Remimazolam alleviates acute lung injury by inhibiting ferroptosis: a multi-omics system pharmacology approach with experimental validation

Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical conditions with significant global health burdens. Ferroptosis, an iron-dependent form of regulated cell death characterized by lipid peroxidation, has been implicated in the pathogenesis of ALI/ARDS. Remimazolam (REM), an ultra-short-acting benzodiazepine sedative, has shown therapeutic potential in ALI/ARDS; however, whether its protective effects are mediated by Ferroptosis modulation remains unclear.

Methods: The anti-injury efficacy of REM was first assessed in LPS induced ALI mice. To investigate the molecular mechanisms underlying the protective effects of REM in ALI/ARDS, an integrated multi-omics approach was employed. Potential targets of REM and ALI/ARDS were systematically identified through pharmacophore-based screening. Publicly available RNA-sequencing (RNA-seq) data (GSE5883) from lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells was retrieved from the GEO data sets. Network pharmacology and transcriptomic analyses were employed to elucidate ferroptosis-related pathways potentially modulated by REM in LPS-induced ALI mice. Subsequent in vivo (ALI mice) and in vitro (MLE-12 cell) experimental validation was performed, wherein ferroptosis-related markers, including iron content, malondialdehyde (MDA), the ratio of reduced to oxidized glutathione (GSH/GSSG), cyclooxygenase-2 (COX2), solute carrier family 7 member 11 (SLC7A11), Glutathione Peroxidase 4 (GPX4), and heme oxygenase-1 (HO-1), were assessed. To further validate REM's HO-1-dependent anti-ferroptotic effects, we treated MLE-12 cells with an HO-1 inhibitor and assessed Ferroptosis biomarkers.

Results: REM treatment reduced pathological lung injury in ALI mice. Network pharmacology analysis revealed that REM's potential targets in ALI/ARDS were enriched in biological processes related to inflammation, oxidative stress, metal ion response, and fatty acid metabolism. RNA-seq analysis further confirmed that REM modulated genes associated with metal ion regulation, lipid metabolism, and oxidative stress. In vivo, REM significantly decreased LPS-induced pulmonary iron overload and MDA levels while simultaneously restoring the GSH/GSSG ratio. Furthermore, REM suppressed LPS-induced upregulation of COX2 and reversed downregulation of SLC7A11 and GPX4. Notably, HO-1 expression was significantly increased in the lung tissues of REM-treated ALI mice compared with ALI mice. In MLE-12 cells, LPS downregulated GPX4, an effect reversed by REM; HO-1 inhibition partially abrogated REM's protection.

Conclusions: This study demonstrated that REM alleviated LPS-induced lung injury by inhibiting Ferroptosis, potentially through upregulation of HO-1 and restoration of the SLC7A11-GSH-GPX4 axis. These findings provide novel mechanistic insights into the anti-ferroptotic effects of REM and highlight its therapeutic potential in ALI/ARDS management.

Acute lung injury; Acute respiratory distress syndrome; Ferroptosis; Heme oxygenase-1; Remimazolam.