Mailuoning Oral Liquid and Its Effective Ingredient Luteolin Ameliorate Lipopolysaccharide‐ and Cigarette Smoke Extract‐Induced Pulmonary Injury by Regulating cGAS – STING – IRF3 – PAI ‐1 Pathway

Mailuoning oral liquid (MLN O) has been used clinically to alleviate inflammatory and thrombotic illnesses, such as thromboangiitis obliterans (TAO), which often present with pulmonary injury in the early stage. However, the mechanisms of action of MLN O and its active ingredients remain unclear. This study aimed to explore the protective effects and mechanisms of MLN O and its effective components against pulmonary injury caused by lipopolysaccharide (LPS) and cigarette smoke extract (CSE). LPS- and CSE-induced rats and HUVECs or HUVECs stimulated by TGF-β1 were used in vivo and in vitro. The components of MLN O in rat plasma were detected with UPLC-QTOF-MS/MS and UPLC-QqQ-MS/MS. Western blotting was used to screen for the most promising and effective ingredient of MLN O. Masson and H&E stainings, immunohistochemistry, ELISA, western blotting, qRT-PCR, and immunofluorescence assays focused on the research of inflammation, fibrosis, and thrombosis. The mechanisms of action of MLN O and luteolin (LUT) were explored using siRNA transfection, ChIP, qRT-PCR, western blotting, immunofluorescence, molecular docking, cellular thermal shift, molecular dynamics simulation, and microscale thermophoresis assays. In total, 35 compounds of MLN O were detected in plasma using UPLC-QTOF-MS/MS. Furthermore, 18 components with higher contents were screened using UPLC-QqQ-MS/MS. The most promising effective ingredient, LUT, was identified by western blotting assay. MLN O and LUT significantly improved inflammation, epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EndMT), fibrosis, and thrombosis by inactivating the cGAS-STING pathway. siRNA transfection and ChIP assays showed that PAI-1 may be a downstream molecule of IRF3 in the cGAS-STING pathway. Most importantly, molecular docking, cellular thermal shift, molecular dynamics simulation, and microscale thermophoresis assays indicated that LUT might directly interact with cGAS. This study is the first to suggest that MLN O and its effective ingredient, LUT, significantly improved LPS- and CSE-induced pulmonary injury by regulating the cGAS-STING-IRF3-PAI-1 pathway, and that LUT may serve as an inhibitor of cGAS in this process. Our study provides a new therapeutic strategy for pulmonary injury and a solid theoretical basis for the clinical application of MLN O.