5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

Hypobaric hypoxia (HH)-induced acute lung injury (ALI) is a growing concern. Tryptophan metabolite 5-Methoxytryptophan (5-MTP) is a recently identified endothelial protective factor. However, the role of 5-MTP in HH-induced ALI remains unclear. Forty healthy male human participants were enrolled in Chongqing (200m) to travel to Golmud (4260m). We found the decreased plasma 5-MTP level was correlated with HH-induced blood oxygen desaturation and acute mountain sickness. ELISA showed 5-MTP levels decreased in the plasma and lungs of ALI mice (male 6-8 weeks C57BL/6J), and also in the supernatant of mouse pulmonary microvascular endothelial cells (PMVECs) after hypoxia. Immunoblotting showed the synthetase Asmt was downregulated in mice lungs and PMVECs after hypoxia, which could be restored by si-Hif1α or NF-κB inhibitor. ChIP assays confirmed that hypoxia enhanced NF-κB p50 binding in Asmt promotor. Application of 5-MTP protected the hyperpermeability in ALI. The transmission electron microscope of mice lungs showed 5-MTP alleviated the endothelial barrier disruption and lipoperoxidation injury. Using limited proteolysis combined with mass spectrometry, we revealed Prdx6-Ser32 as the direct target of 5-MTP, consistent with molecular docking. molecular dynamics simulation, cellular thermal shift assays and microscale thermophoresis confirmed the interaction. 5-MTP prevented the hypoxia-induced decline of Prdx6 and lipoperoxidation. By MG132 and BafA1, we found hypoxia promoted the lysosomal degradation of Prdx6. Immunoblotting and immunofluorescence showed 5-MTP could reduce hypoxia-induced increased lysosomal localization of Prdx6. Prdx6-S32A impaired the protective effects of 5-MTP on PMVECs, and also the lysosomal localization of Prdx6. Adeno-associated viruses were injected intratracheally to confirm Prdx6-S32A also impaired the therapeutic effects of 5-MTP on ALI mice. 5-MTP is a predictive biomarker of hypoxic maladaptation. The restoration of 5-MTP attenuated ALI by inhibiting lipoperoxidation and protecting endothelial barrier via directly interaction with Ser32 site of Prdx6 to prevent its lysosomal degradation. Hypoxia leads to decreased synthesis of 5-Methoxytryptophan (5-MTP) in pulmonary microvascular endothelial cells by downregulating Asmt level via Hif1α induced NF-κB p50 nuclear translocation. 5-MTP can reduce the localization of Prdx6 to lysosomes and increase its protein level by reducing its lysosomal degradation through interacting with Prdx6-S32, without affecting its function of clearing lipid peroxides (LPOs), thus improving the lipid peroxidation and rescuing VE-cadherin level and endothelial barrier function. • 5-MTP is a potential biomarker and therapeutic clue in HH-induced responses. • Hypoxia downregulated Asmt expression in PMVECs via Hif1α induced NF-κB p50 nuclear translocation to decrease the synthesis of 5-MTP • 5-MTP attenuated ALI by inhibiting lipoperoxidation and protecting endothelial barrier. • 5-MTP interacted with Prdx6-S32 to reduce the lysosomal degradation of Prdx6.