Methane inhalation during ex vivo lung perfusion protects donor lungs after cardiac death via modulation of MAPK-related inflammatory and oxidative pathways

BACKGROUND: The scarcity of donor lungs remains a significant challenge in lung transplantation (LTx). Ex vivo lung perfusion (EVLP) presents a promising approach to repair and assess lungs obtained from donation after cardiac death (DCD). Methane is a biologically active gas with reported anti-inflammatory and antioxidant effects and may serve as an adjunctive strategy during EVLP. METHODS: A rat model of warm ischemia was established, followed by EVLP with or without methane supplementation (2.5%). Lung physiology, histological injury, edema, cell death, oxidative stress, and inflammatory cytokines were assessed after EVLP. RNA sequencing and gene set enrichment analysis were performed to explore underlying mechanisms. Donor left lungs were subsequently transplanted into recipient rats to evaluate post-transplant graft function, injury, and signaling pathway activation. RESULTS: Methane supplementation during EVLP was associated with improved oxygenation capacity, lower pulmonary vascular resistance, reduced pulmonary edema, and fewer cell death-related signals. Histological injury after EVLP showed a decreasing trend in methane-treated lungs but did not reach statistical significance compared with standard EVLP. Gene set enrichment analysis suggested downregulation of the MAPK signaling pathway. Methane treated lungs showed reduced phosphorylation of ERK, JNK, p38, and NF-κB p65, while enhancing Nrf2 and HO-1 expression. After transplantation, methane-preserved lungs exhibited attenuated inflammation and oxidative injury, with reduced cytokine levels accompanied by changes in MAPK-NF-κB/Nrf2 signaling. CONCLUSIONS: Methane inhalation during EVLP may improve DCD lung function by reducing inflammation and oxidative injury, and this effect persists after LTx.