRT1. SIRT1 silencing abolished RLX-2's effects, confirming its role in attenuating OS and inflammation. These findings demonstrated RLX-2's efficacy in neonatal ALI via SIRT1-dependent TLR4/NF-κB inhibition, highlighting therapeutic potential.