N-acetyl-lysyltyrosylcysteine amide, a novel systems pharmacology agent, reduces bronchopulmonary dysplasia in hyperoxic neonatal rat pups

Abstract

Bronchopulmonary dysplasia (BPD) is caused primarily by oxidative stress and inflammation. To induce BPD, neonatal rat pups were raised in hyperoxic (>90% O₂) environments from day one (P1) until day ten (P10) and treated with N-acetyl-lysyltyrosylcysteine amide (KYC). In vivo studies showed that KYC improved lung complexity, reduced myeloperoxidase (MPO) positive (+) myeloid cell counts, MPO protein, chlorotyrosine formation, increased endothelial cell CD31 expression, decreased 8-OH-dG and Cox-1/Cox-2, HMGB1, RAGE, TLR4, increased weight gain and improved survival in hyperoxic pups. EPR studies confirmed that MPO reaction mixtures oxidized KYC to a KYC thiyl radical. Adding recombinant HMGB1 to the MPO reaction mixture containing KYC resulted in KYC thiylation of HMGB1. In rat lung microvascular endothelial cell (RLMVEC) cultures, KYC thiylation of RLMVEC proteins was increased the most in RLMVEC cultures treated with MPO + H₂O₂, followed by H₂O₂, and then KYC alone. KYC treatment of hyperoxic pups decreased total HMGB1 in lung lysates, increased KYC thiylation of HMGB1, terminal HMGB1 thiol oxidation, decreased HMGB1 association with TLR4 and RAGE, and shifted HMGB1 in lung lysates from a non-acetylated to a lysyl-acetylated isoform, suggesting that KYC reduced lung cell death and that recruited immune cells had become the primary source of HMGB1 released into the hyperoxic lungs. MPO-dependent and independent KYC-thiylation of Keap1 were both increased in RLMVEC cultures. Treating hyperoxic pups with KYC increased KYC thiylation and S-glutathionylation of Keap1, and Nrf2 activation. These data suggest that KYC is a novel system pharmacological agent that exploits MPO to inhibit toxic oxidant production and is oxidized into a thiyl radical that inactivates HMGB1, activates Nrf2, and increases antioxidant enzyme expression to improve lung complexity and reduce BPD in hyperoxic rat pups.

Keywords: Bronchopulmonary dysplasia; High mobility group box-1; Kelch-like ECH-Associated protein 1; Myeloperoxidase; Nuclear factor erythroid 2-related factor 2.